Council Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) (Text with EEA relevance)

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Contents

Preamble
1. Article 1
2. Article 2
3. Article 3
4. Article 4
ANNEX
ANNEX PART A: METHODS FOR THE DETERMINATION OF PHYSICO-CHEMICAL PROPERTIES
A.1.
1.
1.4.
1.4.1.
1.4.2.
1.4.4.
1.5. TABLE: APPLICABILITY OF THE METHODS A. B. C. D.
1.6.
1.6.1.
1.6.1.1.
1.6.1.2. Apparatus:
1.6.2.
1.6.4.
4.(1)(2)(3)(4)
A.2.
1.
1.4.
1.5. Table 1: Comparison of the methods
1.6.
1.6.4.
1.6.6.
2. Table 2: Temperature — corrections factors fT
A.3.
1.
1.4.
1.4.1.
3. Table: 1.4.1.1.1.4.1.2.1.4.1.3.1.4.2.1.4.3.1.4.4. Applicability of methods
3. Table: 1.4.1.1.1.4.1.2. Applicability of methods
3. Table: 1.4.1.1.1.4.1.2.1.4.1.3.1.4.2. Applicability of methods
4.(1)(2)(3)(4)(5)(6)(7)
A.4.
1.
1.4.
1.5. Table: 1.4.1.1.4.2.1.4.3.1.4.4.1.4.5.1.4.6.1.4.7. Quality criteria
1.6.
1.6.1.
1.6.2.
1.6.2.2.
1.6.4.
1.6.4.2.
1.6.5.
1.6.6.
1.6.6.1. Inert gas:
1.6.6.2. Preparation of the saturator column:
1.6.7.
4.(1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)
A.5.
1.
1.6.
1.6.4.
1.6.4.1.
1.6.4.1.3. Figure Measuring body
1.6.4.2.
1.6.4.2.2. Mounting:
2. Table: Correction of the measured surface tension Only for aqueous solutions, ρ = 1 g/cm3
3.
4.(1)(2)(3)(4)
A.6.
1.
1.4.
1.5.
1.6.
1.6.3.
1.6.3.2. Test procedure:
1.6.3.3. Apparatus
1.6.3.4. Apparatus (see figures 4 and 3)
1.6.4.
2.
3.
A.8.
1.
1.3. Shake-flask method
1.4.
1.5.
1.5.1. Shake-flask method
1.5.2. Shake-flask method
1.5.3. Shake-flask method
1.6.
1.6.2.
1.6.2.1.
1.6.2.3.
1.6.3.
1.6.3.1. Apparatus
1.6.3.2. Calculation of dead time to
2. Shake-flask method
4.(1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)
A.9.
1.
1.5.
1.6.
1.6.3.
1.6.3.2. Abel apparatus:
A.10.
1.
1.6.
1.6.2.
3.
A.11.
1.
1.6.
A.12.
1.
1.3.
1.6.
1.6.1.
1.6.2.
1.6.3.
1.6.4.
A.13.
1.
1.6.
1.6.2.
2.
A.14.
1.
1.1.
1.4.
1.6.
1.6.1.
1.6.2.
1.6.2.2.
1.6.3.
1.6.3.2.
3.
4.(1)(2)(3)(4)
A.15.
1.
1.6.
A.16.
1.
1.6.
1.6.1.
4.
Figure 1 Pattern of 20 mm test cube
Figure 2 Typical temperature/time curve
A.17.
1.
1.6.
1.6.1.
1.6.2.
3.
3.2.
A.18.
1.
1.6.
2.
2.2.
3.(1)(2)(3)(4)
A.19.
1.
1.6.
2.
2.2.
3.(1)(2)(3)(4)
A.20.
1.
1.5.
1.5.4.
2.
3.
3.1.
A.21.
1.
1.6.
1.6.1.
1.6.1.2.
2.
2.1. Table 1 Examples of results
3.
3.2.
4.(1)
Figure 1 Pressure vessel
Figure 2 Support stand
Figure 3 Ignition system
Appendix For additional technical details, the following standards may be consulted for example.
1.
2.
4.
1.2. For solid substances
Appendix 1 Estimation method
INTRODUCTION
ESTIMATION METHOD
CALCULATION PROCEDURE
REPORT
LITERATURE
Appendix 2
Figure 1 Apparatus for determining the vapour pressure curve according to the dynamic method
Figure 2a Apparatus for determining the vapour pressure curve according to the static method (using a U-tube manometer)
Figure 2b Apparatus for determining the vapour pressure curve according to the static method (using a pressure indicator)
Figure 3 Isoteniscope (see reference 7)
Figure 4 Apparatus for determining the vapour pressure curve according to the vapour pressure balance method
Figure 5 Example of apparatus for evaporation at low pressure by effusion methode, with an effusion cell volume of 8 cm3
Figure 6a An example of a flow system for the determination of vapour pressure by the gas saturation method
Figure 6b An example of system for the determination of vapour pressure by the gas saturation method, with a capillary placed after the saturation chamber
Figure 7 Example of the experimental set-up for spinning rotor method
Figure 8 Example of spinning rotor measuring head
Appendix
Figure 1 Column elution method with recirculating pump
Figure 2 A typical microcolumn
Figure 3 A typical microcolumn
Figure 4 Column elution method with levelling vessel
CALCULATION METHODS Principle of the calculation methods
Hansch π-method
Rekker method
Hansch-Leo method
Combined method
Figure 5 Friction sensitivity apparatus
Appendix Examples of other methods for determination of number average molecular weight (Mn) for polymers
1.1.1.1.2.1.31.4
3.(1)(2)(3)(4)(5)(6)(7)(8)(9)
Appendix Guidance for correcting low molecular content for the presence of insoluble polymer
PART B METHODS FOR THE DETERMINATION OF TOXICITY AND OTHER HEALTH EFFECTS
GENERAL INTRODUCTION A.
B.1 bis.
1.
1.4.
1.5.
1.5.3.
1.6.
3.
4.(1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)
B.1 tris.
1.
1.4.
1.5.
1.6.
3.
4.(1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)
B.2.
1.
1.6.
1.6.2.
1.6.3.
3.
3.1.
B.3.
1.
1.6.
1.6.2.
1.6.3. Assessment of toxicity in the other sex
3.
B.4.
1.
1.4.
1.4.1.
1.4.2.
2.
3.
4.(1)(2)(3)(4)(5)(5a)(6)(7)(8)(9)
Table I Erythema and Eschar formation Oedema formation GRADING OF SKIN REACTIONS
B.5.
1.
1.4.
1.4.1.
1.4.2.
1.4.2.3.
1.4.2.7.
2.
3.
4.(1)(2)(3)(4)(5)(6)(6a)(7)(8)(9)(10)
Table I GRADING OF OCULAR LESIONS
Cornea
Iris
Conjunctivae
Chemosis
B.6.
1.
1.5.
1.5.1.
1.5.1.2.
1.5.1.3.
1.5.2.
1.5.2.2.
1.5.2.3.
3. Test report (GMPT and Buehler test)
B.7.
1.
1.4.
1.4.2.
1.4.3.
3. TEST REPORT
B.8.
1.
1.6.
1.6.2.
1.6.3.
3.
3.1.
pt_2
B.9.
1.
1.6.
1.6.2.
1.6.3.
3.
B.10.
1.
1.4.
1.4.1.
1.4.2.
1.4.3.
2.
3. TEST REPORT
4.(1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)
B.11.
1.
1.4.
1.4.1.
1.4.2.
1.5.
2.
3.
4.(1)(2)(3)(4)(5)(6)(7)(8)
B.12.
1.
1.4.
1.4.1.
1.4.2.
1.5.
1.5.2.
2.
3. TEST REPORT
4.(1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)
B.13/14.
1.
1.5.
1.5.1.
1.5.2.
2.
3. TEST REPORT
4.(1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)
B.15.
1.
1.6. Preparations
Metabolic activation
Tester strains
Media
Use of negative and positive controls
Exposure concentration
Incubation conditions
Spontaneous mutation frequencies
Number of replicates
3.
B.16.
1.
1.6. Preparations
Metabolic activation
Tester strains
Media
Use of negative and positive controls
Exposure concentrations
Incubation conditions
Spontaneous mitotic recombination frequencies
Number of replicates
3.
B.17.
1.
1.4.
1.4.1.
1.4.2.
1.4.3.
2.
3. TEST REPORT
4.(1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)
B.18.
1.
1.6. Preparations
Number of cultures
Use of negative and positive controls
Exposure concentrations
Cells
Metabolic activation
Preparation of cultures
Treatment of the cultures with the test substance Primary rat hepatocytes
Autoradiographic determinations
LSC determinations
2.
3.
B.19.
1.
1.6.
1.6.2. Number of cultures
1.6.3. Preparation of cultures
3.
B.20.
1.
1.6. Preparations
Stocks
Test substance
Number of animals
Route of administration
Use of negative and positive controls
Exposure levels
3.
B.21.
1.
1.6. Preparations
Cells
Medium
Test substance
Metabolic activation
Use of negative and positive controls
Exposure concentrations
3.
B.22.
1.
1.6. Preparations
Route of administration
Experimental animals
Number and sex
Use of negative and positive controls
Dose levels
Limit test

THE COMMISSION OF THE EUROPEAN COMMUNITIES,

Having regard to the Treaty establishing the European Community,

Having regard to Regulation (EC) No 1907/2006 of 18 December 2006 of the European Parliament and of the Council concerning the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), establishing a European Chemicals Agency, amending Directive 1999/45/EC and repealing Council Regulation (EEC) No 793/93 and Commission Regulation (EC) No 1488/94 as well as Council Directive 76/769/EEC and Commission Directives 91/155/EEC, 93/67/EEC, 93/105/EC and 2000/21/EC, and in particular Article 13(3)thereof,

Whereas:

(1) Pursuant to Regulation (EC) No 1907/2006, test methods are to be adopted at Community level for the purposes of tests on substances where such tests are required to generate information on intrinsic properties of substances.

(2) Council Directive 67/548/EEC of 27 June 1967 on the approximation of the laws, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous substances, laid down, in Annex V, methods for the determination of the physico-chemical properties, toxicity and ecotoxicity of substances and preparations. Annex V to Directive 67/548/EEC has been deleted by Directive 2006/121/EC of the European Parliament and of the Council with effect from 1 June 2008.

(3) The test methods contained in Annex V to Directive 67/548/EEC should be incorporated into this Regulation.

(4) This Regulation does not exclude the use of other test methods, provided that their use is in accordance with Article 13(3) of Regulation 1907/2006.

(5) The principles of replacement, reduction and refinement of the use of animals in procedures should be fully taken into account in the design of the test methods, in particular when appropriate validated methods become available to replace, reduce or refine animal testing.

(6) The provisions of this Regulation are in accordance with the opinion of the Committee established under Article 133 of Regulation (EC) No 1907/2006,

HAS ADOPTED THIS REGULATION:

Article 1

The test methods to be applied for the purposes of Regulation 1907/2006/EC are set out in the Annex to this Regulation.

Article 2

The Commission shall review, where appropriate, the test methods contained in this Regulation with a view to replacing, reducing or refining testing on vertebrate animals.

Article 3

All references to Annex V to Directive 67/548/EEC shall be construed as references to this Regulation.

Article 4

This Regulation shall enter into force on the day following its publication in the Official Journal of the European Union.

It shall apply from 1 June 2008. Done at Brussels, 30 May 2008. For the Commission Stavros Dimas Member of the Commission

ANNEX

ANNEX PART A: METHODS FOR THE DETERMINATION OF PHYSICO-CHEMICAL PROPERTIES

A.1. MELTING/FREEZING TEMPERATURE

A.1.

1. METHOD The majority of the methods described are based on the OECD Test Guideline (1). The fundamental principles are given in references (2) and (3).

1.

1.1. INTRODUCTION The methods and devices described are to be applied for the determination of the melting temperature of substances, without any restriction with respect to their degree of purity. The selection of the method is dependent on the nature of the substance to be tested. In consequence the limiting factor will be according to, whether or not the substance can be pulverised easily, with difficulty, or not at all. For some substances, the determination of the freezing or solidification temperature is more appropriate and the standards for these determinations have also been included in this method. Where, due to the particular properties of the substance, none of the above parameters can be conveniently measured, a pour point may be appropriate.

1.2. DEFINITIONS AND UNITS The melting temperature is defined as the temperature at which the phase transition from solid to liquid state occurs at atmospheric pressure and this temperature ideally corresponds to the freezing temperature. As the phase transition of many substances takes place over a temperature range, it is often described as the melting range. Conversion of units (K to oC) t = T - 273,15 t Celsius temperature, degree Celsius (oC) T thermodynamic temperature, kelvin (K)

1.3. REFERENCE SUBSTANCES Reference substances do not need to be employed in all cases when investigating a new substance. They should primarily serve to check the performance of the method from time to time and to allow comparison with results from other methods. Some calibration substances are listed in the references (4).

1.4. PRINCIPLE OF THE TEST METHOD The temperature (temperature range) of the phase transition from the solid to the liquid state or from the liquid to the solid state is determined. In practice while heating/cooling a sample of the test substance at atmospheric pressure the temperatures of the initial melting/freezing and the final stage of melting/freezing are determined. Five types of methods are described, namely capillary method, hot stage methods, freezing temperature determinations, methods of thermal analysis, and determination of the pour point (as developed for petroleum oils). In certain cases, it may be convenient to measure the freezing temperature in place of the melting temperature.

1.4.

1.4.1. Capillary method

1.4.1.

1.4.1.1. Melting temperature devices with liquid bath A small amount of the finely ground substance is placed in a capillary tube and packed tightly. The tube is heated, together with a thermometer, and the temperature rise is adjusted to less than about 1 K/min during the actual melting. The initial and final melting temperatures are determined.

1.4.1.2. Melting temperature devices with metal block As described under 1.4.1.1, except that the capillary tube and the thermometer are situated in a heated metal block, and can be observed through holes in the block.

1.4.1.3. Photocell detection The sample in the capillary tube is heated automatically in a metal cylinder. A beam of light is directed through the substance, by way of a hole in the cylinder, to a precisely calibrated photocell. The optical properties of most substances change from opaque to transparent when they are melting. The intensity of light reaching the photocell increases and sends a stop signal to the digital indicator reading out the temperature of a platinum resistance thermometer located in the heating chamber. This method is not suitable for some highly coloured substances.

1.4.2. Hot stages

1.4.2.

1.4.2.1. Kofler hot bar The Kofler hot bar uses two pieces of metal of different thermal conductivity, heated electrically, with the bar designed so that the temperature gradient is almost linear along its length. The temperature of the hot bar can range from 283 to 573 K with a special temperature-reading device including a runner with a pointer and tab designed for the specific bar. In order to determine a melting temperature, the substance is laid, in a thin layer, directly on the surface of the hot bar. In a few seconds a sharp dividing line between the fluid and solid phase develops. The temperature at the dividing line is read by adjusting the pointer to rest at the line.

1.4.2.2. Melt microscope Several microscope hot stages are in use for the determination of melting temperatures with very small quantities of material. In most of the hot stages the temperature is measured with a sensitive thermocouple but sometimes mercury thermometers are used. A typical microscope hot stage melting temperature apparatus has a heating chamber which contains a metal plate upon which the sample is placed on a slide. The centre of the metal plate contains a hole permitting the entrance of light from the illuminating mirror of the microscope. When in use, the chamber is closed by a glass plate to exclude air from the sample area. The heating of the sample is regulated by a rheostat. For very precise measurements on optically anisotropic substances, polarised light may be used.

1.4.2.3. Meniscus method This method is specifically used for polyamides. The temperature at which the displacement of a meniscus of silicone oil, enclosed between a hot stage and a cover-glass supported by the polyamide test specimen, is determined visually.

1.4.3. Method to determine the freezing temperature The sample is placed in a special test tube and placed in an apparatus for the determination of the freezing temperature. The sample is stirred gently and continuously during cooling and the temperature is measured at suitable intervals. As soon as the temperature remains constant for a few readings this temperature (corrected for thermometer error) is recorded as the freezing temperature. Supercooling must be avoided by maintaining equilibrium between the solid and the liquid phases.

1.4.4. Thermal analysis

1.4.4.

1.4.4.1 Differential thermal analysis (DTA) This technique records the difference in temperatures between the substance and a reference material as a function of temperature, while the substance and reference material are subjected to the same controlled temperature programme. When the sample undergoes a transition involving a change of enthalpy, that change is indicated by an endothermic (melting) or exothermic (freezing) departure from the base line of the temperature record.

1.4.4.2 Differential scanning calorimetry (DSC) This technique records the difference in energy inputs into a substance and a reference material, as a function of temperature, while the substance and reference material are subjected to the same controlled temperature programme. This energy is the energy necessary to establish zero temperature difference between the substance and the reference material. When the sample undergoes a transition involving a change of enthalpy, that change is indicated by an endothermic (melting) or exothermic (freezing) departure from the base line of the heat flow record.

1.4.5. Pour point This method was developed for use with petroleum oils and is suitable for use with oily substances with low melting temperatures. After preliminary heating, the sample is cooled at a specific rate and examined at intervals of 3 K for flow characteristics. The lowest temperature at which movement of the substance is observed is recorded as the pour point.

1.5. QUALITY CRITERIA The applicability and accuracy of the different methods used for the determination of the melting temperature/melting range are listed in the following table:

1.5. TABLE: APPLICABILITY OF THE METHODS A. B. C. D.

Capillary methods Method of measurement Substances which can be pulverised Substances which are not readily pulverised Temperature range Estimated accuracy Existing standards Melting temperature devices with liquid bath yes only to a few 273 to 573 K ±0,3 K JIS K 0064 Melting temperature with metal block yes only to a few 293 to >573 K ±0,5 K ISO 1218 (E) Photocell detection yes several with appliance devices 253 to 573 K ±0,5 K Hot stages and freezing methods Method of measurement Substances which can be pulverised Substances which are not readily pulverised Temperature range Estimated accuracy Existing standards Kofler hot bar yes no 283 to > 573 K ± 1K ANSI/ASTM D 3451-76 Melt microscope yes only to a few 273 to > 573 K ±0,5 K DIN 53736 Meniscus method no specifically for polyamides 293 to > 573 K ±0,5 K ISO 1218 (E) Freezing temperature yes yes 223 to 573 K ±0,5 K e.g. BS 4695 Thermal analysis Method of measurement Substances which can be pulverised Substances which are not readily pulverised Temperature range Estimated accuracy Existing standards Differential thermal analysis yes yes 173 to 1273 K up to 600 K ±0,5 K up to 1273 K ±2,0 K ASTM E 537-76 Differential scanning calorimetry yes yes 173 to 1273 K up to 600 K ±0,5 K up to 1273 K ±2,0 K ASTM E 537-76 Pour point Method of measurement Substances which can be pulverised Substances which are not readily pulverised Temperature range Estimated accuracy Existing standards Pour point for petroleum oils and oily substances for petroleum oils and oily substances 223 to 323 K ±0,3 K ASTM D 97-66

1.6. DESCRIPTION OF THE METHODS The procedures of nearly all the test methods have been described in international and national standards (see Appendix 1).

1.6.

1.6.1. Methods with capillary tube When subjected to a slow temperature rise, finely pulverised substances usually show the stages of melting shown in figure 1.

1.6.1.

Figure 1 Stage A Stage B Stage C Stage D Stage E Stage A (beginning of melting): fine droplets adhere uniformly to the inside wall of the capillary tube Stage B a clearance appears between the sample and the inside wall due to shrinkage of the melt Stage C the shrunken sample begins to collapse downwards and liquefies Stage D a complete meniscus is formed at the surface but an appreciate amount of the sample remains solid Stage E (final stage melting): there are no solid particles During the determination of the melting temperature, the temperatures are recorded at the beginning of the melting and at the final stage.

1.6.1.1. Melting temperature devices with liquid bath apparatus Figure 2 shows a type of standardised melting temperature apparatus made of glass (JIS K 0064); all specifications are in millimeters.

1.6.1.1.

Figure 2 A: Measurement vessel B: Stopper C: Vent D: Thermometer E: Auxiliary thermometer F: Bath liquid G: Capillary tube made of glass, 80 to 100 mm in length, 1,0 ± 0,2 mm inner diameter, 0,2 to 0,3 mm wall thickness H: Side tube

Bath liquid:

A suitable liquid should be chosen. The choice of the liquid depends upon the melting temperature to be determined, e.g. liquid paraffin for melting temperatures no higher than 473 K, silicone oil for melting temperatures no higher than 573 K. For melting temperatures above 523 K, a mixture consisting of three parts sulphuric acid and two parts potassium sulphate (in mass ratio) can be used. Suitable precautions should be taken if a mixture such as this is used.

Thermometer:

Only those thermometers should be used which fulfil the requirements of the following or equivalent standards: ASTM E 1-71, DIN 12770, JIS K 8001.

Procedure:

The dry substance is finely pulverised in a mortar and is put into the capillary tube, fused at one end, so that the filling level is approximately 3 mm after being tightly packed. To obtain a uniform packed sample, the capillary tube should be dropped from a height of approximately 700 mm through a glass tube vertically onto a watch glass. The filled capillary tube is placed in the bath so that the middle part of the mercury bulb of the thermometer touches the capillary tube at the part where the sample is located. Usually the capillary tube is introduced into the apparatus about 10 K below the melting temperature. The bath liquid is heated so that the temperature rise is approximately 3 K/min. The liquid should be stirred. At about 10 K below the expected melting temperature the rate of temperature rise is adjusted to a maximum of 1 K/min.

Calculation:

The calculation of the melting temperature is as follows: T = TD + 0,00016 (TD - TE) n where: T corrected melting temperature in K TD temperature reading of thermometer D in K TE temperature reading of thermometer E in K n number of graduations of mercury thread on thermometer D at emergent stem.

1.6.1.2. Melting temperature devices with metal block

1.6.1.2. Apparatus:

This consists of:

a cylindrical metal block, the upper part of which is hollow and forms a chamber (see figure 3),

a metal plug, with two or more holes, allowing tubes to be mounted into the metal block,

a heating system, for the metal block, provided for example by an electrical resistance enclosed in the block,

a rheostat for regulation of power input, if electric heating is used,

four windows of heat-resistant glass on the lateral walls of the chamber, diametrically disposed at right-angles to each other. In front of one of these windows is mounted an eye-piece for observing the capillary tube. The other three windows are used for illuminating the inside of the enclosure by means of lamps,

a capillary tube of heat-resistant glass closed at one end (see 1.6.1.1).

Thermometer:

See standards mentioned in 1.6.1.1. Thermoelectrical measuring devices with comparable accuracy are also applicable.

Figure 3 Thermometer Capillary tube Metal plug Eye-piece Lamp Electrical resistance Metal heating block

1.6.1.3. Photocell detection Apparatus and procedure: The apparatus consists of a metal chamber with automated heating system. Three capillary are filled accordingly to 1.6.1.1 and placed in the oven. Several linear increases of temperature are available for calibrating the apparatus and the suitable temperature rise is electrically adjusted at a pre-selected constant and linear rate. recorders show the actual oven temperature and the temperature of the substance in the capillary tubes.

1.6.2. Hot stages

1.6.2.

1.6.2.1. Kofler hot bar See Appendix.

1.6.2.2. Melt microscope See Appendix.

1.6.2.3. Meniscus method (polyamides) See Appendix. The heating rate through the melting temperature should be less than 1 K/min.

1.6.3. Methods for the determination of the freezing temperature See Appendix.

1.6.4. Thermal analysis

1.6.4.

1.6.4.1. Differential thermal analysis See Appendix.

1.6.4.2. Differential scanning calorimetry See Appendix.

1.6.5. Determination of the pour point See Appendix.

2. DATA A thermometer correction is necessary in some cases.

3. REPORTING The test report shall, if possible, include the following information: The mean of at least two measurements which are in the range of the estimated accuracy (see tables) is reported as the melting temperature. If the difference between the temperature at the beginning and at the final stage of melting is within the limits of the accuracy of the method, the temperature at the final stage of melting is taken as the melting temperature; otherwise the two temperatures are reported. If the substance decomposes or sublimes before the melting temperature is reached, the temperature at which the effect is observed shall be reported. All information and remarks relevant for the interpretation of results have to be reported, especially with regard to impurities and physical state of the substance.

method used,

precise specification of the substance (identity and impurities) and preliminary purification step, if any,

an estimate of the accuracy.

4.(1)(2)(3)(4)

REFERENCES OECD, Paris, 1981, Test Guideline 102, Decision of the Council C(81) 30 final. IUPAC, B. Le Neindre, B. Vodar, eds. Experimental thermodynamics, Butterworths, London 1975, vol. II, p. 803-834. R. Weissberger ed.: Technique of organic Chemistry, Physical Methods of Organic Chemistry, 3rd ed., Interscience Publ., New York, 1959, vol. I, Part I, Chapter VII. IUPAC, Physicochemical measurements: Catalogue of reference materials from national laboratories, Pure and applied chemistry, 1976, vol. 48, p. 505-515.

A.2. BOILING TEMPERATURE

A.2.

1. METHOD The majority of the methods described are based on the OECD Test Guideline (1). The fundamental principles are given in references (2) and (3).

1.

1.1. INTRODUCTION The methods and devices described here can be applied to liquid and low melting substances, provided that these do not undergo chemical reaction below the boiling temperature (for example: auto-oxidation, rearrangement, degradation, etc.). The methods can be applied to pure and to impure liquid substances. Emphasis is put on the methods using photocell detection and thermal analysis, because these methods allow the determination of melting as well as boiling temperatures. Moreover, measurements can be performed automatically. The dynamic method has the advantage that it can also be applied to the determination of the vapour pressure and it is not necessary to correct the boiling temperature to the normal pressure (101,325 kPa) because the normal pressure can be adjusted during the measurement by a manostat. Remarks: The influence of impurities on the determination of the boiling temperature depends greatly upon the nature of the impurity. When there are volatile impurities in the sample, which could affect the results, the substance may be purified.

1.2. DEFINITIONS AND UNITS The normal boiling temperature is defined as the temperature at which the vapour pressure of a liquid is 101,325 kPa. If the boiling temperature is not measured at normal atmospheric pressure, the temperature dependence of the vapour pressure can be described by the Clausius-Clapeyron equation: log p = ΔHv2,3RT+const. where: p the vapour pressure of the substance in pascals Δ Hv its heat of vaporisation in J mol-1 R the universal molar gas constant = 8,314 J mol-1 K-1 T thermodynamic temperature in K The boiling temperature is stated with regard to the ambient pressure during the measurement. Conversions Pressure (units: kPa) 100 kPa 1 bar = 0,1 MPa (bar is still permissible but not recommended) 133 Pa 1 mm Hg = 1 Torr (the units mm Hg and Torr are not permitted) 1 atm standard atmosphere = 101325 Pa (the unit atm is not permitted) Temperature (units: K) t = T - 273,15 t Celsius temperature, degree Celsius (oC) T thermodynamic temperature, kelvin (K)

1.4. PRINCIPLE OF THE TEST METHOD Five methods for the determination of the boiling temperature (boiling range) are based on the measurement of the boiling temperature, two others are based on thermal analysis.

1.4.

1.4.1. Determination by use of the ebulliometer Ebulliometers were originally developed for the determination of the molecular weight by boiling temperature elevation, but they are also suited for exact boiling temperature measurements. A very simple apparatus is described in ASTM D 1120-72 (see Appendix). The liquid is heated in this apparatus under equilibrium conditions at atmospheric pressure until it is boiling.

1.4.2. Dynamic method This method involves the measurement of the vapour recondensation temperature by means of an appropriate thermometer in the reflux while boiling. The pressure can be varied in this method.

1.4.3. Distillation method for boiling temperature This method involves distillation of the liquid and measurement of the vapour recondensation temperature and determination of the amount of distillate.

1.4.4. Method according to Siwoloboff A sample is heated in a sample tube, which is immersed in a liquid in a heat-bath. A fused capillary, containing an air bubble in the lower part, is dipped in the sample tube.

1.4.5. Photocell detection Following the principle according to Siwoloboff, automatic photo-electrical measurement is made using rising bubbles.

1.4.6. Differential thermal analysis This technique records the difference in temperatures between the substance and a reference material as a function of temperature, while the substance and reference material are subjected to the same controlled temperature programme. When the sample undergoes a transition involving a change of enthalpy, that change is indicated by an endothermic departure (boiling) from the base line of the temperature record.

1.4.7. Differential scanning calorimetry This technique records the difference in energy inputs into a substance and a reference material as a function of temperature, while the substance and reference material are subjected to the same controlled temperature programme. This energy is the energy necessary to establish zero temperature difference between the substance and the reference material. When the sample undergoes a transition involving a change of enthalpy, that change is indicated by an endothermic departure (boiling) from the base line of the heat flow record.

1.5. Table 1: Comparison of the methods

QUALITY CRITERIA The applicability and accuracy of the different methods used for the determination of the boiling temperature/boiling range are listed in table 1. Method of measurement Estimated accuracy Existing standard Ebulliometer ±1,4 K (up to 373 K) ±2,5 K (up to 600 K) ASTM D 1120-72 Dynamic method ±0,5 K (up to 600 K) Distillation process (boiling range) ±0,5 K (up to 600 K) ISO/R 918, DIN 53171, BS 4591/71 According to Siwoloboff ± 2 K (up to 600 K) Photocell detection ±0,3 K (up to 373 K) Differential thermal calorimetry ±0,5 K (up to 600 K) ±2,0 K (up to 1273 K) ASTM E 537-76 Differential scanning calorimetry ±0,5 K (up to 600 K) ±2,0 K (up to 1273 K) ASTM E 537-76

1.6. DESCRIPTION OF THE METHODS The procedures of some test methods have been described in international and national standards (see Appendix).

1.6.

1.6.1. Ebulliometer See Appendix.

1.6.2. Dynamic method See test method A.4 for the determination of the vapour pressure. The boiling temperature observed with an applied pressure of 101,325 kPa is recorded.

1.6.3. Distillation process (boiling range) See Appendix.

1.6.4. Method according to Siwoloboff The sample is heated in a melting temperature apparatus in a sample tube, with a diameter of approximately 5 mm (figure 1). Figure 1 shows a type of standardised melting and boiling temperature apparatus (JIS K 0064) (made of glass, all specifications in millimetres). A capillary tube (boiling capillary) which is fused about 1 cm above the lower end is placed in the sample tube. The level to which the test substance is added is such that the fused section of the capillary is below the surface of the liquid. The sample tube containing the boiling capillary is fastened either to the thermometer with a rubber band or is fixed with a support from the side (see figure 2). Figure 2 Principle according to Siwoloboff Figure 3 Modified principle The bath liquid is chosen according to boiling temperature. At temperatures up to 573 K, silicone oil can be used. Liquid paraffin may only be used up to 473 K. The heating of the bath liquid should be adjusted to a temperature rise of 3 K/min at first. The bath liquid must be stirred. At about 10 K below the expected boiling temperature, the heating is reduced so that the rate of temperature rise is less than 1 K/min. Upon approach of the boiling temperature, bubbles begin to emerge rapidly from the boiling capillary. The boiling temperature is that temperature when, on momentary cooling, the string of bubbles stops and fluid suddenly starts rising in the capillary. The corresponding thermometer reading is the boiling temperature of the substance. In the modified principle (figure 3) the boiling temperature is determined in a melting temperature capillary. It is stretched to a fine point about 2 cm in length (a) and a small amount of the sample is sucked up. The open end of the fine capillary is closed by melting, so that a small air bubble is located at the end. While heating in the melting temperature apparatus (b), the air bubble expands. The boiling temperature corresponds to the temperature at which the substance plug reaches the level of the surface of the bath liquid (c).

1.6.4.

Figure 1 A: Measuring vessel B: Stopper C: Vent D: Thermometer E: Auxiliary thermometer F: Bath liquid G: Sample tube, maximum 5 mm outer diameter; containing a capillary tube, approximately 100 mm long, approximately 1 mm long inner diameter and approximately 0,2 to 0,3 mm wall-thickness H: Side tube

1.6.5. Photocell detection The sample is heated in a capillary tube inside a heated metal block. A light beam is directed, via suitable holes in the block, through the substance onto a precisely calibrated photocell. During the increase of the sample temperature, single air bubbles emerge from the boiling capillary. When the boiling temperature is reached the number of bubbles increases greatly. This causes a change in the intensity of light, recorded by a photocell, and gives a stop signal to the indicator reading out the temperature of a platinum resistance thermometer located in the block. This method is especially useful because it allows determinations below room temperature down to 253,15 K (– 20 oC) without any changes in the apparatus. The instrument merely has to be placed in a cooling bath.

1.6.6. Thermal analysis

1.6.6.

1.6.6.1. Differential thermal analysis See Appendix.

1.6.6.2. Differential scanning calorimetry See Appendix.

2. Table 2: Temperature — corrections factors fT

DATA At small deviations from the normal pressure (max. ± 5 kPa) the boiling temperatures are normalised to Tn by means of the following number-value equation by Sidney Young: Tn = T + (fT × Δp) where: Δp (101,325 - p) [note sign] P pressure measurement in kPa fT rate of change of boiling temperature with pressure in K/kPa T measured boiling temperature in K Tn boiling temperature corrected to normal pressure in K The temperature-correction factors, fT, and equations for their approximation are included in the international and national standards mentioned above for many substances. For example, the DIN 53171 method mentions the following rough corrections for solvents included in paints: Temperature T (K) Correction factor fT (K/kPa) 323,15 0,26 348,15 0,28 373,15 0,31 398,15 0,33 423,15 0,35 448,15 0,37 473,15 0,39 498,15 0,41 523,15 0,4 548,15 0,45 573,15 0,47

3. REPORTING The test report shall, if possible, include the following information: The mean of at least two measurements which are in the range of the estimated accuracy (see table 1) is reported as the boiling temperature. The measured boiling temperatures and their mean shall be stated and the pressure(s) at which the measurements were made shall be reported in kPa. The pressure should preferably be close to normal atmospheric pressure. All information and remarks relevant for the interpretation of results have to be reported, especially with regard to impurities and physical state of the substance.

method used,

precise specification of the substance (identity and impurities) and preliminary purification step, if any,

an estimate of the accuracy.

4.(1)(2)(3) REFERENCES OECD, Paris, 1981, Test Guideline 103, Decision of the Council C (81) 30 final. IUPAC, B. Le Neindre, B. Vodar, editions. Experimental thermodynamics, Butterworths, London, 1975, vol. II. R. Weissberger edition: Technique of organic chemistry, Physical methods of organic chemistry, Third Edition, Interscience Publications, New York, 1959, vol. I, Part I, Chapter VIII.

A.3. RELATIVE DENSITY

A.3.

1. METHOD The methods described are based on the OECD Test Guideline (1). The fundamental principles are given in reference (2).

1.

1.1. INTRODUCTION The methods for determining relative density described are applicable to solid and to liquid substances, without any restriction in respect to their degree of purity. The various methods to be used are listed in table 1.

1.2. DEFINITIONS AND UNITS The relative density D204 of solids or liquids is the ratio between the mass of a volume of substance to be examined, determined at 20 oC, and the mass of the same volume of water, determined at 4 oC. The relative density has no dimension. The density, ρ, of a substance is the quotient of the mass, m, and its volume, v. The density, ρ, is given, in SI units, in kg/m3.

1.3. REFERENCE SUBSTANCES (1) (3) Reference substances do not need to be employed in all cases when investigating a new substance. They should primarily serve to check the performance of the method from time to time and to allow comparison with results from other methods.

1.4. PRINCIPLE OF THE METHODS Four classes of methods are used.

1.4.

1.4.1. Buoyancy methods

1.4.1.

1.4.1.1. Hydrometer (for liquid substances) Sufficiently accurate and quick determinations of density may be obtained by the floating hydrometers, which allow the density of a liquid to be deduced from the depth of immersion by reading a graduated scale.

1.4.1.2. Hydrostatic balance (for liquid and solid substances) The difference between the weight of a test sample measured in air and in a suitable liquid (e.g. water) can be employed to determine its density. For solids, the measured density is only representative of the particular sample employed. For the determination of density of liquids, a body of known volume, v, is weighed first in air and then in the liquid.

1.4.1.3. Immersed body method (for liquid substances) (4) In this method, the density of a liquid is determined from the difference between the results of weighing the liquid before and after immersing a body of known volume in the test liquid.

1.4.2. Pycnometer methods For solids or liquids, pycnometers of various shapes and with known volumes may be employed. The density is calculated from the difference in weight between the full and empty pycnometer and its known volume.

1.4.3. Air comparison pycnometer (for solids) The density of a solid in any form can be measured at room temperature with the gas comparison pycnometer. The volume of a substance is measured in air or in an inert gas in a cylinder of variable calibrated volume. For the calculation of density one mass measurement is taken after concluding the volume measurement.

1.4.4. Oscillating densitimeter (5) (6) (7) The density of a liquid can be measured by an oscillating densitimeter. A mechanical oscillator constructed in the form of a U-tube is vibrated at the resonance frequency of the oscillator which depends on its mass. Introducing a sample changes the resonance frequency of the oscillator. The apparatus has to be calibrated by two liquid substances of known densities. These substances should preferably be chosen such that their densities span the range to be measured.

1.5. QUALITY CRITERIA The applicability of the different methods used for the determination of the relative density is listed in the table.

1.6. DESCRIPTION OF THE METHODS The standards given as examples, which are to be consulted for additional technical details, are attached in the Appendix. The tests have to be run at 20 oC, and at least two measurements performed.

2. DATA See standards.

3. Table: 1.4.1.1.1.4.1.2.1.4.1.3.1.4.2.1.4.3.1.4.4. Applicability of methods

REPORTING The test report shall, if possible, include the following information: The relative density, D204, shall be reported as defined in 1.2, along with the physical state of the measured substance. All information and remarks relevant for the interpretation of results have to be reported, especially with regard to impurities and physical state of the substance. Method of measurement Density Maximum possible dynamic viscosity Existing Standards solid liquid Hydrometer yes 5 Pa s ISO 387, ISO 649-2, NF T 20-050 Hydrostatic balance yes ISO 1183 (A) yes 5 Pa s ISO 901 and 758 Immersed body method yes 20 Pa s DIN 53217 Pycnometer ISO 3507 yes ISO 1183(B), NF T 20-053 yes 500 Pa s ISO 758 Air comparison pycnometer yes DIN 55990 Teil 3, DIN 53243 Oscillating densitimer yes 5 Pa s

method used,

precise specification of the substance (identity and impurities) and preliminary purification step, if any.

3. Table: 1.4.1.1.1.4.1.2. Applicability of methods

(a) solids

(b) liquids

3. Table: 1.4.1.1.1.4.1.2.1.4.1.3.1.4.2. Applicability of methods

(a) solids

(b) liquids

4.(1)(2)(3)(4)(5)(6)(7)

REFERENCES OECD, Paris, 1981, Test Guideline 109, Decision of the Council C(81) 30 final. R. Weissberger ed., Technique of Organic Chemistry, Physical Methods of Organic Chemistry, 3rd ed., Chapter IV, Interscience Publ., New York, 1959, vol. I, Part 1. IUPAC, Recommended reference materials for realization of physico-chemical properties, Pure and applied chemistry, 1976, vol. 48, p. 508. Wagenbreth, H., Die Tauchkugel zur Bestimmung der Dichte von Flüssigkeiten, Technisches Messen tm, 1979, vol. II, p. 427-430. Leopold, H., Die digitale Messung von Flüssigkeiten, Elektronik, 1970, vol. 19, p. 297-302. Baumgarten, D., Füllmengenkontrolle bei vorgepackten Erzeugnissen -Verfahren zur Dichtebestimmung bei flüssigen Produkten und ihre praktische Anwendung, Die Pharmazeutische Industrie, 1975, vol. 37, p. 717-726. Riemann, J., Der Einsatz der digitalen Dichtemessung im Brauereilaboratorium, Brauwissenschaft, 1976, vol. 9, p. 253-255.

A.4. VAPOUR PRESSURE

A.4.

1. METHOD The majority of the methods described are based on the OECD Test Guideline (1). The fundamental principles are given in references (2) and (3).

1.

1.1. INTRODUCTION It is useful to have preliminary information on the structure, the melting temperature and the boiling temperature of the substance to perform this test. There is no single measurement procedure applicable to the entire range of vapour pressures. Therefore, several methods are recommended to be used for the measurement of vapour pressure from < 10-4 to 105 Pa. Impurities will usually affect the vapour pressure, and to an extent which depends greatly upon the kind of impurity. Where there are volatile impurities in the sample, which could affect the result, the substance may be purified. It may also be appropriate to quote the vapour pressure for the technical material. Some methods described here use apparatus with metallic parts; this should be considered when testing corrosive substances.

1.2. DEFINITIONS AND UNITS The vapour pressure of a substance is defined as the saturation pressure above a solid or liquid substance. At the thermodynamic equilibrium, the vapour pressure of a pure substance is a function of temperature only. The SI unit of pressure which should be used is the pascal (Pa). Units which have been employed historically, together with their conversion factors, are: 1 Torr (≡ 1 mm Hg) = 1,333 × 102 Pa 1 atmosphere = 1,013 × 105 Pa 1 bar = 105 Pa The SI unit of temperature is the kelvin (K). The universal molar gas constant R is 8,314 J mol-1 K-1 The temperature dependence of the vapour pressure is described by the Clausius-Clapeyron equation: log p = ΔHv2,3RT+const. where: p the vapour pressure of the substance in pascals ΔHv its heat of vaporisation in Jmol-1 R the universal molar gas constant in Jmol-1 K-1 T thermodynamic temperature in K

1.4. PRINCIPLE OF THE TEST METHODS For determining the vapour pressure, seven methods are proposed which can be applied in different vapour pressure ranges. For each method, the vapour pressure is determined at various temperatures. In a limited temperature range, the logarithm of the vapour pressure of a pure substance is a linear function of the inverse of the temperature.

1.4.

1.4.1. Dynamic method In the dynamic method, the boiling temperature which pertains to a specified pressure is measured. Recommended range: 103 up to 105 Pa. This method has also been recommended for the determination of normal boiling temperature and is useful for that purpose up to 600 K.

1.4.2. Static method In the static process, at thermodynamic equilibrium, the vapour pressure established in a closed system is determined at a specified temperature. This method is suitable for one component and multicomponent solids and liquids. Recommended range: 10 up to 105 Pa. This method can also be used in the range 1 to 10 Pa, providing care is taken.

1.4.3. Isoteniscope This standardised method is also a static method but is usually not suitable for multicomponent systems. Additional information is available in ASTM method D-2879-86. Recommended range: from 100 up to 105 Pa.

1.4.4. Effusion method: Vapour pressure balance The quantity of substance leaving a cell per unit time through an aperture of known size is determined under vacuum conditions such that return of substance into the cell is negligible (e.g. by measurement of the pulse generated on a sensitive balance by a vapour jet or by measuring the weight loss). Recommended range: 10-3 to 1 Pa.

1.4.5. Effusion method: By loss of weight or by trapping vaporisate The method is based on the estimation of the mass of test substance flowing out per unit of time of a Knudsen cell (4) in the form of vapour, through a micro-orifice under ultra-vacuum conditions. The mass of effused vapour can be obtained either by determining the loss of mass of the cell or by condensing the vapour at low temperature and determining the amount of volatilised substance using chromatographic analysis. The vapour pressure is calculated by applying the Hertz-Knudsen relation. Recommended range: 10-3 to 1 Pa.

1.4.6. Gas saturation method A stream of inert carrier gas is passed over the substance in such a way that it becomes saturated with its vapour. The amount of material transported by a known amount of carrier gas is measurable either by collection in a suitable trap or by an intrain analytical technique. This is then used to calculate the vapour pressure at a given temperature. Recommended range: 10-4 to 1 Pa. This method can also be used in the range 1 to 10 Pa providing care is taken.

1.4.7. Spinning rotor In the spinning rotor gauge, the actual measuring element is a small steel ball which is suspended in a magnetic field and rotates with high speed. The gas pressure is deduced from the pressure-dependent slow-down of the steel ball. Recommended range: 10-4 to 0,5 Pa.

1.5. Table: 1.4.1.1.4.2.1.4.3.1.4.4.1.4.5.1.4.6.1.4.7. Quality criteria

QUALITY CRITERIA The various methods of determining the vapour pressure are compared as to application, repeatability, reproducibility, measuring range, existing standard. This is done in the following table. Measuring Method Substances Estimated Repeatability Estimated Reproducibility Recommended Range Existing Standard solid liquid Dynamic method Low melting yes Up to 25 % Up to 25 % 103 Pa to 2 × 103 Pa — 1 to 5 % 1 to 5 % 2 × 103 Pa to 105 Pa — Static method yes yes 5 to 10 % 5 to 10 % 10 Pa to 105 Pa NFT 20-048 (5) Isoteniscope yes yes 5 to 10 % 5 to 10 % 102 Pa to 105 Pa ASTM-D 2879-86 Effusion method Vap.Pres.balance yes yes 5 to 20 % 5 to 50 % 10-3 Pa to 1 Pa NFT 20-047(6) Effusion method weigt loss yes yes 10 to 30 % — 10-3 Pa to 1 Pa — Gas saturation method yes yes 10 to 30 % Up to 50 % 10-4 Pa to 1 Pa — Spinning rotor method yes yes 10 to 20 % — 10-4 Pa to 0,5 Pa —

1.6. DESCRIPTION OF THE METHODS

1.6.

1.6.1. Dynamic measurement

1.6.1.

1.6.1.1. Apparatus The measuring apparatus typically consists of a boiling vessel with attached cooler made of glass or metal (figure 1), equipment for measuring the temperature, and equipment for regulating and measuring the pressure. A typical measuring apparatus shown in the drawing is made from heat-resistant glass and is composed of five parts: The large, partially double-walled tube consists of a ground jacket joint, a cooler, a cooling vessel and an inlet. The glass cylinder, with a Cottrell pump, is mounted in the boiling section of the tube and has a rough surface of crushed glass to avoid bumping in the boiling process. The temperature is measured with a suitable temperature sensor (e.g. resistance thermometer, jacket thermocouple) immersed in the apparatus to the point of measurement (No 5, figure 1) through a suitable inlet (e.g. male ground joint). The necessary connections are made to the pressure regulation and measuring equipment. The bulb, which acts as a buffer volume, is connected with the measuring apparatus by means of a capillary tube. The boiling vessel is heated by a heating element (e.g. cartridge heater) inserted into the glass apparatus from below. The heating current required is set and regulated via a thermocouple. The necessary vacuum of between 102 Pa and approximately 105 Pa is produced with a vacuum pump. A suitable valve is used to meter air or nitrogen for pressure regulation (measuring range approximately 102 to 105 Pa) and ventilation. Pressure is measured with a manometer.

1.6.1.2. Measurement procedure The vapour pressure is measured by determining the boiling temperature of the sample at various specified pressures between roughly 103 and 105 Pa. A steady temperature under constant pressure indicates that the boiling temperature has been reached. Frothing substances cannot be measured using this method. The substance is placed in the clean, dry sample vessel. Problems may be encountered with non-powder solids but these can sometimes be solved by heating the cooling jacket. Once the vessel has been filled the apparatus is sealed at the flange and the substance degassed. The lowest desired pressure is then set and the heating is switched on. At the same time, the temperature sensor is connected to a recorder. Equilibrium is reached when a constant boiling temperature is recorded at constant pressure. Particular care must be taken to avoid bumping during boiling. In addition, complete condensation must occur on the cooler. When determining the vapour pressure of low melting solids, care should be taken to avoid the condenser blocking. After recording this equilibrium point, a higher pressure is set. The process is continued in this manner until 105 Pa has been reached (approximately 5 to 10 measuring points in all). As a check, equilibrium points must be repeated at decreasing pressures.

1.6.2. Static measurement

1.6.2.

1.6.2.1. Apparatus The apparatus comprises a container for the sample, a heating and cooling system to regulate the temperature of the sample and measure the temperature. The apparatus also includes instruments to set and measure the pressure. Figures 2a and 2b illustrate the basic principles involved. The sample chamber (figure 2a) is bounded on one side by a suitable high-vacuum valve. A U-tube containing a suitable manometer fluid is attached to the other side. One end of the U-tube branches off to the vacuum pump, the nitrogen cylinder or ventilation valve, and a manometer. A pressure gauge with a pressure indicator can be used instead of a U-tube (figure 2b). In order to regulate the temperature of the sample, the sample vessel together with valve and U-tube or pressure gauge is placed in a bath which is kept at a constant temperature of ±0,2 K. The temperature measurements are taken on the outside wall of the vessel containing the sample or in the vessel itself. A vacuum pump with an upstream cooling trap is used to evacuate the apparatus. In method 2a the vapour pressure of the substance is measured indirectly using a zero indicator. This takes into account the fact that the density of the fluid in the U-tube alters if the temperature changes greatly. The following fluids are suitable for use as zero indicators for the U-tube, depending on the pressure range and the chemical behaviour of the test substance: silicone fluids, phthalates. The test substance must not dissolve noticeably in or react with the U-tube fluid. For the manometer, mercury can be used in the range of normal air pressure to 102 Pa, while silicone fluids and phthalates are suitable for use below 102 Pa down to 10 Pa. Heatable membrane capacity manometers can even be used at below 10-1 Pa. There are also other pressure gauges which can be used below 102 Pa.

1.6.2.2. Measurement procedure Before measuring, all components of the apparatus shown in figure 2 must be cleaned and dried thoroughly. For method 2a, fill the U-tube with the chosen liquid, which must be degassed at an elevated temperature before readings are taken. The test substance is placed in the apparatus, which is then closed and the temperature is reduced sufficiently for degassing. The temperature must be low enough to ensure that the air is sucked out, but — in the case of multiple component system — it must not alter the composition of the material. If required, equilibrium can be established more quickly by stirring. The sample can be supercooled with e.g. liquid nitrogen (taking care to avoid condensation of air or pump fluid) or a mixture of ethanol and dry ice. For low-temperature measurements use a temperature-regulated bath connected to an ultra-cryomat. With the valve over the sample vessel open, suction is applied for several minutes to remove the air. The valve is then closed and the temperature of the sample reduced to the lowest level desired. If necessary, the degassing operation must be repeated several times. When the sample is heated the vapour pressure increases. This alters the equilibrium of the fluid in the U-tube. To compensate for this, nitrogen or air is admitted to the apparatus via a valve until the pressure indicator fluid is at zero again. The pressure required for this can be read off a precision manometer at room temperature. This pressure corresponds to the vapour pressure of the substance at that particular measuring temperature. Method 2b is similar but the vapour pressure is read off directly. The temperature-dependence of vapour pressure is determined at suitably small intervals (approximately 5 to 10 measuring points in all) up to the desired maximum. Low-temperature readings must be repeated as a check. If the values obtained from the repeated readings do not coincide with the curve obtained for increasing temperature, this may be due to one of the following:

1.6.2.2.

1. the sample still contains air (e.g. high-viscosity materials) or low-boiling substances, which is/are released during heating and can be removed by suction following further supercooling;

2. the cooling temperature is not low enough. In this case liquid nitrogen is used as the cooling agent. If either l or 2 is the case, the measurements must be repeated;

3. the substance undergoes a chemical reaction in the temperature range investigated (e.g. decomposition, polymerisation).

1.6.3. Isoteniscope A complete description of this method can be found in reference 7. The principle of the measuring device is shown in figure 3. Similarly to the static method described in 1.6.2, the isoteniscope is appropriate for the investigation of solids or liquids. In the case of liquids, the substance itself serves as the fluid in the auxiliary manometer. A quantity of the liquid, sufficient to fill the bulb and the short leg of the manometer section, is put in the isoteniscope. The isoteniscope is attached to a vacuum system and evacuated, then filled by nitrogen. The evacuation and purge of the system is repeated twice to remove residual oxygen. The filled isoteniscope is placed in an horizontal position so that the sample spreads out into a thin layer in the sample bulb and manometer section (U-part). The pressure of the system is reduced to 133 Pa and the sample gently warmed until it just boils (removal of dissolved fixed gases). The isoteniscope is then placed so that the sample returns to the bulb and short leg of the manometer, so that both are entirely filled with liquid. The pressure is maintained as for degassing; the drawn-out tip of the sample bulb is heated with a small flame until sample vapour released expands sufficiently to displace part of the sample from the upper part of the bulb and manometer arm into the manometer section of the isoteniscope, creating a vapour-filled, nitrogen-free space. The isoteniscope is then placed in a constant temperature bath, and the pressure of nitrogen is adjusted until its pressure equals that of the sample. Pressure balance is indicated by the manometer section of the isoteniscope. At the equilibrium, the vapour pressure of nitrogen equals the vapour pressure of the substance. In the case of solids, depending on the pressure and temperature range, the manometer liquids listed in 1.6.2.1 are used. The degassed manometer liquid is filled into a bulge on the long arm of the isoteniscope. Then the solid to be investigated is placed in the bulb and is degassed at elevated temperature. After that the isoteniscope is inclined so that the manometer liquid can flow into the U-tube. The measurement of vapour pressure as a function of temperature is done according to 1.6.2.

1.6.4. Effusion method: vapour pressure balance

1.6.4.

1.6.4.1. Apparatus Various versions of the apparatus are described in the literature (1). The apparatus described here illustrates the general principle involved (figure 4). Figure 4 shows the main components of the apparatus, comprising a high-vacuum stainless steel or glass container, equipment to produce and measure a vacuum and built-in components to measure the vapour pressure on a balance. The following built-in components are included in the apparatus:

an evaporator furnace with flange and rotary inlet. The evaporator furnace is a cylindrical vessel, made of e.g. copper or a chemically resistant alloy with good thermal conductivity. A glass vessel with a copper wall can also be used. The furnace has a diameter of approximately 3 to 5 cm and is 2 to 5 cm high. There are between one and three openings of different sizes for the vapour stream. The furnace is heated either by a heating spiral around the outside. To prevent heat being dissipated to the base plate, the heater is attached to the base plate by a metal with low thermal conductivity (nickel-silver or chromium-nickel steel), e.g. a nickel-silver pipe attached to a rotary inlet if using a furnace with several openings. This arrangement has the advantage of allowing the introduction of a copper bar. This allows cooling from the outside using a cooling bath,

if the copper furnace lid has three openings of different diameters at 90o to each other, various vapour pressure ranges within the overall measuring range can be covered (openings between approximately 0,30 and 4,50 mm diameter). Large openings are used for low vapour pressure and vice versa. By rotating the furnace the desired opening or an intermediate position in the vapour stream (furnace opening — shield — balance pan) can be set and the stream of molecules is released or deflected through the furnace opening onto the scale pan. In order to measure the temperature of the substance, a thermocouple or resistance thermometer is placed at a suitable point,

above the shield is a balance pan belonging to a highly sensitive microbalance (see below). The balance pan is approximately 30 mm in diameter. Gold-plated aluminium is a suitable material,

the balance pan is surrounded by a cylindrical brass or copper refrigeration box. Depending on the type of balance, it has openings for the balance beam and a shield opening for the stream of molecules and should guarantee complete condensation of the vapour on the balance pan. Heat dissipation to the outside is ensured e.g. by a copper bar connected to the refrigeration box. The bar is routed through the base plate and thermally insulated from it, e.g. with a chromium-nickel steel tube. The bar is immersed in a Dewar flask containing liquid nitrogen under the base plate or liquid nitrogen is circulated through the bar. The refrigeration box is thus kept at approximately - 120 oC. The balance pan is cooled exclusively by radiation and is satisfactory for the pressure range under investigation (cooling approximately 1 hour before the start of measurement),

the balance is positioned above the refrigeration box. Suitable balances are e.g. a highly sensitive 2-arm electronic microbalance (8) or a highly sensitive moving coil instrument (see OECD Test Guideline 104, Edition 12.05.81),

the base plate also incorporates electrical connections for thermocouples (or resistance thermometers) and heating coils,

a vacuum is produced in the vessel using a partial vacuum pump or high-vacuum pump (required vacuum approximately 1 to 2 × 10-3 Pa, obtained after 2 h pumping). The pressure is regulated with a suitable ionisation manometer.

1.6.4.2. Measurement procedure The vessel is filled with the test substance and the lid is closed. The shield and refrigeration box are slid across the furnace. The apparatus is closed and the vacuum pumps are switched on. The final pressure before starting to take measurements should be approximately 10-4 Pa. Cooling of the refrigeration box starts at 10-2 Pa. Once the required vacuum has been obtained, start the calibration series at the lowest temperature required. The corresponding opening in the lid is set, the vapour stream passes through the shield directly above the opening and strikes the cooled balance pan. The balance pan must be big enough to ensure that the entire stream guided through the shield strikes it. The momentum of the vapour stream acts as a force against the balance pan and the molecules condense on its cool surface. The momentum and simultaneous condensation produce a signal on the recorder. Evaluation of the signals provides two pieces of information: p = G 2 πRT×103M where G evaporation rate (kg s-1 m-2) M molar mass (g mol-1) T temperature (K) R universal molar gas constant (Jmol-1 K-1) p vapour pressure (Pa) After the necessary vacuum is reached, the series of measurements is commenced at the lowest desired measuring temperature. For further measurements, the temperature is increased by small intervals until the maximum desired temperature value is reached. The sample is then cooled again and a second curve of the vapour pressure may be recorded. If the second run fails to confirm the results of the first run, then it is possible that the substance may be decomposing in the temperature range being measured.

1.6.4.2.

1. in the apparatus described here the vapour pressure is determined directly from the momentum on the balance pan (it is not necessary to know the molecular weight for this (2)). Geometrical factors such as the furnace opening and the angle of the molecular stream must be taken into account when evaluating the readings;

2. the mass of the condensate can be measured at the same time and the rate of evaporation can be calculated from this. The vapour pressure can also be calculated from the rate of evaporation and molecular weight using the Hertz equation (2).

1.6.5. Effusion method — by loss of weight

1.6.5.

1.6.5.1. Apparatus The effusion apparatus consists of the following basic parts: An electrically heated, aluminium vacuum tank with four stainless steel effusion cells is shown in figure 5 for example. The stainless steel foil of about 0,3 mm thickness has an effusion orifice of 0,2 to 1,0 mm diameter and is attached to the effusion cell by a threaded lid.

a tank that can be thermostated and evacuated and in which the effusion cells are located,

a high vacuum pump (e.g. diffusion pump or turbomolecular pump) with vacuum gauge,

a trap, using liquefied nitrogen or dry ice.

1.6.5.2. Measurement procedure The reference and test substances are filled into each effusion cell, the metal diaphragm with the orifice is secured by the threaded lid, and each cell is weighed to within an accuracy of 0,1 mg. The cell is placed in the thermostated apparatus, which is then evacuated to below one tenth of the expected pressure. At defined intervals of time ranging from 5 to 30 hours, air is admitted into the apparatus, and the loss in mass of the effusion cell is determined by reweighing. In order to ensure that the results are not influenced by volatile impurities, the cell is reweighed at defined time intervals to check that the evaporation rate is constant over at least two such intervals of time. The vapour pressure p in the effusion cell is given by: p = mKAt2 πRTM where p vapour pressure (Pa) m mass of the substance leaving the cell during time t (kg) t time (s) A area of the hole (m2) K correction factor R universal gas constant (Jmol-1 K-1) T temperature (K) M molecular mass (kg mol-1) The correction factor K depends on the ratio of length to radius of the cylindrical orifice: ratio 0,1 0,2 0,6 1,0 2,0 K 0,952 0,909 0,771 0,672 0,514 The above equation may be written: p = E mtTM where: E = 1KA2πR and is the effusion cell constant. This effusion cell constant E may be determined with reference substances (2,9), using the following equation: E = p(r)tmM(r)T where: p(r) vapour pressure of the reference substance (Pa) M(r) molecular mass of the reference substance (kg × mol-1)

1.6.6. Gas saturation method

1.6.6.

1.6.6.1. Apparatus A typical apparatus used to perform this test comprises a number of components given in figure 6a and described below (1).

1.6.6.1. Inert gas:

The carrier gas must not react chemically with the test substance. Nitrogen is usually sufficient for this purpose but occasionally other gases may be required (10). The gas employed must be dry (see figure 6a, key 4: relative humidity sensor).

Flow control:

A suitable gas-control system is required to ensure a constant and selected flow through the saturator column.

Traps to collect vapour:

These are dependent on the particular sample characteristics and the chosen method of analysis. The vapour should be trapped quantitatively and in a form which permits subsequent analysis. For some test substances, traps containing liquids such as hexane or ethylene glycol will be suitable. For others, solid absorbents may be applicable. As an alternative to vapour trapping and subsequent analysis, in-train analytical techniques, like chromatography, may be used to determine quantitatively the amount of material transported by a known amount of carrier gas. Furthermore, the loss of mass of the sample can be measured.

Heat exchanger:

For measurements at different temperatures it may be necessary to include a heat-exchanger in the assembly.

Saturator column:

The test substance is deposited from a solution onto a suitable inert support. The coated support is packed into the saturator column, the dimensions of which and the flow rate should be such that complete saturation of the carrier gas is ensured. The saturator column must be thermostated. For measurements above room temperature, the region between the saturator column and the traps should be heated to prevent condensation of the test substance. In order to lower the mass transport occurring by diffusion, a capillary may be placed after the saturator column (figure 6b).

1.6.6.2. Measurement procedure

1.6.6.2. Preparation of the saturator column:

A solution of the test substance in a highly volatile solvent is added to a suitable amount of support. Sufficient test substance should be added to maintain saturation for the duration of the test. The solvent is totally evaporated in air or in a rotary evaporator, and the thoroughly mixed material is added to the saturator column. After thermostating the sample, dry nitrogen is passed through the apparatus.

Measurement:

The traps or in-train detector are connected to the column effluent line and the time recorded. The flow rate is checked at the beginning and at regular intervals during the experiment, using a bubble meter (or continuously with a mass flow-meter). The pressure at the outlet to the saturator must be measured. This may be done either: The time required for collecting the quantity of test substance that is necessary for the different methods of analysis is determined in preliminary runs or by estimates. As an alternative to collecting the substance for further analysis, in-train quantitative analytical technique may be used (e.g. chromatography). Before calculating the vapour pressure at a given temperature, preliminary runs are to be carried out to determine the maximum flow rate that will completely saturate the carrier gas with substance vapour. This is guaranteed if the carrier gas is passed through the saturator sufficiently slowly so that a lower rate gives no greater calculated vapour pressure. The specific analytical method will be determined by the nature of the substance being tested (e.g. gas chromatography or gravimetry). The quantity of substance transported by a known volume of carrier gas is determined.

(a) by including a pressure gauge between the saturator and traps (this may not be satisfactory because this increases the dead space and the adsorptive surface); or

(b) by determining the pressure drops across the particular trapping system used as a function of flow rate in a separate experiment (this may be not very satisfactory for liquid traps).

1.6.6.3. Calculation of vapour pressure Vapour pressure is calculated from the vapour density, W/V, through the equation: p = WV × RTM where: p vapour pressure (Pa) W mass of evaporated test substance (g) V volume of saturated gas (m3) R universal molar gas constant (Jmol-1 K-1) T temperature (K) M molar mass of test substance (g mol-1) Measured volumes must be corrected for pressure and temperature differences between the flow meter and the thermostated saturator. If the flow meter is located downstream from the vapour trap, corrections may be necessary to account for any vaporised trap ingredients (1).

1.6.7. Spinning rotor (8, 11, 13)

1.6.7.

1.6.7.1. Apparatus The spinning rotor technique can be carried out using a spinning rotor viscosity gauge as shown in figure 8. A schematic drawing of the experimental set-up is shown in figure 7. The measuring apparatus typically consists of a spinning rotor measuring head, placed in a thermostated enclosure (regulated within 0,1 oC). The sample container is placed in a thermostated enclosure (regulated within 0,01 oC), and all other parts of the set-up are kept at a higher temperature to prevent condensation. A high-vacuum pump device is connected to the system by means of high-vacuum valves. The spinning rotor measuring head consists of a steel ball (4 to 5 mm diameter) in a tube. The ball is suspended and stabilised in a magnetic field, generally using a combination of permanent magnets and control coils. The ball is made to spin by rotating fields produced by coils. Pick-up coils, measuring the always present low lateral magnetisation of the ball, allow its spinning rate to be measured.

1.6.7.2. Measurement procedure When the ball has reached a given rotational speed v(o) (usually about 400 revolutions per second), further energising is stopped and deceleration takes place, due to gas friction. The drop of rotational speed is measured as a function of time. As the friction caused by the magnetic suspension is negligible as compared with the gas friction, the gas pressure p is given by: p = πc—rρσ10t × ln v(t)v(o) where: c— average speed of the gas molecules r radius of the ball ρ mass density of the ball σ coefficient of tangential momentum transfer (ε = 1 for an ideal spherical surface of the ball) t time v(t) rotational speed after time t v(o) initial rotational speed This equation may also be written: p = πc—rρ10σ × tn - tn-1tn × tn-1 where tn, tn-1 are the times required for a given number N of revolutions. These time intervals tn and tn-1 succeed one another, and tn > t n-1. The average speed of the gas molecule c— is given by: c— = 8RTπM12 where: T temperature R universal molar gas constant M molar mass

2. DATA The vapour pressure from any of the preceding methods should be determined for at least two temperatures. Three or more are preferred in the range 0 to 50 oC, in order to check the linearity of the vapour pressure curve.

3. REPORTING The test report shall, if possible, include the following information: If a transition (change of state, decomposition) is observed, the following information should be noted: All information and remarks relevant for the interpretation of results have to be reported, especially with regard to impurities and physical state of the substance.

method used,

precise specification of the substance (identity and impurities) and preliminary purification step, if any,

at least two vapour pressure and temperature values, preferably in the range 0 to 50 oC,

all of the raw data,

a log p versus 1/T curve,

an estimate of the vapour pressure at 20 or 25 oC.

nature of the change,

temperature at which the change occurs at atmospheric pressure,

vapour pressure at 10 and 20 oC below the transition temperature and 10 and 20 oC above this temperature (unless the transition is from solid to gas).

4.(1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)

REFERENCES OECD, Paris, 1981, Test Guideline 104, Decision of the Council C(81) 30 final. Ambrose, D. in B. Le Neindre, B. Vodar, (Eds.): Experimental Thermodynamics, Butterworths, London, 1975, vol. II. R. Weissberger ed.: Technique of Organic Chemistry, Physical Methods of Organic Chemistry, 3rd ed. Chapter IX, Interscience Publ., New York, 1959, vol. I, Part I. Knudsen, M. Ann. Phys. Lpz., 1909, vol. 29, 1979; 1911, vol. 34, p. 593. NF T 20-048 AFNOR (September 85) Chemical products for industrial use — Determination of vapour pressure of solids and liquids within range from 10-1 to 105 Pa — Static method. NF T 20-047 AFNOR (September 85) Chemical products for industrial use — Determination of vapour pressure of solids and liquids within range from 10-3 to 1 Pa — Vapour pressure balance method. ASTM D 2879-86, Standard test method for vapour pressure-temperature relationship and initial decomposition temperature of liquids by isoteniscope. G. Messer, P. Rohl, G. Grosse and W. Jitschin. J. Vac. Sci. Technol.(A), 1987, 'Vol. 5 (4), p. 2440. Ambrose, D.; Lawrenson, I.J.; Sprake, C.H.S. J. Chem. Thermodynamics 1975, vol. 7, p. 1173. B.F. Rordorf. Thermochimica Acta, 1985, vol. 85, p. 435. G. Comsa, J.K. Fremerey and B. Lindenau. J. Vac. Sci. Technol., 1980, vol. 17 (2), p. 642. G. Reich. J. Vac. Sci. Technol., 1982, vol. 20 (4), p. 1148. J.K. Fremerey. J. Vac. Sci. Technol.(A), 1985, vol. 3 (3), p. 1715.

A.5. SURFACE TENSION

A.5.

1. METHOD The methods described are based on the OECD Test Guideline (1). The fundamental principles are given in reference (2).

1.

1.1. INTRODUCTION The described methods are to be applied to the measurement of the surface tension of aqueous solutions. It is useful to have preliminary information on the water solubility, the structure, the hydrolysis properties and the critical concentration for micelles formation of the substance before performing these tests. The following methods are applicable to most chemical substances, without any restriction in respect to their degree of purity. The measurement of the surface tension by the ring tensiometer method is restricted to aqueous solutions with a dynamic viscosity of less than approximately 200 mPa s.

1.2. DEFINITIONS AND UNITS The free surface enthalpy per unit of surface area is referred to as surface tension. The surface tension is given as: N/m (SI unit) or mN/m (SI sub-unit) 1 N/m = 103 dynes/cm 1 mN/m = 1 dyne/cm in the obsolete cgs system

1.3. REFERENCE SUBSTANCES Reference substances do not need to be employed in all cases when investigating a new substance. They should primarily serve to check the performance of the method from time to time and to allow comparison with results from other methods. Reference substances which cover a wide range of surface tensions are given in references 1 and 3.

1.4. PRINCIPLE OF THE METHODS The methods are based on the measurement of the maximum force which is necessary to exert vertically, on a stirrup or a ring in contact with the surface of the liquid being examined placed in a measuring cup, in order to separate it from this surface, or on a plate, with an edge in contact with the surface, in order to draw up the film that has formed. Substances which are soluble in water at least at a concentration of 1 mg/l are tested in aqueous solution at a single concentration.

1.5. QUALITY CRITERIA These methods are capable of greater precision than is likely to be required for environmental assessment.

1.6. DESCRIPTION OF THE METHODS A solution of the substance is prepared in distilled water. The concentration of this solution should be 90 % of the saturation solubility of the substance in water; when this concentration exceeds 1 g/l, a concentration of 1 g/l is used for testing. Substances with water solubility lower than 1 mg/l need not be tested.

1.6.

1.6.1. Plate method See ISO 304 and NF T 73-060 (Surface active agents — determination of surface tension by drawing up liquid films).

1.6.2. Stirrup method See ISO 304 and NF T 73-060 (Surface active agents — determination of surface tension by drawing up liquid films).

1.6.3. Ring method See ISO 304 and NF T 73-060 (Surface active agents — determination of surface tension by drawing up liquid films).

1.6.4. OECD harmonised ring method

1.6.4.

1.6.4.1. Apparatus Commercially available tensiometers are adequate for this measurement. They consist of the following elements:

mobile sample table,

force measuring system,

measuring body (ring),

measurement vessel.

1.6.4.1.

1.6.4.1.1. Mobile sample table The mobile sample table is used as a support for the temperature-controlled measurement vessel holding the liquid to be tested. Together with the force measuring system, it is mounted on a stand.

1.6.4.1.2. Force measuring system The force measuring system (see figure) is located above the sample table. The error of the force measurement shall not exceed ± 10-6 N, corresponding to an error limit of ±0,1 mg in a mass measurement. In most cases, the measuring scale of commercially available tensiometers is calibrated in mN/m so that the surface tension can be read directly in mN/m with an accuracy of 0,1 mN/m.

1.6.4.1.3. Measuring body (ring) The ring is usually made of a platinum-iridium wire of about 0,4 mm thickness and a mean circumference of 60 mm. The wire ring is suspended horizontally from a metal pin and a wire mounting bracket to establish the connection to the force measuring system (see figure).

1.6.4.1.3. Figure Measuring body

(All dimensions expressed in millimetres) Mounting bracket Pin Ring

1.6.4.1.4. Measurement vessel The measurement vessel holding the test solution to be measured shall be a temperature-controlled glass vessel. It shall be designed so that during the measurement the temperature of the test solution liquid and the gas phase above its surface remains constant and that the sample cannot evaporate. Cylindrical glass vessels having an inside diameter of not less than 45 mm are acceptable.

1.6.4.2. Preparation of the apparatus

1.6.4.2.

1.6.4.2.1. Cleaning Glass vessels shall be cleaned carefully. If necessary they shall be washed with hot chromo-sulphuric acid and subsequently with syrupy phosphoric acid (83 to 98 % by weight of H3PO4), thoroughly rinsed in tap water and finally washed with double-distilled water until a neutral reaction is obtained and subsequently dried or rinsed with part of the sample liquid to be measured. The ring shall first be rinsed thoroughly in water to remove any substances which are soluble in water, briefly immersed in chromo-sulphuric acid, washed in double-distilled water until a neutral reaction is obtained and finally heated briefly above a methanol flame. Note: Contamination by substances which are not dissolved or destroyed by chromo-sulphuric acid or phosphoric acid, such as silicones, shall be removed by means of a suitable organic solvent.

1.6.4.2.2. Calibration of the apparatus The validation of the apparatus consists of verifying the zero point and adjusting it so that the indication of the instrument allows reliable determination in mN/m.

1.6.4.2.2. Mounting:

The apparatus shall be levelled, for instance by means of a spirit level on the tensiometer base, by adjusting the levelling screws in the base.

Zero point adjustment:

After mounting the ring on the apparatus and prior to immersion in the liquid, the tensiometer indication shall be adjusted to zero and the ring checked for parallelism to the liquid surface. For this purpose, the liquid surface can be used as a mirror.

Calibrations:

The actual test calibration can be accomplished by means of either of two procedures:

(a) Using a mass: procedure using riders of known mass between 0,1 and 1,0 g placed on the ring. The calibration factor, Φa by which all the instrument readings must be multiplied, shall be determined according to equation (1). Φa=σrσa where: σr=mg2b (mN/m) m mass of the rider (g) g gravity acceleration (981 cm s-2 at sea level) b mean circumference of the ring (cm) σa reading of the tensiometer after placing the rider on the ring (mN/m).

(b) Using water: procedure using pure water whose surface tension at, for instance, 23 oC is equal to 72,3 mN/m. This procedure is accomplished faster than the weight calibration but there is always the danger that the surface tension of the water is falsified by traces of contamination by surfactants. The calibration factor, Φb by which all the instrument readings shall be multiplied, shall be determined in accordance with the equation (2): Φb=σoσg where: σo value cited in the literature for the surface tension of water (mN/m) σg measured value of the surface tension of the water (mN/m) both at the same temperature.

1.6.4.3. Preparation of samples Aqueous solutions shall be prepared of the substances to be tested, using the required concentrations in water, and shall not contain any non-dissolved substances. The solution must be maintained at a constant temperature (±0,5 oC). Since the surface tension of a solution in the measurement vessel alters over a period of time, several measurements shall be made at various times and a curve plotted showing surface tension as a function of time. When no further change occurs, a state of equilibrium has been reached. Dust and gaseous contamination by other substances interfere with the measurement. The work shall therefore be carried out under a protective cover.

1.6.5. Test conditions The measurement shall be made at approximately 20 oC and shall be controlled to within ±0,5 oC.

1.6.6. Performance of test The solutions to be measured shall be transferred to the carefully cleaned measurement vessel, taking care to avoid foaming, and subsequently the measurement vessel shall be placed onto the table of the test apparatus. The table-top with measurement vessel shall be raised until the ring is immersed below the surface of the solution to be measured. Subsequently, the table-top shall be lowered gradually and evenly (at a rate of approximately 0,5 cm/min) to detach the ring from the surface until the maximum force has been reached. The liquid layer attached to the ring must not separate from the ring. After completing the measurements, the ring shall be immersed below the surface again and the measurements repeated until a constant surface tension value is reached. The time from transferring the solution to the measurement vessel shall be recorded for each determination. Readings shall be taken at the maximum force required to detach the ring from the liquid surface.

2. Table: Correction of the measured surface tension Only for aqueous solutions, ρ = 1 g/cm3

DATA In order to calculate the surface tension, the value read in mN/m on the apparatus shall be first multiplied by the calibration factor Φa or Φb (depending on the calibration procedure used). This will yield a value which applies only approximately and therefore requires correction. Harkins and Jordan (4) have empirically determined correction factors for surface-tension values measured by the ring method which are dependent on ring dimensions, the density of the liquid and its surface tension. Since it is laborious to determine the correction factor for each individual measurement from the Harkins and Jordan tables, in order to calculate the surface tension for aqueous solutions the simplified procedure of reading the corrected surface-tension values directly from the table may be used. (Interpolation shall be used for readings ranging between the tabular values.) r = 9,55 mm (average ring radius) r = 0,185 mm (ring wire radius) Experimental Value (mN/m) Corrected Value (mN/m) Weight calibration (see 1.6.4.2.2(a)) Water calibration (see 1.6.4.2.2(b)) 20 16,9 18,1 22 18,7 20,1 24 20,6 22,1 26 22,4 24,1 28 24,3 26,1 30 26,2 28,1 32 28,1 30,1 34 29,9 32,1 36 31,8 34,1 38 33,7 36,1 40 35,6 38,2 42 37,6 40,3 44 39,5 42,3 46 41,4 44,4 48 43,4 46,5 50 45,3 48,6 52 47,3 50,7 54 49,3 52,8 56 51,2 54,9 58 53,2 57,0 60 55,2 59,1 62 57,2 61,3 64 59,2 63,4 66 61,2 65,5 68 63,2 67,7 70 65,2 69,9 72 67,2 72,0 74 69,2 — 76 71,2 — 78 73,2 — This table has been compiled on the basis of the Harkins-Jordan correction. It is similar to that in the DIN Standard (DIN 53914) for water and aqueous solutions (density ρ = 1 g/cm3 and is for a commercially available ring having the dimensions R = 9,55 mm (mean ring radius) and r = 0,185 mm (ring wire radius). The table provides corrected values for surface-tension measurements taken after calibration with weights or calibration with water. Alternatively, without the preceding calibration, the surface tension call can be calculated according to the following formula: σ=f × F4πR where: F the force measured on the dynamometer at the breakpoint of the film R the radius of the ring f the correction factor (1)

3. REPORTING

3.

3.1. TEST REPORT The test report shall, if possible, include the following information:

method used,

type of water or solution used,

precise specification of the substance (identity and impurities),

measurement results: surface tension (reading) stating both the individual readings and their arithmetic mean as well as the corrected mean (taking into consideration the equipment factor and the correction table),

concentration of the solution,

test temperature,

age of solution used; in particular the time between preparation and measurement of the solution,

description of time dependence of surface tension after transferring the solution to the measurement vessel,

all information and remarks relevant for the interpretation of results have to be reported, especially with regard to impurities and physical state of the substance.

3.2. INTERPRETATION OF RESULTS Considering that distilled water has a surface tension of 72,75 mN/m at 20 oC, substances showing a surface tension lower than 60 mN/m under the conditions of this method should be regarded as being surface-active materials.

4.(1)(2)(3)(4)

REFERENCES OECD, Paris, 1981, Test Guideline 115, Decision of the Council C(81) 30 final. R. Weissberger ed.: Technique of Organic Chemistry, Physical Methods of Organic Chemistry, 3rd ed., Interscience Publ., New York, 1959, vol. I, Part I, Chapter XIV. Pure Appl. Chem., 1976, vol. 48, p. 511. Harkins, W.D., Jordan, H.F., J. Amer. Chem. Soc., 1930, vol. 52, p. 1751.

A.6. WATER SOLUBILITY

A.6.

1. METHOD The methods described are based on the OECD Test Guideline (1).

1.

1.1. INTRODUCTION It is useful to have preliminary information on the structural formula, the vapour pressure, the dissociation constant and the hydrolysis (as a function of pH) of the substance to perform this test. No single method is available to cover the whole range of solubilities in water. The two test methods described below cover the whole range of solubilities but are not applicable to volatile substances: The water solubility of the test substance can be considerably affected by the presence of impurities.

one which applies to essentially pure substances with low solubilities, (< 10-2 grams per litre), and which are stable in water, referred to as the column elution method,

the other which applies to essentially pure substances with higher solubilities (> 10-2 grams per litre), and which are stable in water, referred to as the flask method.

1.2. DEFINITION AND UNITS The solubility in water of a substance is specified by the saturation mass concentration of the substance in water at a given temperature. The solubility in water is specified in units of mass per volume of solution. The SI unit is kg/m3 (grams per litre may also be used).

1.4. PRINCIPLE OF THE TEST METHOD The approximate amount of the sample and the time necessary to achieve the saturation mass concentration should be determined in a simple preliminary test.

1.4.

1.4.1. Column elution method This method is based on the elution of a test substance with water from a micro-column which is charged with an inert support material, such as glass beads or sand, coated with an excess of test substance. The water solubility is determined when the mass concentration of the eluate is constant. This is shown by a concentration plateau as a function of time.

1.4.2. Flask method In this method, the substance (solids must be pulverised) is dissolved in water at a temperature somewhat above the test temperature. When saturation is achieved the mixture is cooled and kept at the test temperature, stirring as long as necessary to reach equilibrium. Alternatively, the measurement can be performed directly at the test temperature, if it is assured by appropriate sampling that the saturation equilibrium is reached. Subsequently, the mass concentration of the substance in the aqueous solution, which must not contain any undissolved particles, is determined by a suitable analytical method.

1.5. QUALITY CRITERIA

1.5.

1.5.1. Repeatability For the column elution method, < 30 % may be obtainable; for the flask method, < 15 % should be observed.

1.5.2. Sensitivity This depends upon the method of analysis, but mass concentration determinations down to 10-6 grams per litre can be determined.

1.6. DESCRIPTION OF THE METHOD

1.6.

1.6.1. Test conditions The test is preferably run at 20 ±0,5 oC. If a temperature dependence is suspected in the solubility (> 3 % per oC), two other temperatures at least 10 oC above and below the initially chosen temperature should also be used. In this case, the temperature control should be ±0,1 oC. The chosen temperature should be kept constant in all relevant parts of the equipment.

1.6.2. Preliminary test To approximately 0,1 g of the sample (solid substances must be pulverised) in a glass-stoppered 10 ml graduated cylinder, increasing volumes of distilled water at room temperature are added according to the steps shown in the table below: 0,1 g soluble in x ml of water 0,1 0,5 1 2 10 100 > 100 Approximative solubility (grams per litre) > 1000 1000 to 200 200 to 100 100 to 50 50 to 10 10 to 1 < 1 After each addition of the indicated amount of water, the mixture is shaken vigorously for 10 minutes and is visually checked for any undissolved parts of the sample. If, after addition of 10 ml of water, the sample or parts of it remain undissolved, the experiment has to be repeated in a 100 ml measuring cylinder with larger volumes of water. At lower solubilities the time required to dissolve a substance can be considerably longer (at least 24 h should be allowed). The approximate solubility is given in the table under that volume of added water in which complete dissolution of the sample occurs. If the substance is still apparently insoluble, more than 24 h should be allowed (96 h maximum), or further dilution should be undertaken to ascertain whether the column elution or flask solubility method should be used.

1.6.3. Column elution method

1.6.3.

1.6.3.1. Support material, solvent and eluent The support material for the column elution method should be inert. Possible materials which can be employed are glass beads and sand. A suitable volatile solvent of analytical reagent quality should be used to apply the test substance to the support material. Water which has been double distilled in glass or quartz apparatus should be employed as the eluent. Note: Water directly from an organic ion exchanger must not be used.

1.6.3.2. Loading of the support Approximately 600 mg of support material is weighed and transferred to a 50 ml round-bottom flask. A suitable, weighed amount of test substance is dissolved in the chosen solvent. An appropriate amount of this solution is added to the support material. The solvent must be completely evaporated, e.g. in a rotary evaporator; otherwise water saturation of the support is not achieved due to partition effects on the surface of the support material. The loading of support material may cause problems (erroneous results) if the test substance is deposited as an oil or a different crystal phase. The problem should be examined experimentally and the details reported. The loaded support material is allowed to soak for about two hours in approximately 5 ml of water, and then the suspension is added to the microcolumn. Alternatively, dry loaded support material may be poured into the microcolumn, which has been filled with water, and then equilibrated for approximately two hours.

1.6.3.2. Test procedure:

The elution of the substance from the support material can be carried out in one of two different ways:

recirculating pump (see figure 1),

levelling vessel (see figure 4).

1.6.3.3. Column elution method with recirculating pump

1.6.3.3. Apparatus

A schematic arrangement of a typical system is presented in figure 1. A suitable microcolumn is shown in figure 2, although any size is acceptable, provided it meets the criteria for reproducibility and sensitivity. The column should provide for a headspace of at least five bed volumes of water and be able to hold a minimum of five samples. Alternatively, the size can be reduced if make-up solvent is employed to replace the initial five bed volumes removed with impurities. The column should be connected to a recirculating pump capable of controlling flows of approximately 25 ml/h. The pump is connected with polytetrafluoroethylene (P.T.F.E.) and/or glass connections. The column and pump, when assembled, should have provision for sampling the effluent and equilibrating the headspace at atmospheric pressure. The column material is supported with a small (5 mm) plug of glass wool, which also serves to filter out particles. The recirculating pump can be, for example, a peristaltic pump or a membrane pump (care must be taken that no contamination and/or absorption occurs with the tube material).

Measurement procedure

The flow through the column is started. It is recommended that a flow rate of approximately 25 ml/hr be used (this corresponds to 10 bed volumes/hr for the column described). The first five bed volumes (minimum) are discarded to remove water-soluble impurities. Following this, the recirculating pump is allowed to run until equilibration is established, as defined by five successive samples whose concentrations do not differ by more than ± 30 % in a random fashion. These samples should be separated from each other by time intervals corresponding to the passage of at least 10 bed volumes of the eluent.

1.6.3.4. Column elution method with levelling vessel

1.6.3.4. Apparatus (see figures 4 and 3)

Levelling vessel: the connection to the levelling vessel is made by using a ground glass joint which is connected by PTFE tubing. It is recommended that a flow rate of approximately 25 ml/hr be used. Successive eluate fractions should be collected and analysed by the chosen method.

Measurement procedure

Those fractions from the middle eluate range where the concentrations are constant (± 30 %) in at least five consecutive fractions are used to determine the solubility in water. In both cases (using a recirculating pump or a levelling vessel), a second run is to be performed at half the flow rate of the first. If the results of the two runs are in agreement, the test is satisfactory; if there is a higher apparent solubility with the lower flow rate, then the halving of the flow rate must continue until two successive runs give the same solubility. In both cases (using a recirculating pump or a levelling vessel) the fractions should be checked for the presence of colloidal matter by examination for the Tyndall effect (light scattering). Presence of such particles invalidates the results, and the test should be repeated with improvements in the filtering action of the column. The pH of each sample should be recorded. A second run should be performed at the same temperature.

1.6.4. Flask method

1.6.4.

1.6.4.1. Apparatus For the flask method the following material is needed:

normal laboratory glassware and instrumentation,

a device suitable for the agitation of solutions under controlled constant temperatures,

a centrifuge (preferably thermostated), if required with emulsions, and

equipment for analytical determination.

1.6.4.2. Measurement procedure The quantity of material necessary to saturate the desired volume of water is estimated from the preliminary test. The volume of water required will depend on the analytical method and the solubility range. About five times the quantity of material determined above is weighed into each of three glass vessels fitted with glass stoppers (e.g. centrifuge tubes, flasks). The chosen volume of water is added to each vessel, and the vessels are tightly stoppered. The closed vessels are then agitated at 30 oC. (A shaking or stirring device capable of operating at constant temperature should be used, e.g. magnetic stirring in a thermostatically controlled water bath). After one day, one of the vessels is removed and re-equilibrated for 24 hours at the test temperature with occasional shaking. The contents of the vessel are then centrifuged at the test temperature, and the concentration of test substance in the clear aqueous phase is determined by a suitable analytical method. The other two flasks are treated similarly after initial equilibration at 30 oC for two and three days, respectively. If the concentration results from at least the last two vessels agree with the required reproducibility, the test is satisfactory. The whole test should be repeated, using longer equilibration times, if the results from vessels 1, 2 and 3 show a tendency to increasing values. The measurement procedure can also be performed without pre-incubation at 30 oC. In order to estimate the rate of establishment of the saturation equilibrium, samples are taken until the stirring time no longer influences the concentration of the test solution. The pH of each sample should be recorded.

1.6.5. Analysis A substance-specific analytical method is preferred for these determinations, since small amounts of soluble impurities can cause large errors in the measured solubility. Examples of such methods are: gas or liquid chromatography, titration methods, photometric methods, voltammetric methods.

2. DATA

2.

2.1. COLUMN ELUTION METHOD The mean value from at least five consecutive samples taken from the saturation plateau should be calculated for each run, as should the standard deviation. The results should be given in units of mass per volume of solution. The means calculated on two tests using different flows are compared and should have a repeatability of less than 30 %.

2.2. FLASK METHOD The individual results should be given for each of the three flasks and those results deemed to be constant (repeatability of less than 15 %) should be averaged and given in units of mass per volume of solution. This may require the reconversion of mass units to volume units, using the density when the solubility is very high (> 100 grams per litre).

3. REPORTING

3.

3.1. COLUMN ELUTION METHOD The test report shall, if possible, include the following information:

the results of the preliminary test,

precise specification of the substance (identity and impurities),

the individual concentrations, flow rates and pH of each sample,

the means and standard deviations from at least five samples from the saturation plateau of each run,

the average of the two successive, acceptable runs,

the temperature of the water during the saturation process,

the method of analysis employed,

the nature of the support material employed,

loading of support material,

solvent used,

evidence of any chemical instability of the substance during the test and the method used,

all information relevant for the interpretation of the results, especially with regard to impurities and physical state of the substance.

3.2. FLASK METHOD The test report shall, if possible, include the following information:

the results of the preliminary test,

precise specification of the substance (identity and impurities),

the individual analytical determinations and the average where more than one value was determined for each flask,

the pH of each sample,

the average of the value for the different flasks which were in agreement,

the test temperature,

the analytical method employed,

evidence of any chemical instability of the substance during the test and the method used,

all information relevant for the interpretation of the results, especially with regard to impurities and physical state of the substance.

4.(1)(2)(3) REFERENCES OECD, Paris, 1981, Test Guideline 105, Decision of the Council C(81) 30 final. NF T 20-045 (AFNOR) (September 85) Chemical products for industrial use — Determination of water solubility of solids and liquids with low solubility — Column elution method. NF T 20-046 (AFNOR) (September 85) Chemical products for industrial use — Determination of water solubility of solids and liquids with high solubility — Flask method.

A.8. PARTITION COEFFICIENT

A.8.

1. METHOD The shake flask method described is based on the OECD Test Guideline (1).

1.

1.1. INTRODUCTION It is useful to have preliminary information on structural formula, dissociation constant, water solubility, hydrolysis, n-octanol solubility and surface tension of the substance to perform this test. Measurements should be made on ionisable substances only in their non-ionised form (free acid or free base) produced by the use of an appropriate buffer with a pH of at least one pH unit below (free acid) or above (free base) the pK. This test method includes two separate procedures: the shake flask method and high performance liquid chromatography (HPLC). The former is applicable when the log Pow value (see below for definitions) falls within the range - 2 to 4 and the latter within the range 0 to 6. Before carrying out either of the experimental procedures a preliminary estimate of the partition coefficient should first be obtained. The shake-flask method applies only to essentially pure substances soluble in water and n-octanol. It is not applicable to surface active materials (for which a calculated value or an estimate based on the individual n-octanol and water solubilities should be provided). The HPLC method is not applicable to strong acids and bases, metal complexes, surface-active materials or substances which react with the eluent. For these materials, a calculated value or an estimate based on individual n-octanol and water solubilities should be provided. The HPLC method is less sensitive to the presence of impurities in the test compound than is the shake-flask method. Nevertheless, in some cases impurities can make the interpretation of the results difficult because peak assignment becomes uncertain. For mixtures which give an unresolved band, upper and lower limits of log P should be stated.

1.2. DEFINITION AND UNITS The partition coefficient (P) is defined as the ratio of the equilibrium concentrations (ci) of a dissolved substance in a two-phase system consisting of two largely immiscible solvents. In the case n-octanol and water: Pow=cn-octanolcwater The partition coefficient (P) therefore is the quotient of two concentrations and is usually given in the form of its logarithm to base 10 (log P).

1.3. REFERENCE SUBSTANCES

1.3. Shake-flask method

Reference substances do not need to be employed in all cases when investigating a new substance. They should primarily serve to check the performance of the method from time to time and to allow comparison with results from other methods.

HPLC method

In order to correlate the measured HPLC data of a compound with its P value, a calibration graph of log P versus chromatographic data using at least six reference points has to be established. It is for the user to select the appropriate reference substances. Whenever possible, at least one reference compound should have a Pow above that of the test substance, and another a Pow below that of the test substance. For log P values less than 4, the calibration can be based on data obtained by the shake-flask method. For log P values greater than 4, the calibration can be based on validated literature values if these are in agreement with calculated values. For better accuracy, it is preferable to choose reference compounds which are structurally related to the test substance. Extensive lists of values of log Pow for many groups of chemicals are available (2)(3). If data on the partition coefficients of structurally related compounds are not available, then a more general calibration, established with other reference compounds, may be used. A list of recommended reference substances and their Pow values is given in Appendix 2.

1.4. PRINCIPLE OF THE METHOD

1.4.

1.4.1. Shake-flask method In order to determine a partition coefficient, equilibrium between all interacting components of the system must be achieved, and the concentrations of the substances dissolved in the two phases must be determined. A study of the literature on this subject indicates that several different techniques can be used to solve this problem, i.e. the thorough mixing of the two phases followed by their separation in order to determine the equilibrium concentration for the substance being examined.

1.4.2. HPLC method HPLC is performed on analytical columns packed with a commercially available solid phase containing long hydrocarbon chains (e.g. C8, C18) chemically bound onto silica. Chemicals injected onto such a column move along it at different rates because of the different degrees of partitioning between the mobile phase and the hydrocarbon stationary phase. Mixtures of chemicals are eluted in order of their hydrophobicity, with water-soluble chemicals eluted first and oil-soluble chemicals last, in proportion to their hydrocarbon-water partition coefficient. This enables the relationship between the retention time on such a (reverse phase) column and the n-octanol/water partition coefficient to be established. The partition coefficient is deduced from the capacity factor k, given by the expression: k=tr-toto in which, tr = retention time of the test substance, and to = average time a solvent molecule needs to pass through the column (dead-time). Quantitative analytical methods are not required and only the determination of elution times is necessary.

1.5. QUALITY CRITERIA

1.5.

1.5.1. Repeatability

1.5.1. Shake-flask method

In order to assure the accuracy of the partition coefficient, duplicate determinations are to be made under three different test conditions, whereby the quantity of substance specified as well as the ratio of the solvent volumes may be varied. The determined values of the partition coefficient expressed as their common logarithms should fall within a range of ±0,3 log units.

HPLC method

In order to increase the confidence in the measurement, duplicate determinations must be made. The values of log P derived from individual measurements should fall within a range of ±0,1 log units.

1.5.2. Sensitivity

1.5.2. Shake-flask method

The measuring range of the method is determined by the limit of detection of the analytical procedure. This should permit the assessment of values of log Pow in the range of - 2 to 4 (occasionally when conditions apply, this range may be extended to log Pow up to 5) when the concentration of the solute in either phase is not more than 0,01 mol per litre.

HPLC method

The HPLC method enables partition coefficients to be estimated in the log Pow range 0 to 6. Normally, the partition coefficient of a compound can be estimated to within ± l log unit of the shake-flask value. Typical correlations can be found in the literature (4)(5)(6)(7)(8). Higher accuracy can usually be achieved when correlation plots are based on structurally-related reference compounds (9).

1.5.3. Specificity

1.5.3. Shake-flask method

The Nernst Partition Law applies only at constant temperature, pressure and pH for dilute solutions. It strictly applies to a pure substance dispersed between two pure solvents. If several different solutes occur in one or both phases at the same time, this may affect the results. Dissociation or association of the dissolved molecules result in deviations from the Nernst Partition Law. Such deviations are indicated by the fact that the partition coefficient becomes dependent upon the concentration of the solution. Because of the multiple equilibria involved, this test method should not be applied to ionisable compounds without applying a correction. The use of buffer solutions in place of water should be considered for such compounds; the pH of the buffer should be at least 1 pH unit from the pKa of the substance and bearing in mind the relevance of this pH for the environment.

1.6. DESCRIPTION OF THE METHOD

1.6.

1.6.1. Preliminary estimate of the partition coefficient The partition coefficient is estimated preferably by using a calculation method (see Appendix 1), or where appropriate, from the ratio of the solubilities of the test substance ill the pure solvents (10).

1.6.2. Shake-flask method

1.6.2.

1.6.2.1. Preparation n-Octanol: the determination of the partition coefficient should be carried out with high purity analytical grade reagent. Water: water distilled or double distilled in glass or quartz apparatus should be employed. For ionisable compounds, buffer solutions in place of water should be used if justified. Note: Water taken directly from an ion exchanger should not be used.

1.6.2.1.

1.6.2.1.1. Pre-saturation of the solvents Before a partition coefficient is determined, the phases of the solvent system are mutually saturated by shaking at the temperature of the experiment. To do this, it is practical to shake two large stock bottles of high purity analytical grade n-octanol or water each with a sufficient quantity of the other solvent for 24 hours on a mechanical shaker and then to let them stand long enough to allow the phases to separate and to achieve a saturation state.

1.6.2.1.2. Preparation for the test The entire volume of the two-phase system should nearly fill the test vessel. This will help prevent loss of material due to volatilisation. The volume ratio and quantities of substance to be used are fixed by the following: Three tests are carried out. In the first, the calculated volume ratio of n-octanol to water is used; in the second, this ratio is divided by two; and in the third, this ratio is multiplied by two (e.g. 1:1, 1:2, 2:1).

the preliminary assessment of the partition coefficient (see above),

the minimum quantity of test substance required for the analytical procedure, and

the limitation of a maximum concentration in either phase of 0,01 mol per litre.

1.6.2.1.3. Test substance A stock solution is prepared in n-octanol pre-saturated with water. The concentration of this stock solution should be precisely determined before it is employed in the determination of the partition coefficient. This solution should be stored under conditions which ensure its stability.

1.6.2.2. Test conditions The test temperature should be kept constant (± 1 oC) and lie in the range of 20 to 25 oC.

1.6.2.3. Measurement procedure

1.6.2.3.

1.6.2.3.1. Establishment of the partition equilibrium Duplicate test vessels containing the required, accurately measured amounts of the two solvents together with the necessary quantity of the stock solution should be prepared for each of the test conditions. The n-octanol phases should be measured by volume. The test vessels should either be placed in a suitable shaker or shaken by hand. When using a centrifuge tube, a recommended method is to rotate the tube quickly through 180o about its transverse axis so that any trapped air rises through the two phases. Experience has shown that 50 such rotations are usually sufficient for the establishment of the partition equilibrium. To be certain, 100 rotations in five minutes are recommended.

1.6.2.3.2. Phase separation When necessary, in order to separate the phases, centrifugation of the mixture should be carried out. This should be done in a laboratory centrifuge maintained at room temperature, or, if a non-temperature controlled centrifuge is used, the centrifuge tubes should be kept for equilibration at the test temperature for at least one hour before analysis.

1.6.2.4. Analysis For the determination of the partition coefficient, it is necessary to determine the concentrations of the test substance in both phases. This may be done by taking an aliquot of each of the two phases from each tube for each test condition and analyzing them by the chosen procedure. The total quantity of substance present in both phases should be calculated and compared with the quantity of the substance originally introduced. The aqueous phase should be sampled by a procedure that minimises the risk of including traces of n-octanol: a glass syringe with a removable needle can be used to sample the water phase. The syringe should initially be partially filled with air. Air should be gently expelled while inserting the needle through the n-octanol layer. An adequate volume of aqueous phase is withdrawn into the syringe. The syringe is quickly removed from the solution and the needle detached. The contents of the syringe may then be used as the aqueous sample. The concentration in the two separated phases should preferably be determined by a substance-specific method. Examples of analytical methods which may be appropriate are:

photometric methods,

gas chromatography,

high-performance liquid chromatography.

1.6.3. HPLC method

1.6.3.

1.6.3.1. Preparation

1.6.3.1. Apparatus

A liquid chromatograph, fitted with a pulse-free pump and a suitable detection device, is required. The use of an injection valve with injection loops is recommended. The presence of polar groups in the stationary phase may seriously impair the performance of the HPLC column. Therefore, stationary phases should have the minimal percentage of polar groups (11). Commercial microparticulate reverse-phase packings or ready-packed columns can be used. A guard column may be positioned between the injection system and the analytical column.

Mobile phase

HPLC grade methanol and HPLC grade water are used to prepare the eluting solvent, which is degassed before use. Isocratic elution should be employed. Methanol/water ratios with a minimum water content of 25 % should be used. Typically a 3:1 (v/v) methanol-water mixture is satisfactory for eluting compounds of log P 6 within an hour, at a flow rate of 1 ml/min. For compounds of high log P it may be necessary to shorten the elution time (and those of the reference compounds) by decreasing the polarity of the mobile phase or the column length. Substances with very low solubility in n-octanol tend to give abnormally low log Pow values with the HPLC method; the peaks of such compounds sometimes accompany the solvent front. This is probably due to the fact that the partitioning process is too slow to reach the equilibrium in the time normally taken by an HPLC separation. Decreasing the flow rate and/or lowering the methanol/water ratio may then be effective to arrive at a reliable value. Test and reference compounds should be soluble in the mobile phase in sufficient concentrations to allow their detection. Only in exceptional cases may additives be used with the methanol-water mixture, since additives will change the properties of the column. For chromatograms with additives it is mandatory to use a separate column of the same type. If methanol-water is not appropriate, other organic solvent-water mixtures call be used, e.g. ethanol-water or acetonitrile-water. The pH of the eluent is critical for ionisable compounds. It should be within the operating pH range of the column, which is usually between 2 and 8. Buffering is recommended. Care must be taken to avoid salt precipitation and column deterioration which occur with some organic phase/buffer mixtures. HPLC measurements with silica-based stationary phases above pH 8 are not advisable since the use of an alkaline, mobile phase may cause rapid deterioration in the performance of the column.

Solutes

The reference compounds should be the purest available. Compounds to be used for test or calibration purposes are dissolved in the mobile phase if possible.

Test conditions

The temperature during the measurements should not vary by more than ± 2 K.

1.6.3.2. Measurement

1.6.3.2. Calculation of dead time to

The dead time to can be determined by using either a homologous series (e.g. n-alkyl methyl ketones) or unretained organic compounds (e.g. thiourea or formamide). For calculating the dead time to by using a homologous series, a set of at least seven members of a homologous series is injected and the respective retention times are determined. The raw retention times tr (nc + 1) are plotted as a function of tr(nc) and the intercept a and slope b of the regression equation: tr(nc + 1) = a + b tr(nc) are determined (nc = number of carbon atoms). The dead time to is then given by: to = a/(1 - b)

Calibration graph

The next step is to construct a correlation plot of log k values versus log p for appropriate reference compounds. In practice, a set of between 5 and 10 standard reference compounds whose log p is around the expected range are injected simultaneously and the retention times are determined, preferably on a recording integrator linked to the detection system. The corresponding logarithms of the capacity factors, log k, are calculated and plotted as a function of the log p determined by the shake-flask method. The calibration is performed at regular intervals, at least once daily, so that possible changes in column performance can be allowed for.

Determination of the capacity factor of the test substance

The test substance is injected in as small a quantity of mobile phase as possible. The retention time is determined (in duplicate), permitting the calculation of the capacity factor k. From the correlation graph of the reference compounds, the partition coefficient of the test substance can be interpolated. For very low and very high partition coefficients, extrapolation is necessary. In those cases particular care has to be taken of the confidence limits of the regression line.

2. DATA

2. Shake-flask method

The reliability of the determined values of P can be tested by comparison of the means of the duplicate determinations with the overall mean.

3. REPORTING The test report shall, if possible, include the following information:

precise specification of the substance (identity and impurities),

when the methods are not applicable (e.g. surface active material), a calculated value or an estimate based on the individual n-octanol and water solubilities should be provided,

all information and remarks relevant for the interpretation of results, especially with regard to impurities and physical state of the substance.

the result of the preliminary estimation, if any,

temperature of the determination,

data on the analytical procedures used in determining concentrations,

time and speed of centrifugation, if used,

the measured concentrations in both phases for each determination (this means that a total of 12 concentrations will be reported),

the weight of the test substance, the volume of each phase employed in each test vessel and the total calculated amount of test substance present in each phase after equilibration,

the calculated values of the partition coefficient (P) and the mean should be reported for each set of test conditions as should the mean for all determinations. If there is a suggestion of concentration dependency of the partition coefficient, this should be noted in the report,

the standard deviation of individual P values about their mean should be reported,

the mean P from all determinations should also be expressed as its logarithm (base 10),

the calculated theoretical Pow when this value has been determined or when the measured value is > 104,

pH of water used and of the aqueous phase during the experiment,

if buffers are used, justification for the use of buffers in place of water, composition, concentration and pH of the buffers, pH of the aqueous phase before and after the experiment.

the result of the preliminary estimation, if any,

test and reference substances, and their purity,

temperature range of the determinations,

pH at which the determinations are made,

details of the analytical and guard column, mobile phase and means of detection,

retention data and literature log P values for reference compounds used in calibration,

details of fitted regression line (log k versus log P),

average retention data and interpolated log P value for the test compound,

description of equipment and operating conditions,

elution profiles,

quantities of test and references substances introduced in the column,

dead-time and how it was measured.

4.(1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)

REFERENCES OECD, Paris, 1981, Test Guideline 107, Decision of the Council C(81) 30 final. C. Hansch and A.J. Leo, Substituent Constants for Correlation Analysis in Chemistry and Biology, John Wiley, New York, 1979. Log P and Parameter Database, A tool for the quantitative prediction of bioactivity (C. Hansch, chairman, A.J. Leo, dir.) — Available from Pomona College Medical Chemistry Project 1982, Pomona College, Claremont, California 91711. L. Renberg, G. Sundström and K. Sundh-Nygärd, Chemosphere, 1980, vol. 80, p. 683. H. Ellgehausen, C. D'Hondt and R. Fuerer, Pestic. Sci., 1981, vol. 12, p. 219. B. McDuffie, Chemosphere, 1981, vol. 10, p. 73. W.E. Hammers et al., J. Chromatogr., 1982, vol. 247, p. 1. J.E. Haky and A.M. Young, J. Liq. Chromat., 1984, vol. 7, p. 675. S. Fujisawa and E. Masuhara, J. Biomed. Mat. Res., 1981, vol. 15, p. 787. O. Jubermann, Verteilen und Extrahieren, in Methoden der Organischen Chemie (Houben Weyl), Allgemeine Laboratoriumpraxis (edited by E. Muller), Georg Thieme Verlag, Stuttgart, 1958, Band I/1, p. 223-339. R.F. Rekker and H.M. de Kort, Euro. J. Med. Chem., 1979, vol. 14, p. 479. A. Leo, C. Hansch and D. Elkins, Partition coefficients and their uses. Chem. Rev., 1971, vol. 71, p. 525. R.F. Rekker, The Hydrophobic Fragmental Constant, Elsevier, Amsterdam, 1977. NF T 20-043 AFNOR (1985). Chemical products for industrial use — Determination of partition coefficient — Flask shaking method. C.V. Eadsforth and P. Moser, Chemosphere, 1983, vol. 12, p. 1459. A. Leo, C. Hansch and D. Elkins, Chem. Rev., 1971, vol. 71, p. 525. C. Hansch, A. Leo, S.H. Unger, K.H. Kim, D. Nikaitani and E.J. Lien, J. Med. Chem., 1973, vol. 16, p. 1207. W.B. Neely, D.R. Branson and G.E. Blau, Environ. Sci. Technol., 1974, vol. 8, p. 1113. D.S. Brown and E.W. Flagg, J. Environ. Qual., 1981, vol. 10, p. 382. J.K. Seydel and K.J. Schaper, Chemische Struktur und biologische Aktivität von Wirkstoffen, Verlag Chemie, Weinheim, New York, 1979. R. Franke, Theoretical Drug Design Methods, Elsevier, Amsterdam, 1984. Y.C. Martin, Quantitative Drug Design, Marcel Dekker, New York, Base1, 1978. N.S. Nirrlees, S.J. Noulton, C.T. Murphy, P.J. Taylor; J. Med. Chem., 1976, vol. 19, p. 615.

A.9. FLASH-POINT

A.9.

1. METHOD

1.

1.1. INTRODUCTION It is useful to have preliminary information on the flammability of the substance before performing this test. The test procedure is applicable to liquid substances whose vapours can be ignited by ignition sources. The test methods listed in this text are only reliable for flash-point ranges which are specified in the individual methods. The possibility of chemical reactions between the substance and the sample holder should be considered when selecting the method to be used.

1.2. DEFINITIONS AND UNITS The flash-point is the lowest temperature, corrected to a pressure of 101,325 kPa, at which a liquid evolves vapours, under the conditions defined in the test method, in such an amount that a flammable vapour/air mixture is produced in the test vessel. Units: oC t = T - 273,15 (t in oC and T in K)

1.4. PRINCIPLE OF THE METHOD The substance is placed in a test vessel and heated or cooled to the test temperature according to the procedure described in the individual test method. Ignition trials are carried out in order to ascertain whether or not the sample flashed at the test temperature.

1.5. QUALITY CRITERIA

1.5.

1.5.1. Repeatability The repeatability varies according to flash-point range and the test method used; maximum 2 oC.

1.5.2. Sensitivity The sensitivity depends on the test method used.

1.5.3. Specificity The specificity of some test methods is limited to certain flash-point ranges and subject to substance-related data (e.g. high viscosity).

1.6. DESCRIPTION OF THE METHOD

1.6.

1.6.1. Preparations A sample of the test substance is placed in a test apparatus according to 1.6.3.1 and/or 1.6.3.2. For safety, it is recommended that a method utilising a small sample size, circa 2 cm3, be used for energetic or toxic substances.

1.6.2. Test conditions The apparatus should, as far as is consistent with safety, be placed in a draught-free position.

1.6.3. Performance of the test

1.6.3.

1.6.3.1. Equilibrium method See ISO 1516, ISO 3680, ISO 1523, ISO 3679.

1.6.3.2. Non-equilibrium method

1.6.3.2. Abel apparatus:

See BS 2000 part 170, NF M07-011, NF T66-009.

Abel-Pensky apparatus:

See EN 57, DIN 51755 part 1 (for temperatures from 5 to 65 oC), DIN 51755 part 2 (for temperatures below 5 oC), NF M07-036.

Tag apparatus:

See ASTM D 56.

Pensky-Martens apparatus:

See ISO 2719, EN 11, DIN 51758, ASTM D 93, BS 2000-34, NF M07-019.

Remarks:

When the flash-point, determined by a non-equilibrium method in 1.6.3.2, is found to be 0 ± 2 oC, 21 ± 2 oC or 55 ± 2 oC, it should be confirmed by an equilibrium method using the same apparatus. Only the methods which can give the temperature of the flash-point may be used for a notification. To determine the flash-point of viscous liquids (paints, gums and similar) containing solvents, only apparatus and test methods suitable for determining the flash-point of viscous liquids may be used. See ISO 3679, ISO 3680, ISO 1523, DIN 53213 part 1.

2. DATA

3. REPORTING The test report shall, if possible, include the following information:

the precise specification of the substance (identification and impurities),

the method used should be stated as well as any possible deviations,

the results and any additional remarks relevant for the interpretation of results.

4. REFERENCES None.

A.10. FLAMMABILITY (SOLIDS)

A.10.

1. METHOD

1.

1.1. INTRODUCTION It is useful to have preliminary information on potentially explosive properties of the substance before performing this test. This test should only be applied to powdery, granular or paste-like substances. In order not to include all substances which can be ignited but only those which burn rapidly or those whose burning behaviour is in any way especially dangerous, only substances whose burning velocity exceeds a certain limiting value are considered to be highly flammable. It can be especially dangerous if incandescence propagates through a metal powder because of the difficulties in extinguishing a fire. Metal powders should be considered highly flammable if they support spread of incandescence throughout the mass within a specified time.

1.2. DEFINITION AND UNITS Burning time expressed in seconds.

1.3. REFERENCE SUBSTANCES Not specified.

1.4. PRINCIPLE OF THE METHOD The substance is formed into an unbroken strip or powder train about 250 mm long and a preliminary screening test performed to determine if, on ignition by a gas flame, propagation by burning with flame or smouldering occurs. If propagation over 200 mm of the train occurs within a specified time then a full test programme to determine the burning rate is carried out.

1.5. QUALITY CRITERIA Not stated.

1.6. DESCRIPTION OF METHOD

1.6.

1.6.1. Preliminary screening test The substance is formed into an unbroken strip or powder train about 250 mm long by 20 mm wide by 10 mm high on a non-combustible, non-porous and low heat-conducting base plate. A hot flame from a gas burner (minimum diameter 5 mm) is applied to one end of the powder train until the powder ignites or for a maximum of two minutes (five minutes for powders of metals or metal-alloys). It should be noted whether combustion propagates along 200 mm of the train within the 4 minutes test period (or 40 minutes for metal powders). If the substance does not ignite and propagate combustion either by burning with flame or smouldering along 200 mm of the powder train within the four minutes (or 40 minutes) test period, then the substance should not be considered as highly flammable and no further testing is required. If the substance propagates burning of a 200 mm length of the powder train in less than four minutes, or less than 40 minutes for metal powders, the procedure described below (point 1.6.2. and following) should be carried out.

1.6.2. Burning rate test

1.6.2.

1.6.2.1. Preparation Powdery or granular substances are loosely filled into a mould 250 mm long with a triangular cross-section of inner height 10 mm and width 20 mm. On both sides of the mould in a longitudinal direction two metal plates are mounted as lateral limitations which project 2 mm beyond the upper edge of the triangular cross section (figure). The mould is then dropped three times from a height of 2 cm onto a solid surface. If necessary the mould is then filled up again. The lateral limitations are then removed and the excess substance scraped off. A non-combustible, non-porous and low heat-conducting base plate is placed on top of the mould, the apparatus inverted and the mould removed. Paste-like substances are spread on a non-combustible, non-porous and low heat-conducting base plate in the form of a rope 250 mm in length with a cross section of about 1 cm2.

1.6.2.2. Test conditions In the case a moisture-sensitive substance, the test should be carried out as quickly as possible after its removal from the container.

1.6.2.3. Performance of the test Arrange the pile across the draught in a fume cupboard. The air-speed should be sufficient to prevent fumes escaping into the laboratory and should not be varied during the test. A draught screen should be erected around the apparatus. A hot flame from a gas burner (minimum diameter of 5 mm) is used to ignite the pile at one end. When the pile has burned a distance of 80 mm, the rate of burning over the next 100 mm is measured. The test is performed six times, using a clean cool plate each time, unless a positive result is observed earlier.

2. DATA The burning time from the preliminary screening test (1.6.1) and the shortest burning time in up to six tests (1.6.2.3) are relevant for evaluation.

3. REPORTING

3.

3.1. TEST REPORT The test report shall, if possible, include the following information:

the precise specification of the substance (identification and impurities),

a description of the substance to be tested, its physical state including moisture content,

results from the preliminary screening test and from the burning rate test if performed,

all additional remarks relevant to the interpretation of results.

3.2. INTERPRETATION OF THE RESULT Powdery, granular or paste-1ike substances are to be considered as highly flammable when the time of burning in any tests carried out according to the test procedure described in 1.6.2 is less than 45 seconds. Powders of metals or metal-alloys are considered to be highly flammable when they can be ignited and the flame or the zone of reaction spreads over the whole sample in 10 minutes or less.

4. REFERENCES NF T 20-042 (September 85) Chemical products for industrial use. Determination of the flammability of solids.

A.11. FLAMMABILITY (GASES)

A.11.

1. METHOD

1.

1.1. INTRODUCTION This method allows a determination of whether gases mixed with air at room temperature (circa 20 oC) and atmospheric pressure are flammable and, if so, over what range of concentrations. Mixtures of increasing concentrations of the test gas with air are exposed to an electrical spark and it is observed whether ignition occurs.

1.2. DEFINITION AND UNITS The range of flammability is the range of concentration between the lower and the upper explosion limits. The lower and the upper explosion limits are those limits of concentration of the flammable gas in admixture with air at which propagation of a flame does not occur.

1.3. REFERENCE SUBSTANCES Not specified.

1.4. PRINCIPLE OF THE METHOD The concentration of gas in air is increased step by step and the mixture is exposed at each stage to an electrical spark.

1.5. QUALITY CRITERIA Not stated.

1.6. DESCRIPTION OF THE METHOD

1.6.

1.6.1. Apparatus The test vessel is an upright glass cylinder having a minimum inner diameter of 50 mm and a minimum height of 300 mm. The ignition electrodes are separated by a distance of 3 to 5 mm and are placed 60 mm above the bottom of the cylinder. The cylinder is fitted with a pressure-release opening. The apparatus has to be shielded to restrict any explosion damage. A standing induction spark of 0,5 sec. duration, which is generated from a high voltage transformer with an output voltage of 10 to 15 kV (maximum of power input 300 W), is used as the ignition source. An example of a suitable apparatus is described in reference (2).

1.6.2. Test conditions The test must be performed at room temperature (circa 20 oC).

1.6.3. Performance of the test Using proportioning pumps, a known concentration of gas in air is introduced into the glass cylinder. A spark is passed through the mixture and it is observed whether or not a flame detaches itself from the ignition source and propagates independently. The gas concentration is varied in steps of 1 % vol. until ignition occurs as described above. If the chemical structure of the gas indicates that it would be non-flammable and the composition of the stoichiometric mixture with air can be calculated, then only mixtures in the range from 10 % less than the stoichiometric composition to 10 % greater than this composition need be tested in 1 % steps.

2. DATA The occurrence of flame propagation is the only relevant information data for the determination of this property.

3. REPORTING The test report shall, if possible, include the following information:

the precise specification of the substance (identification and impurities),

a description, with dimensions, of the apparatus used,

the temperature at which the test was performed,

the tested concentrations and the results obtained,

the result of the test: non-flammable gas or highly flammable gas,

if it is concluded that the gas is non-flammable then the concentration range over which it was tested in 1 % steps should be stated,

all information and remarks relevant to the interpretation of results have to be reported.

4.(1)(2) REFERENCES NF T 20-041 (September 85) Chemical products for industrial use. Determination of the flammability of gases. W. Berthold, D. Conrad, T. Grewer, H. Grosse-Wortmann Entwicklung einer Standard-Apparatur zur Messung von Explosionsgrenzen. Chem.-Ing.- Tech. 1984, vo1. 56, 2, 126-127. , T. Redeker und H. Schacke, p. 126-127.

A.12. FLAMMABILITY (CONTACT WITH WATER)

A.12.

1. METHOD

1.

1.1. INTRODUCTION This test method can be used to determine whether the reaction of a substance with water or damp air leads to the development of dangerous amounts of gas or gases which may be highly flammable. The test method can be applied to both solid and liquid substances. This method is not applicable to substances which spontaneously ignite when in contact with air.

1.2. DEFINITIONS AND UNITS Highly flammable: substances which, in contact with water or damp air, evolve highly flammable gases in dangerous quantities at a minimum rate of 1 litre/kg per hour.

1.3. PRINCIPLE OF THE METHOD The substance is tested according to the step by step sequence described below; if ignition occurs at any step, no further testing is necessary. If it is known that the substance does not react violently with water then proceed to step 4 (1.3.4).

1.3.

1.3.1. Step 1 The test substance is placed in a trough containing distilled water at 20 oC and it is noted whether or not the evolved gas ignites.

1.3.2. Step 2 The test substance is placed on a filter paper floating on the surface of a dish containing distilled water at 20 oC and it is noted whether or not the evolved gas ignites. The filter paper is merely to keep the substance in one place to increase the chances of ignition.

1.3.3. Step 3 The test substance is made into a pile approximately 2 cm high and 3 cm diameter. A few drops of water are added to the pile and it is noted whether or not the evolved gas ignites.

1.3.4. Step 4 The test substance is mixed with distilled water at 20 oC and the rate of evolution of gas is measured over a period of seven hours, at one-hour intervals. If the rate of evolution is erratic, or is increasing, after seven hours, the measuring time should be extended to a maximum time of five days. The test may be stopped if the rate at any time exceeds 1 litre/kg per hour.

1.4. REFERENCE SUBSTANCES Not specified.

1.5. QUALITY CR1TERIA Not stated.

1.6. DESCRIPTION OF METHODS

1.6.

1.6.1. Step 1

1.6.1.

1.6.1.1. Test conditions The test is performed at room temperature (circa 20 oC).

1.6.1.2. Performance of the test A small quantity (approximately 2 mm diameter) of the test substance should be placed in a trough containing distilled water. A note should be made of whether (i) any gas is evolved and (ii) if ignition of the gas occurs. If ignition of the gas occurs then no further testing of the substance is needed because the substance is regarded as hazardous.

1.6.2. Step 2

1.6.2.

1.6.2.1. Apparatus A filter-paper is floated flat on the surface of distilled water in any suitable vessel, e.g. a 100 mm diameter evaporating dish.

1.6.2.2. Test conditions The test is performed at room temperature (circa 20 oC).

1.6.2.3. Performance of the test A small quantity of the test substance (approximately 2 mm diameter) is placed onto the centre of the filter-paper. A note should be made of whether (i) any gas is evolved and (ii) if ignition of the gas occurs. If ignition of the gas occurs then no further testing of the substance is needed because the substance is regarded as hazardous.

1.6.3. Step 3

1.6.3.

1.6.3.1. Test conditions The test is performed at room temperature (circa 20 oC).

1.6.3.2. Performance of the test The test substance is made into a pile approximately 2 cm high and 3 cm diameter with an indentation in the top. A few drops of water are added to the hollow and a note is made of whether (i) any gas is evolved and (ii) if ignition of the gas occurs. If ignition of the gas occurs then no further testing of the substance is needed because the substance is regarded as hazardous.

1.6.4. Step 4

1.6.4.

1.6.4.1. Apparatus The apparatus is set up as shown in the figure.

1.6.4.2. Test conditions Inspect the container of the test substance for any powder < 500 μm (particle size). If the powder constitutes more than 1 % w/w of the total, or if the sample is friable, then the whole of the substance should be ground to a powder before testing to allow for a reduction in particle size during storage and handling; otherwise the substance is to be tested as received. The test should be performed at room temperature (circa 20 oC) and atmospheric pressure.

1.6.4.3. Performance of the test 10 to 20 ml of water are put into the dropping funnel of the apparatus and 10 g of substance are put in the conical flask. The volume of gas evolved can be measured by any suitable means. The tap of the dropping funnel is opened to let the water into the conical flask and a stop watch is started. The gas evolution is measured each hour during a seven hour period. If, during this period, the gas evolution is erratic, or if, at the end of this period, the rate of gas evolution is increasing, then measurements should be continued for up to five days. If, at any time of measurement, the rate of gas evolution exceeds 1 litre/kg per hour, the test can be discontinued. This test should be performed in triplicate. If the chemical identity of the gas is unknown, the gas should be analysed. When the gas contains highly flammable components and it is unknown whether the whole mixture is highly flammable, a mixture of the same composition has to be prepared and tested according to the method A.11.

2. DATA The substance is considered hazardous if:

spontaneous ignition takes place in any step of the test procedure, or

there is evolution of flammable gas at a rate greater than 1 litre/kg of the substance per hour.

3. REPORTING The test report shall, if possible, include the following information:

the precise specification of the substance (identification and impurities),

details of any initial preparation of the test substance,

the results of the tests (steps 1, 2, 3 and 4),

the chemical identity of gas evolved,

the rate of evolution of gas if step 4 (1.6.4) is performed,

any additional remarks relevant to the interpretation of the results.

4.(1)(2) REFERENCES Recommendations on the transport of dangerous goods, test and criteria, 1990, United Nations, New York. NF T 20-040 (September 85) Chemical products for industrial use. Determination of the flammability of gases formed by the hydrolysis of solid and liquid products.

A.13. PYROPHORIC PROPERTIES OF SOLIDS AND LIQUIDS

A.13.

1. METHOD

1.

1.1. INTRODUCTION The test procedure is applicable to solid or liquid substances, which, in small amounts, will ignite spontaneously a short time after coming into contact with air at room temperature (circa 20 oC). Substances which need to be exposed to air for hours or days at room temperature or at elevated temperatures before ignition occurs are not covered by this test method.

1.2. DEFINITIONS AND UNITS Substances are considered to have pyrophoric properties if they ignite or cause charring under the conditions described in 1.6. The auto-flammability of liquids may also need to be tested using method A.15. Auto-ignition temperature (liquids and gases).

1.3. REFERENCE SUBSTANCES Not specified.

1.4. PRINCIPLE OF THE METHOD The substance, whether solid or liquid, is added to an inert carrier and brought into contact with air at ambient temperature for a period of five minutes. If liquid substances do not ignite then they are absorbed onto filter paper and exposed to air at ambient temperature (circa 20 oC) for five minutes. If a solid or liquid ignites, or a liquid ignites or chars a filter paper, then the substance is considered to be pyrophoric.

1.5. QUALITY CRITERIA Repeatability: because of the importance in relation to safety, a single positive result is sufficient for the substance to be considered pyrophoric.

1.6. DESCRIPTION OF THE TEST METHOD

1.6.

1.6.1. Apparatus A porcelain cup of circa 10 cm diameter is filled with diatomaceous earth to a height of about 5 mm at room temperature (circa 20 oC). Note: Diatomaceous earth or any other comparable inert substance which is generally obtainable shall be taken as representative of soil onto which the test substance might be spilled in the event of an accident. Dry filter paper is required for testing liquids which do not ignite on contact with air when in contact with an inert carrier.

1.6.2. Performance of the test

1.6.2.

(a) Powdery solids 1 to 2 cm3 of the substance to be tested is poured from circa 1 m height onto a non-combustible surface and it is observed whether the substance ignites during dropping or within five minutes of settling. The test is performed six times unless ignition occurs;

(b) liquids Circa 5 cm3 of the liquid to be tested is poured into the prepared porcelain cup and it is observed whether the substance ignites within five minutes. If no ignition occurs in the six tests, perform the following tests: A 0,5 ml test sample is delivered from a syringe to an indented filter paper and it is observed whether ignition or charring of the filter paper occurs within five minutes of the liquid being added. The test is performed three times unless ignition or charring occurs.

2. DATA

2.

2.1. TREATMENT OF RESULTS Testing can be discontinued as soon as a positive result occurs in any of the tests.

2.2. EVALUATION If the substance ignites within five minutes when added to an inert carrier and exposed to air, or a liquid substance chars or ignites a filter paper within five minutes when added and exposed to air, it is considered to be pyrophoric.

3. REPORTING The test report shall, if possible, include the following information:

the precise specification of the substance (identification and impurities),

the results of the tests,

any additional remark relevant to the interpretation of the results.

4.(1)(2) REFERENCES NF T 20-039 (September 85) Chemical products for industrial use. Determination of the spontaneous flammability of solids and liquids. Recommendations on the Transport of Dangerous Goods, Test and criteria, 1990, United Nations, New York.

A.14. EXPLOSIVE PROPERTIES

A.14.

1. METHOD

1.

1.1. INTRODUCTION The method provides a scheme of testing to determine whether a solid or a pasty substance presents a danger of explosion when submitted to the effect of a flame (thermal sensitivity), or to shock or friction (sensitivity to mechanical stimuli), and whether a liquid substance presents a danger of explosion when submitted to the effect of a flame or shock. The method comprises three parts: The method yields data to assess the likelihood of initiating an explosion by means of certain common stimuli. The method is not intended to ascertain whether a substance is capable of exploding under any conditions. The method is appropriate for determining whether a substance will present a danger of explosion (thermal and mechanical sensitivity) under the particular conditions specified in the directive. It is based on a number of types of apparatus which are widely used internationally (1) and which usually give meaningful results. It is recognised that the method is not definitive. Alternative apparatus to that specified may be used provided that it is internationally recognised and the results can be adequately correlated with those from the specified apparatus. The tests need not be performed when available thermodynamic information (e.g. heat of formation, heat of decomposition) and/or absence of certain reactive groups (2) in the structural formula establishes beyond reasonable doubt that the substance is incapable of rapid decomposition with evolution of gases or release of heat (i.e. the material does not present any risk of explosion). A test of mechanical sensitivity with respect to friction is not required for liquids.

1.1.

(a) a test of thermal sensitivity (1);

(b) a test of mechanical sensitivity with respect to shock (1);

1.2. DEFINITIONS AND UNITS Explosive: Substances which may explode under the effect of flame or which are sensitive to shock or friction in the specified apparatus (or are more mechanically sensitive than 1,3-dinitrobenzene in alternative apparatus).

1.3. REFERENCE SUBSTANCES 1,3-dinitrobenzene, technical crystalline product sieved to pass 0,5 mm, for the friction and shock methods. Perhydro-1,3,5-trinitro-1,3,5-triazine (RDX, hexogen, cyclonite — CAS 121-82-4), recrystallised from aqueous cyclohexanone, wet-sieved through a 250 μm and retained on a 150 μm sieve and dried at 103 ± 2 oC (for four hours) for the second series of friction and shock tests.

1.4. PRINCIPLE OF THE METHOD Preliminary tests are necessary to establish safe conditions for the performance of the three tests of sensitivity.

1.4.

1.4.1. Safety-in-handling tests (3) For safety reasons, before performing the main tests, very small samples (circa 10 mg) of the substance are subjected to heating without confinement in a gas flame, to shock in any convenient form of apparatus and to friction by the use of a mallet against an anvil or any form of friction machine. The objective is to ascertain if the substance is so sensitive and explosive that the prescribed sensitivity tests, particularly that of thermal sensitivity, should be performed with special precautions so as to avoid injury to the operator.

1.4.2. Thermal sensitivity The method involves heating the substance in a steel tube, closed by orifice plates with differing diameters of hole, to determine whether the substance is liable to explode under conditions of intense heat and defined confinement.

1.4.3. Mechanical sensitivity (shock) The method involves subjecting the substance to the shock from a specified mass dropped from a specified height.

1.4.4. Mechanical sensitivity (friction) The method involves subjecting solid or pasty substances to friction between standard surfaces under specified conditions of load and relative motion.

1.5. QUALITY CRITERIA Not stated.

1.6. DESCRIPTION OF METHOD

1.6.

1.6.1. Thermal sensitivity (effect of a flame)

1.6.1.

1.6.1.1. Apparatus The apparatus consists of a non-reusable steel tube with its re-usable closing device (figure 1), installed in a heating and protective device. Each tube is deep-drawn from sheet steel (see Appendix) and has an internal diameter of 24 mm, a length of 75 mm and wall thickness of 0,5 mm. The tubes are flanged at the open end to enable them to be closed by the orifice plate assembly. This consists of a pressure-resistant orifice plate, with a central hole, secured firmly to a tube using a two-part screw joint (nut and threaded collar). The nut and threaded collar are made from chromium-manganese steel (see Appendix) which is spark-free up to 800 oC. The orifice plates are 6 mm thick, made from heat-resistant steel (see Appendix), and are available with a range of diameters of opening.

1.6.1.2. Test conditions Normally the substance is tested as received although in certain cases, e.g. if pressed, cast or otherwise condensed, it may be necessary to test the substance after crushing. For solids, the mass of material to be used in each test is determined using a two-stage dry run procedure. A tared tube is filled with 9 cm3 of substance and the substance tamped with 80 N force applied to the total cross-section of the tube. For reasons of safety or in cases where the physical form of the sample can be changed by compression other filling procedures may be used; e.g. if the substance is very friction sensitive then tamping is not appropriate. If the material is compressible then more is added and tamped until the tube is filled to 55 mm from the top. The total mass used to fill the tube to the 55 mm level is determined and two further increments, each tamped with 80 N force, are added. Material is then either added with tamping, or taken out, as required, to leave the tube filled to a level 15 mm from the top. A second dry run is performed, starting with a tamped quantity of a third of the total mass found in the first dry run. Two more of these increments are added with 80 N tamping and the level of the substance in the tube adjusted to 15 mm from the top by addition or subtraction of material as required. The amount of solid determined in the second dry run is used for each trial; filling being performed in three equal amounts, each compressed to 9 cm3 by whatever force is necessary. (This may be facilitated by the use of spacing rings). Liquids and gels are loaded into the tube to a height of 60 mm taking particular care with gels to prevent the formation of voids. The threaded collar is slipped onto the tube from below, the appropriate orifice plate is inserted and the nut tightened after applying some molybdenum disulphide based lubricant. It is essential to check that none of the substance is trapped between the flange and the plate, or in the threads. Heating is provided by propane taken from an industrial cylinder, fitted with a pressure regulator (60 to 70 mbar), through a meter and evenly distributed (as indicated by visual observation of the flames from the burners) by a manifold to four burners. The burners are located around the test chamber as shown in figure 1. The four burners have a combined consumption of about 3,2 litres of propane per minute. Alternative fuel gases and burners may be used but the heating rate must be as specified in figure 3. For all apparatus, the heating rate must be checked periodically using tubes filled with dibutyl phthalate as indicated in figure 3.

1.6.1.3. Performance of the tests Each test is performed until either the tube is fragmented or the tube has been heated for five minutes. A test resulting in the fragmentation of the tube into three or more pieces, which in some cases may be connected to each other by narrow strips of metal as illustrated in figure 2, is evaluated as giving an explosion. A test resulting in fewer fragments or no fragmentation is regarded as not giving an explosion. A series of three tests with a 6,0 mm diameter orifice plate is first performed and, if no explosions are obtained, a second series of three tests is performed with a 2,0 mm diameter orifice plate. If an explosion occurs during either test series then no further tests are required.

1.6.1.4. Evaluation The test result is considered positive if an explosion occurs in either of the above series of tests.

1.6.2. Mechanical sensitivity (shock)

1.6.2.

1.6.2.1. Apparatus (figure 4) The essential parts of a typical fall hammer apparatus are a cast steel block with base, anvil, column, guides, drop weights, release device and a sample holder. The steel anvil 100 mm (diameter) × 70 mm (height) is screwed to the top of a steel block 230 mm (length) × 250 mm (width) × 200 mm (height) with a cast base 450 mm (length) × 450 mm (width) × 60 mm (height). A column, made from seamless drawn steel tube, is secured in a holder screwed on to the back of the steel block. Four screws anchor the apparatus to a solid concrete block 60 × 60 × 60 cm such that the guide rails are absolutely vertical and the drop weight falls freely. 5 and 10 kg weights, made from solid steel, are available for use. The striking head of each weight is of hardened steel, HRC 60 to 63, and has a minimum diameter of 25 mm. The sample under test is enclosed in a shock device consisting of two coaxial solid steel cylinders, one above the other, in a hollow cylindrical steel guide ring. The solid steel cylinders should be of 10 (-0,003, -0,005) mm diameter and 10 mm height and have polished surfaces, rounded edges (radius of curvature 0,5 mm) and a hardness of HRC 58 to 65. The hollow cylinder must have an external diameter of 16 mm, a polished bore of 10 (+0,005, +0,010) mm and a height of 13 mm. The shock device is assembled on an intermediate anvil (26 mm diameter and 26 mm height) made of steel and centred by a ring with perforations to allow escape of fumes.

1.6.2.2. Test conditions The sample volume should be 40 mm3, or a volume to suit any alternative apparatus. Solid substances should be tested in the dry state and prepared as follows: Substances normally supplied as pastes should be tested in the dry state where possible or, in any case, following removal of the maximum possible amount of diluent. Liquid substances are tested with a 1 mm gap between the upper and lower steel cylinders.

1.6.2.2.

(a) powdered substances are sieved (sieve size 0,5 mm); all that has passed through the sieve is used for testing;

(b) pressed, cast or otherwise condensed substances are broken into small pieces and sieved; the sieve fraction from 0,5 to 1 mm diameter is used for testing and should be representative of the original substance.

1.6.2.3. Performance of the tests A series of six tests are performed dropping the 10 kg mass from 0,40 m (40 J). If an explosion is obtained during the six tests at 40 J, a further series of six tests, dropping a 5 kg mass from 0,15 m (7,5 J), must be performed. In other apparatus, the sample is compared with the chosen reference substance using an established procedure (e.g. up-and-down technique etc.).

1.6.2.4. Evaluation The test result is considered positive if an explosion (bursting into flame and/or a report is equivalent to explosion) occurs at least once in any of the tests with the specified shock apparatus or the sample is more sensitive than 1,3-dinitrobenzene or RDX in an alternative shock test.

1.6.3. Mechanical sensitivity (friction)

1.6.3.

1.6.3.1. Apparatus (figure 5) The friction apparatus consists of a cast steel base plate on which is mounted the friction device. This consists of a fixed porcelain peg and moving porcelain plate. The porcelain plate is held in a carriage which runs in two guides. The carriage is connected to an electric motor via a connecting rod, an eccentric cam and suitable gearing such that the porcelain plate is moved, once only, back and forth beneath the porcelain peg for a distance of 10 mm. The porcelain peg may be loaded with, for example, 120 or 360 newtons. The flat porcelain plates are made from white technical porcelain (roughness 9 to 32 μm) and have the dimensions 25 mm (length) × 25 mm (width) × 5 mm (height). The cylindrical porcelain peg is also made of white technical porcelain and is 15 mm long, has a diameter of 10 mm and roughened spherical end surfaces with a radius of curvature of 10 mm.

1.6.3.2. Test conditions The sample volume should be 10 mm3 or a volume to suit any alternative apparatus. Solid substances are tested in the dry state and prepared as follows: Substances normally supplied as pastes should be tested in the dry state where possible. If the substance cannot be prepared in the dry state, the paste (following removal of the maximum possible amount of diluent) is tested as a 0,5 mm thick, 2 mm wide, 10 mm long film, prepared with a former.

1.6.3.2.

(a) powdered substances are sieved (sieve size 0,5 mm); all that has passed through the sieve is used for testing;

(b) pressed, cast or otherwise condensed substances are broken into small pieces and sieved; the sieve fraction < 0,5 mm diameter is used for testing.

1.6.3.3. Performance of the tests The porcelain peg is brought onto the sample under test and the load applied. When carrying out the test, the sponge marks of the porcelain plate must lie transversely to the direction of the movement. Care must be taken that the peg rests on the sample, that sufficient test material lies under the peg and also that the plate moves correctly under the peg. For pasty substances, a 0,5 mm thick gauge with a 2 × 10 mm slot is used to apply the substance to the plate. The porcelain plate has to move 10 mm forwards and backwards under the porcelain peg in a time of 0,44 seconds. Each part of the surface of the plate and peg must only be used once; the two ends of each peg will serve for two trials and the two surfaces of a plate will each serve for three trials. A series of six tests are performed with a 360 N loading. If a positive event is obtained during these six tests, a further series of six tests must be performed with a 120 N loading. In other apparatus, the sample is compared with the chosen reference substance using an established procedure (e.g. up-and-down technique, etc.).

1.6.3.4. Evaluation The test result is considered positive if an explosion (crepitation and/or a report or bursting into flame are equivalent to explosion) occurs at least once in any of the tests with the specified friction apparatus or satisfies the equivalent criteria in an alternative friction test.

2. DATA In principle, a substance is considered to present a danger of explosion in the sense of the directive if a positive result is obtained in the thermal, shock or friction sensitivity test.

3. REPORTING

3.

3.1. TEST REPORT The test report shall, if possible, include the following information:

identity, composition, purity, moisture content, etc. of the substance tested,

the physical form of the sample and whether or not it has been crushed, broken and/or sieved,

observations during the thermal sensitivity tests (e.g. sample mass, number of fragments, etc.),

observations during the mechanical sensitivity tests (e.g. formation of considerable amounts of smoke or complete decomposition without a report, flames, sparks, report, crepitation, etc.),

results of each type of test,

if alternative apparatus has been used, scientific justification as well as evidence of correlation between results obtained with specified apparatus and those obtained with equivalent apparatus must be given,

any useful comments such as reference to tests with similar products which might be relevant to a proper interpretation of the results,

all additional remarks relevant for the interpretation of the results.

3.2. INTERPRETATION AND EVALUATION OF RESULTS The test report should mention any results which are considered false, anomalous or unrepresentative. If any of the results should be discounted, an explanation and the results of any alternative or supplementary testing should be given. Unless an anomalous result can be explained, it must be accepted at face value and used to classify the substance accordingly.

4.(1)(2)(3)(4)

REFERENCES Recommendations on the Transport of Dangerous Goods: Tests and criteria, 1990, United Nations, New York. Bretherick, L., Handbook of Reactive Chemical Hazards, 4th edition, Butterworths, London, ISBN 0-750-60103-5, 1990. Koenen, H., Ide, K.H. and Swart, K.H., Explosivstoffe, 1961, vol. 3, 6-13 and 30-42. NF T 20-038 (September 85) Chemical products for industrial use — Determination of explosion risk.

A.15. AUTO-IGNITION TEMPERATURE (LIQUIDS AND GASES)

A.15.

1. METHOD

1.

1.1. INTRODUCTION Explosive substances and substances which ignite spontaneously in contact with air at ambient temperature should not be submitted to this test. The test procedure is applicable to gases, liquids and vapours which, in the presence of air, can be ignited by a hot surface. The auto-ignition temperature can be considerably reduced by the presence of catalytic impurities, by the surface material or by a higher volume of the test vessel.

1.2. DEFINITIONS AND UNITS The degree of auto-ignitability is expressed in terms of the auto-ignition temperature. The auto-ignition temperature is the lowest temperature at which the test substance will ignite when mixed with air under the conditions defined in the test method.

1.3. REFERENCE SUBSTANCES Reference substances are cited in the standards (see 1.6.3). They should primarily serve to check the performance of the method from time to time and to allow comparison with results from other methods.

1.4. PRINCIPLE OF THE METHOD The method determines the minimum temperature of the inner surface of an enclosure that will result in ignition of a gas, vapour or liquid injected into the enclosure.

1.5. QUALITY CRITERIA The repeatability varies according to the range of auto-ignition temperatures and the test method used. The sensitivity and specificity depend on the test method used.

1.6. DESCRIPTION OF THE METHOD

1.6.

1.6.1. Apparatus The apparatus is described in the method referred to in 1.6.3.

1.6.2. Test conditions A sample of the test substance is tested according to the method referred to in 1.6.3.

1.6.3. Performance of the test See IEC 79-4, DIN 51794, ASTM-E 659-78, BS 4056, NF T 20-037.

2. DATA Record the test-temperature, atmospheric pressure, quantity of sample used and time-1ag until ignition occurs.

3. REPORTING The test report shall, if possible, include the following information:

the precise specification of the substance (identification and impurities),

the quantity of sample used, atmospheric pressure,

the apparatus used,

the results of measurements (test temperatures, results concerning ignition, corresponding time-lags),

all additional remarks relevant to the interpretation of results.

4. REFERENCES None.

A.16. RELATIVE SELF-IGNITION TEMPERATURE FOR SOLIDS

A.16.

1. METHOD

1.

1.1. INTRODUCTION Explosive substances and substances which ignite spontaneously in contact with air at ambient temperature should not be submitted to this test. The purpose of this test is to provide preliminary information on the auto-flammability of solid substances at elevated temperatures. If the heat developed either by a reaction of the substance with oxygen or by exothermic decomposition is not lost rapidly enough to the surroundings, self-heating leading to self-ignition occurs. Self-ignition therefore occurs when the rate of heat-production exceeds the rate of heat loss. The test procedure is useful as a preliminary screening test for solid substances. In view of the complex nature of the ignition and combustion of solids, the self-ignition temperature determined according to this test method should be used for comparison purposes only.

1.2. DEFINITIONS AND UNITS The self-ignition temperature as obtained by this method is the minimum ambient temperature expressed in oC at which a certain volume of a substance will ignite under defined conditions.

1.3. REFERENCE SUBSTANCE None.

1.4. PRINCIPLE OF THE METHOD A certain volume of the substance under test is placed in an oven at room temperature; the temperature/time curve relating to conditions in the centre of the sample is recorded while the temperature of the oven is increased to 400 oC, or to the melting point if lower, at a rate of 0,5 oC/min. For the purpose of this test, the temperature of the oven at which the sample temperature reaches 400 oC by self-heating is called the self-ignition temperature.

1.5. QUALITY CRITERIA None.

1.6. DESCRIPTION OF THE METHOD

1.6.

1.6.1. Apparatus

1.6.1.

1.6.1.1. Oven A temperature-programmed laboratory oven (volume about 2 litres) fitted with natural air circulation and explosion relief. In order to avoid a potential explosion risk, any decomposition gases must not be allowed to come into contact with the electric heating elements.

1.6.1.2. Wire mesh cube A piece of stainless steel wire mesh with 0,045 mm openings should be cut according to the pattern in figure 1. The mesh should be folded and secured with wire into an open-topped cube.

1.6.1.3. Thermocouples Suitable thermocouples.

1.6.1.4. Recorder Any two-channel recorder calibrated from 0 to 600 oC or corresponding voltage.

1.6.2. Test conditions Substances are tested as received.

1.6.3. Performance of the test The cube is filled with the substance to be tested and is tapped gently, adding more of the substance until the cube is completely full. The cube is then suspended in the centre of the oven at room temperature. One thermocouple is placed at the centre of the cube and the other between the cube and the oven wall to record the oven temperature. The temperatures of the oven and sample are continuously recorded while the temperature of the oven is increased to 400 oC, or to the melting point if lower, at a rate of 0,5 oC/min. When the substance ignites the sample thermocouple will show a very sharp temperature rise above the oven temperature.

2. DATA The temperature of the oven at which the sample temperature reaches 400 oC by self-heating is relevant for evaluation (see figure 2).

3. REPORTING The test report shall, if possible, include the following information:

a description of the substance to be tested,

the results of measurement including the temperature/time curve,

all additional remarks relevant for the interpretation of the results.

4. REFERENCES NF T 20-036 (September 85) Chemical products for industrial use. Determination of the relative temperature of the spontaneous flammability of solids.

4. Figure 1 Pattern of 20 mm test cube

Figure 2 Typical temperature/time curve

sample temperature oven temperature ignition temperature Time

A.17. OXIDISING PROPERTIES (SOLIDS)

A.17.

1. METHOD

1.

1.1. INTRODUCTION It is useful to have preliminary information on any potentially explosive properties of the substance before performing this test. This test is not applicable to liquids, gases, explosive or highly flammable substances, or organic peroxides. This test need not be performed when examination of the structural formula establishes beyond reasonable doubt that the substance is incapable of reacting exothermically with a combustible material. In order to ascertain if the test should be performed with special precautions, a preliminary test should be performed.

1.2. DEFINITION AND UNITS Burning time: reaction time, in seconds, taken for the reaction zone to travel along a pile, following the procedure described in 1.6. Burning rate: expressed in millimetres per second. Maximum burning rate: the highest value of the burning rates obtained with mixtures containing 10 to 90 % by weight of oxidiser.

1.3. REFERENCE SUBSTANCE Barium nitrate (analytical grade) is used as reference substance for the test and the preliminary test. The reference mixture is that mixture of barium nitrate with powdered cellulose, prepared according to 1.6, which has the maximum burning rate (usually a mixture with 60 % barium nitrate by weight).

1.4. PRINCIPLE OF THE METHOD A preliminary test is carried out in the interests of safety. No further testing is required when the preliminary test clearly indicates that the test substance has oxidising properties. When this is not the case, the substance should then be subject to the full test. In the full test, the substance to be tested and a defined combustible substance will be mixed in various ratios. Each mixture is then formed into a pile and the pile is ignited at one end. The maximum burning rate determined is compared with the maximum burning rate of the reference mixture.

1.5. QUALITY CRITERIA If required, any method of grinding and mixing is valid provided that the difference in the maximum rate of burning in the six separate tests differs from the arithmetic mean value by no more than 10 %.

1.6. DESCRIPTION OF THE METHOD

1.6.

1.6.1. Preparation

1.6.1.

1.6.1.1. Test substance Reduce the test sample to a particle size < 0,125 mm using the following procedure: sieve the test substance, grind the remaining fraction, repeat the procedure until the whole test portion has passed the sieve. Any grinding and sieving method satisfying the quality criteria may be used. Before preparing the mixture the substance is dried at 105 oC, until constant weight is obtained. If the decomposition temperature of the substance to be tested is below 105 oC, the substance has to be dried at a suitable lower temperature.

1.6.1.2. Combustible substance Powdered cellulose is used as a combustible substance. The cellulose should be a type used for thin-layer chromatography or column chromatography. A type with fibre-lengths of more than 85 % between 0,020 and 0,075 mm has proved to be suitable. The cellulose powder is passed through a sieve with a mesh-size of 0,125 mm. The same batch of cellulose is to be used throughout the test. Before preparing the mixture, the powdered cellulose is dried at 105 oC until constant weight is obtained. If wood-meal is used in the preliminary test, then prepare a soft-wood wood-meal by collecting the portion which passes through a sieve mesh of 1,6 mm, mix thoroughly, then dry at 105 oC for four hours in a layer not more than 25 mm thick. Cool and store in an air-tight container filled as full as practicable until required, preferably within 24 hours of drying.

1.6.1.3. Ignition source A hot flame from a gas burner (minimum diameter 5 mm) should be used as the ignition source. If another ignition source is used (e.g. when testing in an inert atmosphere), the description and the justification should be reported.

1.6.2. Performance of the test Note: Mixtures of oxidisers with cellulose or wood-meal must be treated as potentially explosive and handled with due care.

1.6.2.

1.6.2.1. Preliminary test The dried substance is thoroughly mixed with the dried cellulose or wood-meal in the proportions 2 of test substance to 1 of cellulose or wood-meal by weight and the mixture is formed into a small cone-shaped pile of dimensions 3,5 cm (diameter of base) × 2,5 cm (height) by filling, without tamping, a cone-shaped former (e.g. a laboratory glass funnel with the stem plugged). The pile is placed on a cool, non-combustible, non-porous and low heat-conducting base plate. The test should be carried out in a fume cupboard as in 1.6.2.2. The ignition source is put in contact with the cone. The vigour and duration of the resultant reaction are observed and recorded. The substance is to be considered as oxidising if the reaction is vigorous. In any case where the result is open to doubt, it is then necessary to complete the full train test described below.

1.6.2.2. Train test Prepare oxidiser cellulose-mixtures containing 10 to 90 % weight of oxidiser in 10 % increments. For borderline cases, intermediate oxidiser cellulose mixtures should be used to obtain the maximum burning rate more precisely. The pile is formed by means of a mould. The mould is made of metal, has a length of 250 mm and a triangular cross-section with an inner height of 10 mm and an inner width of 20 mm. On both sides of the mould, in the longitudinal direction, two metal plates are mounted as lateral limitations which project 2 mm beyond the upper edge of the triangular cross-section (figure). This arrangement is loosely filled with a slight excess of mixture. After dropping the mould once from a height of 2 cm onto a solid surface, the remaining excess substance is scraped off with an obliquely positioned sheet. The lateral limitations are removed and the remaining powder is smoothed, using a roller. A non-combustible, non-porous and low heat-conducting base plate is then placed on the top of the mould, the apparatus inverted and the mould removed. Arrange the pile across the draught in a fume cupboard. The air-speed should be sufficient to prevent fumes escaping into the laboratory and should not be varied during the test. A draught screen should be erected around the apparatus. Due to hygroscopicity of cellulose and of some substances to be tested, the test should be carried out as quickly as possible. Ignite one end of the pile by touching with the flame. Measure the time of reaction over a distance of 200 mm after the reaction zone has propagated an initial distance of 30 mm. The test is performed with the reference substance and at least once with each one of the range of mixtures of the test substance with cellulose. If the maximum burning rate is found to be significantly greater than that from the reference mixture, the test can be stopped; otherwise the test should be repeated five times for each of the three mixtures giving the fastest burning rate. If the result is suspected of being a false positive, then the test should be repeated using an inert substance with a similar particle size, such as kieselguhr, in place of cellulose. Alternatively, the test substance cellulose mixture, having the fastest burning rate, should be retested in an inert atmosphere (< 2 % v/v oxygen content).

2. DATA For safety reasons the maximum burning rate — not the mean value — shall be considered to be the characteristic oxidising property of the substance under test. The highest value of burning rate within a run of six tests of a given mixture is relevant for evaluation. Plot a graph of the highest value of burning rate for each mixture versus the oxidiser concentration. From the graph take the maximum burning rate. The six measured values of burning rate within a run obtained from the mixture with the maximum burning rate must not differ from the arithmetic mean value by more than 10 %; otherwise the methods of grinding and mixing must be improved. Compare the maximum burning rate obtained with the maximum burning rate of the reference mixture (see 1.3). If tests are conducted in an inert atmosphere, the maximum reaction rate is compared with that from the reference mixture in an inert atmosphere.

3. REPORT

3.

3.1. TEST REPORT The test report shall, if possible, include the following information:

the identity, composition, purity, moisture content etc. of the substance tested,

any treatment of the test sample (e.g. grinding, drying),

the ignition source used in the tests,

the results of measurements,

the mode of reaction (e.g. flash burning at the surface, burning through the whole mass, any information concerning the combustion products, etc.),

all additional remarks relevant for the interpretation of results, including a description of the vigour (flaming, sparking, fuming, slow smouldering, etc.) and approximate duration produced in the preliminary safety/screening test for both test and reference substance,

the results from tests with an inert substance, if any,

the results from tests in an inert atmosphere, if any.

3.2. INTERPRETATION OF THE RESULT A substance is to be considered as an oxidising substance when: In order to avoid a false positive, the results obtained when testing the substance mixed with an inert material and/or when testing under an inert atmosphere should also be considered when interpreting the results.

3.2.

(a) in the preliminary test, there is a vigorous reaction;

(b) in the full test, the maximum burning rate of the mixtures tested is higher than or equal to the maximum burning rate of the reference mixture of cellulose and barium nitrate.

4. REFERENCES NF T 20-035 (September 85) Chemical products for industrial use. Determination of the oxidising properties of solids.

A.18. NUMBER-AVERAGE MOLECULAR WEIGHT AND MOLECULAR WEIGHT DISTRIBUTION OF POLYMERS

A.18.

1. METHOD This Gel Permeation Chromatographic method is a replicate of the OECD TG 118 (1996). The fundamental principles and further technical information are given in reference (1).

1.

1.1. INTRODUCTION Since the properties of polymers are so varied, it is impossible to describe one single method setting out precisely the conditions for separation and evaluation which cover all eventualities and specificities occurring in the separation of polymers. In particular, complex polymer systems are often not amenable to gel permeation chromatography (GPC). When GPC is not practicable, the molecular weight may be determined by means of other methods (see Appendix). In such cases, full details and justification should be given for the method used. The method described is based on DIN Standard 55672 (1). Detailed information about how to carry out the experiments and how to evaluate the data can be found in this DIN Standard. In case modifications of the experimental conditions are necessary, these changes must be justified. Other standards may be used, if fully referenced. The method described uses polystyrene samples of known polydispersity for calibration and it may have to be modified to be suitable for certain polymers, e.g. water soluble and long-chain branched polymers.

1.2. DEFINITIONS AND UNITS The number-average molecular weight Mn and the weight average molecular weight Mw are determined using the following equations: Mn=Σni=1HiΣni=1Hi/Mi Mw=Σni=1Hi × MiΣni=1Hi where, Hi is the level of the detector signal from the baseline for the retention volume Vi, Mi is the molecular weight of the polymer fraction at the retention volume Vi, and n is the number of data points. The breadth of the molecular weight distribution, which is a measure of the dispersity of the system, is given by the ratio Mw/Mn.

1.3. REFERENCE SUBSTANCES Since GPC is a relative method, calibration must be undertaken. Narrowly distributed, linearly constructed polystyrene standards with known average molecular weights Mn and Mw and a known molecular weight distribution are normally used for this. The calibration curve can only be used in the determination of the molecular weight of the unknown sample if the conditions for the separation of the sample and the standards have been selected in an identical manner. A determined relationship between the molecular weight and elution volume is only valid under the specific conditions of the particular experiment. The conditions include, above all, the temperature, the solvent (or solvent mixture), the chromatography conditions and the separation column or system of columns. The molecular weights of the sample determined in this way are relative values and are described as polystyrene equivalent molecular weights. This means that dependent on the structural and chemical differences between the sample and the standards, the molecular weights can deviate from the absolute values to a greater or a lesser degree. If other standards are used, e.g. polyethylene glycol, polyethylene oxide, polymethyl methacrylate, polyacrylic acid, the reason should be stated.

1.4. PRINCIPLE OF THE TEST METHOD Both the molecular weight distribution of the sample and the average molecular weights (Mn, Mw) can be determined using GPC. GPC is a special type of liquid chromatography in which the sample is separated according to the hydrodynamic volumes of the individual constituents (2). Separation is effected as the sample passes through a column which is filled with a porous material, typically an organic gel. Small molecules can penetrate the pores whereas large molecules are excluded. The path of the large molecules is thereby shorter and these are eluted first. The medium-sized molecules penetrate some of the pores and are eluted later. The smallest molecules, with a mean hydrodynamic radius smaller than the pores of the gel, can penetrate all of the pores. These are eluted last. In an ideal situation, the separation is governed entirely by the size of the molecular species, but in practice it is difficult to avoid at least some absorption effects interfering. Uneven column packing and dead volumes can worsen the situation (2). Detection is effected by, e.g. refractive index or UV-absorption, and yields a simple distribution curve. However, to attribute actual molecular weight values to the curve, it is necessary to calibrate the column by passing down polymers of known molecular weight and, ideally, of broadly similar structure e.g. various polystyrene standards. Typically a Gaussian curve results, sometimes distorted by a small tail to the low molecular weight side, the vertical axis indicating the quantity, by weight, of the various molecular weight species eluted, and the horizontal axis the log molecular weight.

1.5. QUALITY CRITERIA The repeatability (Relative Standard Deviation: RSD) of the elution volume should be better than 0,3 %. The required repeatability of the analysis has to be ensured by correction via an internal standard if a chromatogram is evaluated time-dependently and does not correspond to the above mentioned criterion (1). The polydispersities are dependent on the molecular weights of the standards. In the case of polystyrene standards typical values are: Mp < 2000 Mw/Mn < 1,20 2000 ≤ Mp ≤ 106 Mw/Mn < 1,05 Mp > 106 Mw/Mn < 1,20 (Mp is the molecular weight of the standard at the peak maximum)

1.6. DESCRIPTION OF THE TEST METHOD

1.6.

1.6.1. Preparation of the standard polystyrene solutions The polystyrene standards are dissolved by careful mixing in the chosen eluent. The recommendations of the manufacturer must be taken into account in the preparation of the solutions. The concentrations of the standards chosen are dependent on various factors, e.g. injection volume, viscosity of the solution and sensitivity of the analytical detector. The maximum injection volume must be adapted to the length of the column, in order to avoid overloading. Typical injection volumes for analytical separations using GPC with a column of 30 cm × 7,8 mm are normally between 40 and 100 μl. Higher volumes are possible, but they should not exceed 250 μl. The optimal ratio between the injection volume and the concentration must be determined prior to the actual calibration of the column.

1.6.2. Preparation of the sample solution In principle, the same requirements apply to the preparation of the sample solutions. The sample is dissolved in a suitable solvent, e.g. tetrahydrofuran (THF), by shaking carefully. Under no circumstances should it be dissolved using an ultrasonic bath. When necessary, the sample solution is purified via a membrane filter with a pore size of between 0,2 and 2 μm. The presence of undissolved particles must be recorded in the final report as these may be due to high molecular weight species. An appropriate method should be used to determine the percentage by weight of the undissolved particles. The solutions should be used within 24 hours.

1.6.3. Apparatus It must be ensured that the GPC system is inert with regard to the utilised solvents (e.g. by the use of steel capillaries for THF solvent).

solvent reservoir,

degasser (where appropriate),

pump,

pulse dampener (where appropriate),

injection system,

chromatography columns,

detector,

flowmeter (where appropriate),

data recorder-processor,

waste vessel.

1.6.4. Injection and solvent delivery system A defined volume of the sample solution is loaded onto the column either using an auto-sampler or manually in a sharply defined zone. Withdrawing or depressing the plunger of the syringe too quickly, if done manually, can cause changes in the observed molecular weight distribution. The solvent-delivery system should, as far as possible, be pulsation-free ideally incorporating a pulse dampener. The flow rate is of the order of 1 ml/min.

1.6.5. Column Depending on the sample, the polymer is characterised using either a simple column or several columns connected in sequence. A number of porous column materials with defined properties (e.g. pore size, exclusion limits) are commercially available. Selection of the separation gel or the length of the column is dependent on both the properties of the sample (hydrodynamic volumes, molecular weight distribution) and the specific conditions for separation such as solvent, temperature and flow rate (1)(2)(3).

1.6.6. Theoretical plates The column or the combination of columns used for separation must be characterised by the number of theoretical plates. This involves, in the case of THF as elution solvent, loading a solution of ethyl benzene or other suitable non-polar solute onto a column of known length. The number of theoretical plates is given by the following equation: N=5,54VeW1/22 or N=16VeW2 where, N the number of theoretical plates Ve the elution volume at the peak maximum W the baseline peak width W1/2 the peak width at half height

1.6.7. Separation efficiency In addition to the number of theoretical plates, which is a quantity determining the bandwidth, a part is also played by the separation efficiency, this being determined by the steepness of the calibration curve. The separation efficiency of a column is obtained from the following relationship: Ve,Mx-Ve,(10Mx)cross sectional area of the column≥6,0 cm3cm2 where, Ve, Mx the elution volume for polystyrene with the molecular weight Mx Ve,(10.Mx) the elution volume for polystyrene with a ten times greater molecular weight The resolution of the system is commonly defined as follows: R1,2=2×Ve1-Ve2W1+W2×1log10M2/M1 where, Ve1, Ve2 the elution volumes of the two polystyrene standards at the peak maximum W1, W2 the peak widths at the base-line M1, M2 the molecular weights at the peak maximum (should differ by a factor of 10) The R-value for the column system should be greater than 1.7 (4).

1.6.8. Solvents All solvents must be of high purity (for THF purity of 99,5 % is used). The solvent reservoir (if necessary in an inert gas atmosphere) must be sufficiently large for the calibration of the column and several sample analyses. The solvent must be degassed before it is transported to the column via the pump.

1.6.9. Temperature control The temperature of the critical internal components (injection loop, columns, detector and tubing) should be constant and consistent with the choice of solvent.

1.6.10. Detector The purpose of the detector is to record quantitatively the concentration of sample eluted from the column. In order to avoid unnecessary broadening of peaks the cuvette volume of the detector cell must be kept as small as possible. It should not be larger than 10 μl except for light scattering and viscosity detectors. Differential refractometry is usually used for detection. However, if required by the specific properties of the sample or the elution solvent, other types of detectors can be used, e.g. UV/VIS, IR, viscosity detectors, etc.

2. DATA AND REPORTING

2.

2.1. DATA The DIN Standard (1) should be referred to for the detailed evaluation criteria as well as for the requirements relating to the collecting and processing of data. For each sample, two independent experiments must be carried out. They have to be analysed individually. Mn, Mw, Mw/Mn and Mp must be provided for every measurement. It is necessary to indicate explicitly that the measured values are relative values equivalent to the molecular weights of the standard used. After determination of the retention volumes or the retention times (possibly corrected using an internal standard), log Mp values (Mp being the peak maxima of the calibration standard) are plotted against one of those quantities. At least two calibration points are necessary per molecular weight decade, and at least five measurement points are required for the total curve, which should cover the estimated molecular weight of the sample. The low molecular weight end-point of the calibration curve is defined by n-hexyl benzene or another suitable non-polar solute. The number average and the weight-average molecular weights are generally determined by means of electronic data processing, based on the formulas of section 1.2. In case manual digitisation is used, ASTM D 3536-91 can be consulted (3). The distribution curve must be provided in the form of a table or as figure (differential frequency or sum percentages against log M). In the graphic representation, one molecular weight decade should be normally about 4 cm in width and the peak maximum should be about 8 cm in height. In the case of integral distribution curves the difference in the ordinate between 0 and 100 % should be about 10 cm.

2.2. TEST REPORT The test report must include the following information:

2.2.

2.2.1. Test substance:

available information about test substance (identity, additives, impurities),

description of the treatment of the sample, observations, problems.

2.2.2. Instrumentation:

reservoir of eluent, inert gas, degassing of the eluent, composition of the eluent, impurities,

pump, pulse dampener, injection system,

separation columns (manufacturer, all information about the characteristics of the columns, such as pore size, kind of separation material, etc., number, length and order of the columns used),

number of the theoretical plates of the column (or combination), separation efficiency (resolution of the system),

information on symmetry of the peaks,

column temperature, kind of temperature control,

detector (measurement principle, type, cuvette volume),

flowmeter if used (manufacturer, measurement principle),

system to record and process data (hardware and software).

2.2.3. Calibration of the system:

detailed description of the method used to construct the calibration curve,

information about quality criteria for this method (e.g. correlation coefficient, error sum of squares, etc.),

information about all extrapolations, assumptions and approximations made during the experimental procedure and the evaluation and processing of data,

all measurements used for constructing the calibration curve have to be documented in a table which includes the following information for each calibration point:

name of the sample,

manufacturer of the sample,

characteristic values of the standards Mp, Mn, Mw, Mw/Mn, as provided by the manufacturer or derived by subsequent measurements, together with details about the method of determination,

injection volume and injection concentration,

Mp value used for calibration,

elution volume or corrected retention time measured at the peak maxima,

Mp calculated at the peak maximum,

percentage error of the calculated Mp and the calibration value.

2.2.4. Evaluation:

evaluation on a time basis: methods used to ensure the required reproducibility (method of correction, internal standard, etc.),

information about whether the evaluation was effected on the basis of the elution volume or the retention time,

information about the limits of the evaluation if a peak is not completely analysed,

description of smoothing methods, if used,

preparation and pre-treatment procedures of the sample,

the presence of undissolved particles, if any,

injection volume (μl) and injection concentration (mg/ml),

observations indicating effects which lead to deviations from the ideal GPC profile,

detailed description of all modifications in the testing procedures,

details of the error ranges,

any other information and observations relevant for the interpretation of the results.

3.(1)(2)(3)(4)

REFERENCES DIN 55672(1995) Gelpermeationschromatographie (GPC) mit Tetrahydrofuran (THF) als Elutionsmittel, Teil 1. Yau, W.W., Kirkland, J.J., and Bly, D.D. eds., (1979) Modern Size Exclusion Liquid Chromatography, J. Wiley and Sons. ASTM D 3536-91, (1991). Standard Test Method for Molecular Weight Averages and Molecular Weight Distribution by Liquid Exclusion Chromatography (Gel Permeation Chromatography-GPC) American Society for Testing and Materials, Philadelphia, Pennsylvania. ASTM D 5296-92, (1992) Standard Test Method for Molecular Weight Averages and Molecular Weight Distribution of Polystyrene by High Performance Size-Exclusion Chromatography. American Society for Testing and Materials, Philadelphia, Pennsylvania.

A.19. LOW MOLECULAR WEIGHT CONTENT OF POLYMERS

A.19.

1. METHOD This Gel Permeation Chromatographic method is a replicate of the OECD TG 119 (1996). The fundamental principles and further technical information are given in the references.

1.

1.2. DEFINITIONS AND UNITS Low molecular weight is arbitrarily defined as a molecular weight below 1000 dalton. The number-average molecular weight Mn and the weight average molecular weight Mw are determined using the following equations: Mn=Σni=1HiΣni=1Hi/Mi Mw=Σni=1Hi × MiΣni=1Hi where, Hi the level of the detector signal from the baseline for the retention volume Vi, Mi the molecular weight of the polymer fraction at the retention volume Vi, and n is the number of data points The breadth of the molecular weight distribution, which is a measure of the dispersity of the system, is given by the ratio Mw/Mn.

1.4. PRINCIPLE OF THE TEST METHOD Both the molecular weight distribution of the sample and the average molecular weights (Mn, Mw) can be determined using GPC. GPC is a special type of liquid chromatography in which the sample is separated according to the hydrodynamic volumes of the individual constituents (2). Separation is effected as the sample passes through a column which is filled with a porous material, typically an organic gel. Small molecules can penetrate the pores whereas large molecules are excluded. The path of the large molecules is thereby shorter and these are eluted first. The medium-sized molecules penetrate some of the pores and are eluted later. The smallest molecules, with a mean hydrodynamic radius smaller than the pores of the gel, can penetrate all of the pores. These are eluted last. In an ideal situation, the separation is governed entirely by the size of the molecular species, but in practice it is difficult to avoid at least some absorption effects interfering. Uneven column packing and dead volumes can worsen the situation (2). Detection is effected by e.g. refractive index or UV-absorption and yields a simple distribution curve. However, to attribute actual molecular weight values to the curve, it is necessary to calibrate the column by passing down polymers of known molecular weight and, ideally, of broadly similar structure, e.g. various polystyrene standards. Typically a Gaussian curve results, sometimes distorted by a small tail to the low molecular weight side, the vertical axis indicating the quantity, by weight, of the various molecular weight species eluted, and the horizontal axis the log molecular weight. The low molecular weight content is derived from this curve. The calculation can only be accurate if the low molecular weight species respond equivalently on a per mass basis to the polymer as a whole.

1.5. QUALITY CRITERIA The repeatability (Relative Standard Deviation: RSD) of the elution volume should be better than 0,3 %. The required repeatability of the analysis has to be ensured by correction via an internal standard if a chromatogram is evaluated time-dependently and does not correspond to the above mentioned criterion (1). The polydispersities are dependent on the molecular weights of the standards. In the case of polystyrene standards typical values are: Mp < 2000 Mw/Mn < 1,20 2000< Mp< 106 Mw/Mn < 1,05 Mp > 106 Mw/Mn < 1,20 (Mp is the molecular weight of the standard at the peak maximum)

1.6. DESCRIPTION OF THE TEST METHOD

1.6.

1.6.3. Correction for content of impurities and additives Correction of the content of species of M < 1000 for the contribution from non-polymer specific components present (e.g. impurities and/or additives) is usually necessary, unless the measured content is already < 1 %. This is achieved by direct analysis of the polymer solution or the GPC eluate. In cases where the eluate, after passage through the column, is too dilute for a further analysis it must be concentrated. It may be necessary to evaporate the eluate to dryness and dissolve it again. Concentration of the eluate must be effected under conditions which ensure that no changes occur in the eluate. The treatment of the eluate after the GPC step is dependent on the analytical method used for the quantitative determination.

1.6.4. Apparatus GPC apparatus comprises the following components: It must be ensured that the GPC system is inert with regard to the utilised solvents (e.g. by the use of steel capillaries for THF solvent).

solvent reservoir,

degasser (where appropriate),

pump,

pulse dampener (where appropriate),

injection system,

chromatography columns,

detector,

flowmeter (where appropriate),

data recorder-processor,

waste vessel.

1.6.5. Injection and solvent delivery system A defined volume of the sample solution is loaded onto the column either using an auto-sampler or manually in a sharply defined zone. Withdrawing or depressing the plunger of the syringe too quickly, if done manually, can cause changes in the observed molecular weight distribution. The solvent-delivery system should, as far as possible, be pulsation-free ideally incorporating a pulse dampener. The flow rate is of the order of 1 ml/min.

1.6.6. Column Depending on the sample, the polymer is characterised using either a simple column or several columns connected in sequence. A number of porous column materials with defined properties (e.g. pore size, exclusion limits) are commercially available. Selection of the separation gel or the length of the column is dependent on both the properties of the sample (hydrodynamic volumes, molecular weight distribution) and the specific conditions for separation such as solvent, temperature and flow rate (1) (2) (3).

1.6.7. Theoretical plates The column or the combination of columns used for separation must be characterised by the number of theoretical plates. This involves, in the case of THF as elution solvent, loading a solution of ethyl benzene or other suitable non-polar solute onto a column of known length. The number of theoretical plates is given by the following equation: N=5,54VeW1/22 or N=16VeW2 where, N the number of theoretical plates Ve the elution volume at the peak maximum W the baseline peak width W1/2 the peak width at half height

1.6.8. Separation efficiency In addition to the number of theoretical plates, which is a quantity determining the bandwidth, a part is also played by the separation efficiency, this being determined by the steepness of the calibration curve. The separation efficiency of a column is obtained from the following relationship: Ve,Mx-Ve,(10Mx)cross sectional area of the column≥6,0cm3cm2 where, Ve, Mx the elution volume for polystyrene with the molecular weight Mx Ve,(10.Mx) the elution volume for polystyrene with a ten times greater molecular weight The resolution of the system is commonly defined as follows: R1,2=2×Ve1-Ve2W1+W2×1log10M2/M1 where, Ve1, Ve2 the elution volumes of the two polystyrene standards at the peak maximum W1, W2 the peak widths at the base-1ine M1, M2 the molecular weights at the peak maximum (should differ by a factor of 10). The R-value for the column system should be greater than 1,7 (4).

1.6.9. Solvents All solvents must be of high purity (for THF purity of 99,5 % is used). The solvent reservoir (if necessary in an inert gas atmosphere) must be sufficiently large for the calibration of the column and several sample analyses. The solvent must be degassed before it is transported to the column via the pump.

1.6.10. Temperature control The temperature of the critical internal components (injection loop, columns, detector and tubing) should be constant and consistent with the choice of solvent.

1.6.11. Detector The purpose of the detector is to record quantitatively the concentration of sample eluted from the column. In order to avoid unnecessary broadening of peaks the cuvette volume of the detector cell must be kept as small as possible. It should not be larger than 10 μl except for light scattering and viscosity detectors. Differential refractometry is usually used for detection. However, if required by the specific properties of the sample or the elution solvent, other types of detectors can be used, e.g. UV/VIS, IR, viscosity detectors, etc.

2. DATA AND REPORTING

2.

2.1. DATA The DIN Standard (1) should be referred to for the detailed evaluation criteria as well as for the requirements relating to the collecting and processing of data. For each sample, two independent experiments must be carried out. They have to be analysed individually. In all cases it is essential to determine also data from blanks, treated under the same conditions as the sample. It is necessary to indicate explicitly that the measured values are relative values equivalent to the molecular weights of the standard used. After determination of the retention volumes or the retention times (possibly corrected using an internal standard), log Mp values (Mp being the peak maxima of the calibration standard) are plotted against one of those quantities. At least two calibration points are necessary per molecular weight decade, and at least five measurement points are required for the total curve, which should cover the estimated molecular weight of the sample. The low molecular weight end-point of the calibration curve is defined by n-hexyl benzene or another suitable non-polar solute. The portion of the curve corresponding to molecular weights below 1000 is determined and corrected as necessary for impurities and additives. The elution curves are generally evaluated by means of electronic data processing. In case manual digitisation is used, ASTM D 3536-91 can be consulted (3). If any insoluble polymer is retained on the column, its molecular weight is likely to be higher than that of the soluble fraction, and if not considered would result in an overestimation of the low molecular weight content. Guidance for correcting the low molecular weight content for insoluble polymer is provided in the Appendix. The distribution curve must be provided in the form of a table or as figure (differential frequency or sum percentages against log M). In the graphic representation, one molecular weight decade should be normally about 4 cm in width and the peak maximum should be about 8 cm in height. In the case of integral distribution curves the difference in the ordinate between 0 and 100 % should be about 10 cm.

2.2. TEST REPORT The test report must include the following information:

2.2.

2.2.1. Test substance:

available information about test substance (identity, additives, impurities),

description of the treatment of the sample, observations, problems.

2.2.2. Instrumentation:

reservoir of eluent, inert gas, degassing of the eluent, composition of the eluent, impurities,

pump, pulse dampener, injection system,

separation columns (manufacturer, all information about the characteristics of the columns, such as pore size, kind of separation material, etc., number, length and order of the columns used),

number of the theoretical plates of the column (or combination), separation efficiency (resolution of the system),

information on symmetry of the peaks,

column temperature, kind of temperature control,

detector (measurement principle, type, cuvette volume),

flowmeter if used (manufacturer, measurement principle),

system to record and process data (hardware and software).

2.2.3. Calibration of the system:

detailed description of the method used to construct the calibration curve,

information about quality criteria for this method (e.g. correlation coefficient, error sum of squares, etc.),

information about all extrapolations, assumptions and approximations made during the experimental procedure and the evaluation and processing of data,

all measurements used for constructing the calibration curve have to be documented in a table which includes the following information for each calibration point:

name of the sample,

manufacturer of the sample,

characteristic values of the standards Mp, Mn, Mw, Mw/Mn, as provided by the manufacturer or derived by subsequent measurements, together with details about the method of determination,

injection volume and injection concentration,

Mp value used for calibration,

elution volume or corrected retention time measured at the peak maxima,

Mp calculated at the peak maximum,

percentage error of the calculated Mp and the calibration value.

2.2.4. Information on the low molecular weight polymer content:

description of the methods used in the analysis and the way in which the experiments were conducted,

information about the percentage of the low molecular weight species content (w/w) related to the total sample,

information about impurities, additives and other non-polymer species in percentage by weight related to the total sample.

2.2.5. Evaluation:

evaluation on a time basis: all methods to ensure the required reproducibility (method of correction, internal standard etc.),

information about whether the evaluation was effected on the basis of the elution volume or the retention time,

information about the limits of the evaluation if a peak is not completely analysed,

description of smoothing methods, if used,

preparation and pre-treatment procedures of the sample,

the presence of undissolved particles, if any,

injection volume (μl) and injection concentration (mg/ml),

observations indicating effects which lead to deviations from the ideal GPC profile,

detailed description of all modifications in the testing procedures,

details of the error ranges,

any other information and observations relevant for the interpretation of the results.

3.(1)(2)(3)(4)

REFERENCES DIN 55672 (1995) Gelpermeationschromatographie (GPC) mit Tetrahydrofuran (THF) als Elutionsmittel, Teil 1. Yau, W.W., Kirkland, J.J., and Bly, D.D. eds. (1979) Modern Size Exclusion Liquid Chromatography, J. Wiley and Sons. ASTM D 3536-91, (1991) Standard Test method for Molecular Weight Averages and Molecular Weight Distribution by Liquid Exclusion Chromatography (Gel Permeation Chromatography-GPC). American Society for Testing and Materials, Philadelphia, Pennsylvania. ASTM D 5296-92, (1992) Standard Test method for Molecular Weight Averages and Molecular Weight Distribution of Polystyrene by High Performance Size-Exclusion Chromatography. American Society for Testing and Materials, Philadelphia, Pennsylvania.

A.20. SOLUTION/EXTRACTION BEHAVIOUR OF POLYMERS IN WATER

A.20.

1. METHOD The method described is a replicate of the revised version of OECD TG 120 (1997). Further technical information is given in reference (1).

1.

1.1. INTRODUCTION For certain polymers, such as emulsion polymers, initial preparatory work may be necessary before the method set out hereafter can be used. The method is not applicable to liquid polymers and to polymers that react with water under the test conditions. When the method is not practical or not possible, the solution/extraction behaviour may be investigated by means of other methods. In such cases, full details and justification should be given for the method used.

1.2. REFERENCE SUBSTANCES None.

1.3. PRINCIPLE OF THE TEST METHOD The solution/extraction behaviour of polymers in an aqueous medium is determined using the flask method (see A.6 Water Solubility, Flask method) with the modifications described below.

1.4. QUALITY CRITERIA None.

1.5. DESCRIPTION OF THE TEST METHOD

1.5.

1.5.1. Equipment The following equipment is required for the method:

crushing device, e.g. grinder for the production of particles of known size,

apparatus for shaking with possibility of temperature control,

membrane filter system,

appropriate analytical equipment,

standardised sieves.

1.5.2. Sample preparation A representative sample has first to be reduced to a particle size between 0,125 and 0,25 mm using appropriate sieves. Cooling may be required for the stability of the sample or for the grinding process. Materials of a rubbery nature can be crushed at liquid nitrogen temperature (1). If the required particle size fraction is not attainable, action should be taken to reduce the particle size as much as possible, and the result reported. In the report, it is necessary to indicate the way in which the crushed sample was stored prior to the test

1.5.3. Procedure Three samples of 10 g of the test substance are weighed into each of three vessels fitted with glass stoppers and 1000 ml of water is added to each vessel. If handling an amount of 10 g polymer proves impracticable, the next highest amount which can be handled should be used and the volume of water adjusted accordingly. The vessels are tightly stoppered and then agitated at 20 oC. A shaking or stirring device capable of operating at constant temperature should be used. After a period of 24 hours, the content of each vessel is centrifuged or filtered and the concentration of polymer in the clear aqueous phase is determined by a suitable analytical method. If suitable analytical methods for the aqueous phase are not available, the total solubility/extractivity can be estimated from the dry weight of the filter residue or centrifuged precipitate. It is usually necessary to differentiate quantitatively between the impurities and additives on the one hand and the low molecular weight species on the other hand. In the case of gravimetric determination, it is also important to perform a blank run using no test substance in order to account for residues arising from the experimental procedure. The solution/extraction behaviour of polymers in water at 37 oC at pH 2 and pH 9 may be determined in the same way as described for the conduct of the experiment at 20 oC. The pH values can be achieved by the addition of either suitable buffers or appropriate acids or bases such as hydrochloric acid, acetic acid, analytical grade sodium or potassium hydroxide or NH3. Depending on the method of analysis used, one or two tests should be performed. When sufficiently specific methods are available for direct analysis of the aqueous phase for the polymer component, one test as described above should suffice. However, when such methods are not available and determination of the solution/extraction behaviour of the polymer is limited to indirect analysis by determining only the total organic carbon content (TOC) of the aqueous extract, an additional test should be conducted. This additional test should also be done in triplicate, using ten times smaller polymer samples and the same amounts of water as those used in the first test.

1.5.4. Analysis

1.5.4.

1.5.4.1. Test conducted with one sample size Methods may be available for direct analysis of polymer components in the aqueous phase. Alternatively, indirect analysis of dissolved/extracted polymer components, by determining the total content of soluble parts and correcting for non polymer-specific components, could also be considered. Analysis of the aqueous phase for the total polymeric species is possible: either by a sufficiently sensitive method, e.g.: or by vacuum evaporation to dryness of the aqueous extract and spectroscopic (IR, UV, etc.) or AAS/ICP analysis of the residue. If analysis of the aqueous phase as such is not practicable, the aqueous extract should be extracted with a water-immiscible organic solvent e.g. a chlorinated hydrocarbon. The solvent is then evaporated and the residue analysed as above for the notified polymer content. Any components in this residue which are identified as being impurities or additives are to be subtracted for the purpose of determining the degree of solution/extraction of the polymer itself. When relatively large quantities of such materials are present, it may be necessary to subject the residue to e.g. HPLC or GC analysis to differentiate the impurities from the monomer and monomer-derived species present so that the true content of the latter can be determined. In some cases, simple evaporation of the organic solvent to dryness and weighing the dry residue may be sufficient.

TOC using persulphate or dichromate digestion to yield CO2 followed by estimation by IR or chemical analysis,

Atomic Absorption Spectrometry (AAS) or its Inductively Coupled Plasma (ICP) emission equivalent for silicon or metal containing polymers,

UV absorption or spectrofluorimetry for aryl polymers,

LC-MS for low molecular weight samples,

1.5.4.2. Test conducted with two different sample sizes All aqueous extracts are analysed for TOC. A gravimetric determination is performed on the undissolved/not extracted part of the sample. If, after centrifugation or filtering of the content of each vessel, polymer residues remain attached to the wall of the vessel, the vessel should be rinsed with the filtrate until the vessel is cleared from all visible residues. Following which, the filtrate is again centrifuged or filtered. The residues remaining on the filter or in the centrifuge tube are dried at 40 oC under vacuum and weighed. Drying is continued until a constant weight is reached.

2. DATA

2.

2.1. TEST CONDUCTED WITH ONE SAMPLE SIZE The individual results for each of the three flasks and the average values should be given and expressed in units of mass per volume of the solution (typically mg/l) or mass per mass of polymer sample (typically mg/g). Additionally, the weight loss of the sample (calculated as the weight of the solute divided by the weight of the initial sample) should also be given. The relative standard deviations (RSD) should be calculated. Individual figures should be given for the total substance (polymer + essential additives, etc.) and for the polymer only (i.e. after subtracting the contribution from such additives).

2.2. TEST CONDUCTED WITH TWO DIFFERENT SAMPLE SIZES The individual TOC values of the aqueous extracts of the two triplicate experiments and the average value for each experiment should be given expressed as units of mass per volume of solution (typically mgC/l), as well as in units of mass per weight of the initial sample (typically mgC/g). If there is no difference between the results at the high and the low sample/water ratios, this may indicate that all extractable components were indeed extracted. In such a case, direct analysis would normally not be necessary. The individual weights of the residues should be given and expressed in percentage of the initial weights of the samples. Averages should be calculated per experiment. The differences between 100 and the percentages found represent the percentages of soluble and extractable material in the original sample.

3. REPORTING

3.

3.1. TEST REPORT The test report must include the following information:

3.1.

3.1.1. Test substance:

available information about test substance (identity, additives, impurities, content of low molecular weight species).

3.1.2. Experimental conditions:

description of the procedures used and experimental conditions,

description of the analytical and detection methods.

3.1.3. Results:

results of solubility/extractivity in mg/l; individual and mean values for the extraction tests in the various solutions, broken down in polymer content and impurities, additives, etc.,

results of solubility/extractivity in mg/g of polymer,

TOC values of aqueous extracts, weight of the solute and calculated percentages, if measured,

the pH of each sample,

information about the blank values,

where necessary, references to the chemical instability of the test substance, during both the testing process and the analytical process,

all information which is important for the interpretation of the results.

4.(1) REFERENCES DIN 53733 (1976) Zerkleinerung von Kunststofferzeugnissen für Prüfzwecke.

A.21. OXIDISING PROPERTIES (LIQUIDS)

A.21.

1. METHOD

1.

1.1. INTRODUCTION This test method is designed to measure the potential for a liquid substance to increase the burning rate or burning intensity of a combustible substance, or to form a mixture with a combustible substance which spontaneously ignites, when the two are thoroughly mixed. It is based on the UN test for oxidising liquids (1) and is equivalent to it. However, as this method A.21 is primarily designed to satisfy the requirements of Regulation (EC) No 1907/2006, comparison with only one reference substance is required. Testing and comparison to additional reference substances may be necessary when the results of the test are expected to be used for other purposes. This test need not be performed when examination of the structural formula establishes beyond reasonable doubt that the substance is incapable of reacting exothermically with a combustible material. It is useful to have preliminary information on any potential explosive properties of the substance before performing this test. This test is not applicable to solids, gases, explosive or highly flammable substances, or organic peroxides. This test may not need to be performed when results for the test substance in the UN test for oxidising liquids (1) are already available.

1.2. DEFINITIONS AND UNITS Mean pressure rise time is the mean of the measured times for a mixture under test to produce a pressure rise from 690 kPa to 2070 kPa above atmospheric.

1.3. REFERENCE SUBSTANCE 65 % (w/w) aqueous nitric acid (analytical grade) is required as a reference substance. Optionally, if the experimenter foresees that the results of this test may eventually be used for other purposes, testing of additional reference substances may also be appropriate.

1.4. PRINCIPLE OF THE TEST METHOD The liquid to be tested is mixed in a 1 to 1 ratio, by mass, with fibrous cellulose and introduced into a pressure vessel. If during mixing or filling spontaneous ignition occurs, no further testing is necessary. If spontaneous ignition does not occur the full test is carried out. The mixture is heated in a pressure vessel and the mean time taken for the pressure to rise from 690 kPa to 2070 kPa above atmospheric is determined. This is compared with the mean pressure rise time for the 1:1 mixture of the reference substance(s) and cellulose.

1.5. QUALITY CRITERIA In a series of five trials on a single substance no results should differ by more than 30 % from the arithmetic mean. Results that differ by more than 30 % from the mean should be discarded, the mixing and filling procedure improved and the testing repeated.

1.6. DESCRIPTION OF THE METHOD

1.6.

1.6.1. Preparation

1.6.1.

1.6.1.1. Combustible substance Dried, fibrous cellulose with a fibre length between 50 and 250 μm and a mean diameter of 25 μm, is used as the combustible material. It is dried to constant weight in a layer not more than 25 mm thick at 105 oC for four hours and kept in a desiccator, with desiccant, until cool and required for use. The water content of the dried cellulose should be less than 0,5 % by dry mass. If necessary, the drying time should be prolonged to achieve this. The same batch of cellulose is to be used throughout the test.

1.6.1.2. Apparatus

1.6.1.2.

1.6.1.2.1. Pressure vessel A pressure vessel is required. The vessel consists of a cylindrical steel pressure vessel 89 mm in length and 60 mm in external diameter (see figure 1). Two flats are machined on opposite sides (reducing the cross-section of the vessel to 50 mm) to facilitate holding whilst fitting up the firing plug and vent plug. The vessel, which has a bore of 20 mm diameter is internally rebated at either end to a depth of 19 mm and threaded to accept 1'' British Standard Pipe (BSP) or metric equivalent. A pressure take-off, in the form of a side arm, is screwed into the curved face of the pressure vessel 35 mm from one end and at 90o to the machined flats. The socket for this is bored to a depth of 12 mm and threaded to accept the 1/2" BSP (or metric equivalent) thread on the end of the side-arm. If necessary, an inert seal is fitted to ensure a gas-tight seal. The side-arm extends 55 mm beyond the pressure vessel body and has a bore of 6 mm. The end of the side-arm is rebated and threaded to accept a diaphragm type pressure transducer. Any pressure-measuring device may be used provided that it is not affected by the hot gases or the decomposition products and is capable of responding to rates of pressure rise of 690-2070 kPa in not more than 5 ms. The end of the pressure vessel farthest from the side-arm is closed with a firing plug which is fitted with two electrodes, one insulated from, and the other earthed to, the plug body. The other end of the pressure vessel is closed by a bursting disk (bursting pressure approximately 2200 kPa) held in place with a retaining plug which has a 20 mm bore. If necessary, an inert seal is used with the firing plug to ensure a gas-tight fit. A support stand (figure 2) holds the assembly in the correct attitude during use. This usually comprises a mild steel base plate measuring 235 mm × 184 mm × 6 mm and a 185 mm length of square hollow section (S.H.S.) 70 mm × 70 mm × 4 mm. A section is cut from each of two opposite sides at one end of the length of S.H.S. so that a structure having two flat sided legs surmounted by 86 mm length of intact box section results. The ends of these flat sides are cut to an angle of 60o to the horizontal and welded to the base plate. A slot measuring 22 mm wide × 46 mm deep is machined in one side of the upper end of the base section such that when the pressure vessel assembly is lowered, firing plug end first, into the box section support, the side-arm is accommodated in the slot. A piece of steel 30 mm wide and 6 mm thick is welded to the lower internal face of the box section to act as a spacer. Two 7 mm thumb screws, tapped into the opposite face, serve to hold the pressure vessel firmly in place. Two 12 mm wide strips of 6 mm thick steel, welded to the side pieces abutting the base of the box section, support the pressure vessel from beneath.

1.6.1.2.2. Ignition system The ignition system consists of a 25 cm long Ni/Cr wire with a diameter 0,6 mm and a resistance of 3,85 ohm/m. The wire is wound, using a 5 mm diameter rod, in the shape of a coil and is attached to the firing plug electrodes. The coil should have one of the configurations shown in figure 3. The distance between the bottom of the vessel and the underside of the ignition coil should be 20 mm. If the electrodes are not adjustable, the ends of the ignition wire between the coil and the bottom of the vessel should be insulated by a ceramic sheath. The wire is heated by a constant current power supply able to deliver at least 10 A.

1.6.2. Performance of the test The apparatus, assembled complete with pressure transducer and heating system but without the bursting disk in position, is supported firing plug end down. 2,5 g of the liquid to be tested is mixed with 2,5 g of dried cellulose in a glass beaker using a glass stirring rod. For safety, the mixing should be performed with a safety shield between the operator and mixture. If the mixture ignites during mixing or filling, no further testing is necessary. The mixture is added, in small portions with tapping, to the pressure vessel making sure that the mixture is packed around the ignition coil and is in good contact with it. It is important that the coil is not distorted during the packing process as this may lead to erroneous results. The bursting disk is placed in position and the retaining plug is screwed in tightly. The charged vessel is transferred to the firing support stand, bursting disk uppermost, which should be located in a suitable, armoured fume cupboard or firing cell. The power supply is connected to the external terminals of the firing plug and 10 A applied. The time between the start of mixing and switching on the power should not exceed 10 minutes. The signal produced by the pressure transducer is recorded on a suitable system which allows both evaluation and the generation of a permanent record of the time pressure profile obtained (e.g. a transient recorder coupled to a chart recorder). The mixture is heated until the bursting disk ruptures or until at least 60 s have elapsed. If the bursting disk does not rupture, the mixture should be allowed to cool before carefully dismantling the apparatus, taking precautions to allow for any pressurisation which may occur. Five trials are performed with the test substance and the reference substance(s). The time taken for the pressure to rise from 690 kPa to 2070 kPa above atmospheric is noted. The mean pressure rise time is calculated. In some cases, substances may generate a pressure rise (too high or too low), caused by chemical reactions not characterising the oxidising properties of the substance. In these cases, it may be necessary to repeat the test with an inert substance, e.g. diatomite (kieselguhr), in place of the cellulose in order to clarify the nature of the reaction.

2. DATA Pressure rise times for both the test substance and the reference substance(s). Pressure rise times for the tests with an inert substance, if performed.

2. 2.1. Table 1 Examples of results

TREATMENT OF RESULTS The mean pressure rise times for both the test substance and the reference substances(s) are calculated. The mean pressure rise time for the tests with an inert substance (if performed) is calculated. Some examples of results are shown in Table 1. Substance Mean pressure rise time for a 1:1 mixture with celulose (ms) Ammonium dichromate, saturated aqueous solution 20800 Calcium nitrate, saturated aqueous solution 6700 Ferric nitrate, saturated aqueous solution 4133 Lithium perchlorate, saturated aqueous solution 1686 Magnesium perchlorate, saturated aqueous solution 777 Nickel nitrate, saturated aqueous solution 6250 Nitric acid, 65 % 4767 Perchloric acid, 50 % 121 Perchloric acid, 55 % 59 Potassium nitrate, 30 % aqueous solution 26690 Silver nitrate, saturated aqueous solution Sodium chlorate, 40 % aqueous solution 2555 Sodium nitrate, 45 % aqueous solution 4133 Inert substance Water: cellulose

3. REPORT

3.

3.1. TEST REPORT The test report should include the following information:

the identity, composition, purity, etc. of the substance tested,

the concentration of the test substance,

the drying procedure of the cellulose used,

the water content of the cellulose used,

the results of the measurements,

the results from tests with an inert substance, if any,

the calculated mean pressure rise times,

any deviations from this method and the reasons for them,

all additional information or remarks relevant to the interpretation of the results.

3.2. INTERPRETATION OF THE RESULTS The test results are assessed on the basis of: A liquid substance is to be considered as an oxidiser when: In order to avoid a false positive result, if necessary, the results obtained when testing the substance with an inert material should also be considered when interpreting the results.

3.2.

(a) whether the mixture of test substance and cellulose spontaneously ignites; and

(b) the comparison of the mean time taken for the pressure to rise from 690 kPa to 2070 kPa with that of the reference substance(s).

(a) a 1:1 mixture, by mass, of the substance and cellulose spontaneously ignites; or

(b) a 1:1 mixture, by mass, of the substance and cellulose exhibits a mean pressure rise time less than or equal to the mean pressure rise time of a 1:1 mixture, by mass, of 65 % (w/w) aqueous nitric acid and cellulose.

4.(1) REFERENCES Recommendations on the Transport of Dangerous Goods, Manual of Tests and Criteria. 3rd revised edition. UN Publication No: ST/SG/AC.10/11/Rev. 3, 1999, page 342. Test O.2: Test for oxidising liquids. Note: either of these configurations may be used.

4.(1)

Figure 1 Pressure vessel

(A) Pressure vessel body (B) Bursting disk retaining plug (C) Firing plug (D) Soft lead washer (E) Bursting disc (F) Side arm (G) Pressure transducer head (H) Washer (J) Insulated electrode (K) Earthed electrode (L) Insulation (M) Steel cone (N) Washer distorting groove

Figure 2 Support stand

Figure 3 Ignition system

(A) Ignition coil (B) Insulation (C) Electrodes (D) Firing plug

Appendix For additional technical details, the following standards may be consulted for example.

1. Capillary methods

1.

1.1. Melting temperature devices with liquid bath ASTM E 324-69 Standard test method for relative initial and final melting points and the melting range of organic chemicals BS 4634 Method for the determination of melting point and/or melting range DIN 53181 Bestimmung des Schmelzintervalles von Harzen nach Kapillarverfarehn JIS K 00-64 Testing methods for melting point of chemical products

1.2. Melting temperature devices with metal block DIN 53736 Visuelle Bestimmung der Schmelztemperatur von teilkristallinen Kunststoffen ISO 1218 (E) Plastics — polyamides — determination of melting point

2. Hot stages

2.

2.1. Kofler hot bar ANSI/ASTM D 3451-76 Standard recommended practices for testing polymeric powder coatings

2.2. Melt microscope DIN 53736 Visuelle Bestimmung der Schmelztemperatur von teilkristallinen Kunststoffen

2.3. Meniscus method (polyamides) ISO 1218 (E) Plastics — polyamides — determination of melting point ANSI/ASTM D 2133-66 Standard specification for acetal resin injection moulding and extrusion materials NF T 51-050 Résines de polyamides. Détermination du point de fusion méthode du ménisque

3. Methods for the determination of the freezing temperature BS 4633 Method for the determination of crystallising point BS 4695 Method for Determination of Melting Point of petroleum wax (Cooling Curve) DIN 51421 Bestimmung des Gefrierpunktes von Flugkraftstoffen, Ottokraftstoffen und Motorenbenzolen ISO 2207 Cires de pétrole: détermination de la température de figeage DIN 53175 Bestimmung des Erstarrungspunktes von Fettsäuren NF T 60-114 Point de fusion des paraffines NF T 20-051 Méthode de détermination du point de cristallisation (point de congélation) ISO 1392 Method for the determination of the freezing point

4. Thermal analysis

4.

4.1. Differential thermal analysis ASTM E 537-76 Standard method for assessing the thermal stability of chemicals by methods of differential thermal analysis ASTM E 473-85 Standard definitions of terms relating to thermal analysis ASTM E 472-86 Standard practice for reporting thermoanalytical data DIN 51005 Thermische Analyse, Begriffe

4.2. Differential scanning calorimetry ASTM E 537-76 Standard method for assessing the thermal stability of chemicals by methods of differential thermal analysis ASTM E 473-85 Standard definitions of terms relating to thermal analysis ASTM E 472-86 Standard practice for reporting thermoanalytical data DIN 51005 Thermische Analyse, Begriffe

5. Determination of the pour point NBN 52014 Echantillonnage et analyse des produits du pétrole: Point de trouble et point d'écoulement limite — Monsterneming en ontleding van aardolieproducten: Troebelingspunt en vloeipunt ASTM D 97-66 Standard test method for pour point of petroleum oils ISO 3016 Petroleum oils — Determination of pour point

Appendix For additional technical details, the following standards may be consulted for example.

1.1.1. Ebulliometer Melting temperature devices with liquid bath ASTM D 1120-72 Standard test method for boiling point of engine anti-freezes

2. Distillation process (boiling range) ISO/R 918 Test Method for Distillation (Distillation Yield and Distillation Range) BS 4349/68 Method for determination of distillation of petroleum products BS 4591/71 Method for the determination of distillation characteristics DIN 53171 Losungsmittel für Anstrichstoffe, Bestimmung des Siedeverlaufes NF T 20-608 Distillation: détermination du rendement et de l'intervalle de distillation

3. Differential thermal analysis and differential scanning calorimetry ASTM E 537-76 Standard method for assessing the thermal stability of chemicals by methods of differential thermal analysis ASTM E 473-85 Standard definitions of terms relating to thermal analysis ASTM E 472-86 Standard practice for reporting thermoanalytical data DIN 51005 Thermische Analyse, Begriffe

Appendix For additional technical details, the following standards may be consulted for example.

1. Buoyancy methods

1.

1.1. Hydrometer DIN 12790, ISO 387 Hydrometer; general instructions DIN 12791 Part I: Density hydrometers; construction, adjustment and use Part II: Density hydrometers; standardised sizes, designation Part III: Use and test ISO 649-2 Laboratory glassware: Density hydrometers for general purpose NF T 20-050 Chemical products for industrial use — Determination of density of liquids — Areometric method DIN 12793 Laboratory glassware: range find hydrometers

1.2. Hydrostatic balance

1.2. For solid substances

ISO 1183 Method A: Methods for determining the density and relative density of plastics excluding cellular plastics NF T 20-049 Chemical products for industrial use — Determination of the density of solids other than powders and cellular products — Hydrostatic balance method ASTM-D-792 Specific gravity and density of plastics by displacement DIN 53479 Testing of plastics and elastomers; determination of density

For liquid substances

ISO 901 ISO 758 DIN 51757 Testing of mineral oils and related materials; determination of density ASTM D 941-55, ASTM D 1296-67 and ASTM D 1481-62 ASTM D 1298 Density, specific gravity or API gravity of crude petroleum and liquid petroleum products by hydrometer method BS 4714 Density, specific gravity or API gravity of crude petroleum and liquid petroleum products by hydrometer method

1.3. Immersed body method DIN 53217 Testing of paints, varnishes and similar coating materials; determination of density; immersed body method

2. Pycnometer methods

2.

2.1. For liquid substances ISO 3507 Pycnometers ISO 758 Liquid chemical products; determination of density at 20 oC DIN 12797 Gay-Lussac pycnometer (for non-volatile liquids which are not too viscous) DIN 12798 Lipkin pycnometer (for liquids with a kinematic viscosity of less than 100 10-6 m2 s-1 at 15 oC) DIN 12800 Sprengel pycnometer (for liquids as DIN 12798) DIN 12801 Reischauer pycnometer (for liquids with a kinematic viscosity of less than 100. 10-6 m2 s-1 at 20 oC, applicable in particular also to hydrocarbons and aqueous solutions as well as to liquids with higher vapour pressure, approximately 1 bar at 90 oC) DIN 12806 Hubbard pycnometer (for viscous liquids of all types which do not have too high a vapour pressure, in particular also for paints, varnishes and bitumen) DIN 12807 Bingham pycnometer (for liquids, as in DIN 12801) DIN 12808 Jaulmes pycnometer (in particular for ethanol — water mixture) DIN 12809 Pycnometer with ground-in thermometer and capillary side tube (for liquids which are not too viscous) DIN 53217 Testing of paints, varnishes and similar products; determination of density by pycnometer DIN 51757 Point 7: Testing of mineral oils and related materials; determination of density ASTM D 297 Section 15: Rubber products — chemical analysis ASTM D 2111 Method C: Halogenated organic compounds BS 4699 Method for determination of specific gravity and density of petroleum products (graduated bicapillary pycnometer method) BS 5903 Method for determination of relative density and density of petroleum products by the capillary — stoppered pycnometer method NF T 20-053 Chemical products for industrial use — Determination of density of solids in powder and liquids — Pyknometric method

2.2. For solid substances ISO 1183 Method B: Methods for determining the density and relative density of plastics excluding cellular plastics NF T 20-053 Chemical products for industrial use — Determination of density of solids in powder and liquids — Pyknometric method DIN 19683 Determination of the density of soils

3. Air comparison pycnometer DIN 55990 Part 3: Prüfung von Anstrichstoffen und ähnlichen Beschichtungsstoffen; Pulverlack; Bestimmung der Dichte DIN 53243 Anstrichstoffe; chlorhaltige Polymere; Prüfung

Appendix 1 Estimation method

INTRODUCTION

Calculated values of the vapour pressure can be used:

for deciding which of the experimental methods is appropriate,

for providing an estimate or limit value in cases where the experimental method cannot be applied due to technical reasons (including where the vapour pressure is very low),

to help identify those cases where omitting experimental measurement is justified because the vapour pressure is likely to be < 10-5 Pa at ambient temperature.

ESTIMATION METHOD

The vapour pressure of liquids and solids can be estimated by use of the modified Watson Correlation (a). The only experimental data required is the normal boiling point. The method is applicable over the pressure range from 105 Pa to 10-5 Pa. Detailed information on the method is given in Handbook of Chemical Property Estimation Methods (b).

CALCULATION PROCEDURE

According to (b) the vapour pressure is calculated as follows: ln Pvp ≈ ΔHvbΔZbRTb1-3-2 TTbmTTb-2m3-2 TTbm-1ln TTb where: T = temperature of interest Tb = normal boiling point Pvp = vapour pressure at temperature T ΔHvb = heat of vaporisation ΔZb = compressibility factor (estimated at 0,97) m = empirical factor depending on the physical state at the temperature of interest further: ΔHvbTb=KF8,75+R ln Tb where KF is an empirical factor considering the polarity of the substance. For several compound types, KF factors are listed in reference (b). Quite often, data are available in which a boiling point at reduced pressure is given. In such a case, according to (b), the vapour pressure is calculated as follows: ln Pvp ≈ ln P1+Δ Hv1Δ ZbRT11-3-2 TT1m T1T-2m3-2 TT1m-1 ln TT1 where T1 is the boiling point at the reduced pressure P1.

REPORT

When using the estimation method, the report shall include a comprehensive documentation of the calculation.

LITERATURE

(a) K.M. Watson, Ind. Eng. Chem., 1943, vol. 35, p. 398.

(b) W.J. Lyman, W.F. Reehl, D.H. Rosenblatt. Handbook of Chemical Property Estimation Methods, Mc Graw-Hill, 1982.

Appendix 2

Figure 1 Apparatus for determining the vapour pressure curve according to the dynamic method

1 = Thermocouple 2 = Vacuum buffer volume 3 = Pressure gauge 4 = Vacuum 5 = Measuring point 6 = Heating element circa 150 W

Figure 2a Apparatus for determining the vapour pressure curve according to the static method (using a U-tube manometer)

1. Test substance 2. Vapour phase 3. High vacuum valve 4. U-tube (auxiliary manometer) 5. Manometer 6. Temperature bath 7. Temperature measuring device 8. To vacuum pump 9. Ventilation

Figure 2b Apparatus for determining the vapour pressure curve according to the static method (using a pressure indicator)

1. Test substance 2. Vapour phase 3. High vacuum valve 4. Pressure gauge 5. Pressure indicator 6. Temperature bath 7. Temperature measuring device

Figure 3 Isoteniscope (see reference 7)

1. To pressure control and measurement system 2. 8 mm OD tube 3. Dry nitrogen in pressure system 4. Sample vapour 5. Small tip 6. Liquid sample

Figure 4 Apparatus for determining the vapour pressure curve according to the vapour pressure balance method

1. Test substance 2. Vapour phase with vapour stream 3. Evaporation furnace with rotary inlet 3a. Furnace lid with opening 4. Furnace heating (refrigeration) 5. Measurement of temperature of sample 6. Refrigeration box 7. Shield 8. Refrigeration bar for refrigeration box 9. Balance pan 10. Microbalance 11. To recorder 12. To high-vacuum pump

Figure 5 Example of apparatus for evaporation at low pressure by effusion methode, with an effusion cell volume of 8 cm3

1 Connection to vacuum 2 Wells for platinum resistance thermometer or temperature measurement and control (2) 3 Lid for vacuum tank 4 O-ring 5 Aluminium vacuum tank 6 Device for installing and removing the effusion cells 7 Threaded lid 8 Butterfly nuts (6) 9 Bolts (6) 10 Stainless steel effusion cells 11 Heater cartridges (6)

Figure 6a An example of a flow system for the determination of vapour pressure by the gas saturation method

1 = Flow regulator 2 = Heat exchanger 3 = Needle valves 4 = Relative humidity sensor 5 = Saturation columns 6 = PTFE joints 7 = Flow meter 8 = Trap (absorber) 9 = Oil trap 10 = Fritted bubbler Vent Manifold

Figure 6b An example of system for the determination of vapour pressure by the gas saturation method, with a capillary placed after the saturation chamber

1. Thermal mass flowmeter 2. Manometer 3. Temperature-controlled chamber 4. Thermostating coil for carrier gas 5. Thermometer (Pt 100) 6. Gas saturation chamber 7. Capillary 8. Absorption vessels 9. Gas meter 10. Cold trap

Figure 7 Example of the experimental set-up for spinning rotor method

Vapour pressure apparatus A. spinning rotor sensor head; B. sample cell; C. thermostat; D. vacuum line (turbo pump); E. air thermostat.

Figure 8 Example of spinning rotor measuring head

1. Ball; 2. Evacuated tubular extension of 6; 3. Permanent magnets (2); 4. Coils (2) for vertical stabilization; 5. Driving coils (4); 6. Connection flange.

Appendix

Figure 1 Column elution method with recirculating pump

Atmospheric equilibration Flow meter Microcolumn Thermostatically controlled circulating pump Recirculating pump Two-way valve for sampling

Figure 2 A typical microcolumn

(All dimensions in millimetres) (Connection for ground glass joint) Headspace Interior 5 Exterior 19 Plug of glass wool Stopcock with two-way action

Figure 3 A typical microcolumn

(All dimensions in millimetres) Connection for ground glass joint Headspace Interior 5 Exterior 19 Stopcock

Figure 4 Column elution method with levelling vessel

1 = Levelling vessel (e.g. 2,5 litre flask) 2 = Column (see figure 3) 3 = Fraction collector 4 = Thermostat 5 = Teflon tubing 6 = Ground glass joint 7 = Water line (between thermostat and column, inner diameter: approximately 8 mm)

Appendix 1 Calculation/estimation methods

INTRODUCTION

A general introduction to calculation methods, data and examples are provided in the Handbook of Chemical Property Estimation Methods (a). Calculated values of Pow can be used:

for deciding which of the experimental methods is appropriate (shake-flask range: log Pow: - 2 to 4, HPLC range: log Pow: 0 to 6),

for selecting the appropriate test conditions (e.g. reference substances for HPLC procedures, volume ratio n-octanol/water for shake flask method),

as a laboratory internal check on possible experimental errors,

for providing a Pow-estimate in cases where the experimental methods cannot be applied for technical reasons.

ESTIMATION METHOD Preliminary estimate of the partition coefficient

The value of the partition coefficient can be estimated by the use of the solubilities of the test substance in the pure solvents: For this: Pestimate = saturation cn-octanolsaturation cwater

CALCULATION METHODS Principle of the calculation methods

All calculation methods are based on the formal fragmentation of the molecule into suitable substructures for which reliable log Pow-increments are known. The log Pow of the whole molecule is then calculated as the sum of its corresponding fragment values plus the sum of correction terms for intramolecular interactions. Lists of fragment constants and correction terms ate available (b)(c)(d)(e);. Some are regularly updated (b).

Quality criteria

In general, the reliability of the calculation method decreases with increasing complexity of the compound under study. In the case of simple molecules with low molecular weight and one or two functional groups, a deviation of 0,1 to 0,3 log Pow units between the results of the different fragmentation methods and the measured value can be expected. In the case of more complex molecules the margin of error can be greater. This will depend on the reliability and availability of fragment constants, as well as on the ability to recognise intramolecular interactions (e.g. hydrogen bonds) and the correct use of the correction terms (less of a problem with the computer software CLOGP-3) (b). In the case of ionising compounds the correct consideration of the charge or degree of ionisation is important.

Calculation procedures

Hansch π-method

The original hydrophobic substituent constant, π, introduced by Fujira et al. (f) is defined as: πx = log Pow (PhX) - log Pow (PhH) where Pow (PhX) is the partition coefficient of an aromatic derivative and Pow (PhH) that of the parent compound (e.g. πCl = log Pow (C6H5Cl) - log Pow (C6H6) = 2,84 - 2,13 = 0,71). According to its definition the π-method is applicable predominantly for aromatic substitution. π-values for a large number of substituents have been tabulated (b)(c)(d). They are used for the calculation of log Pow for aromatic molecules or substructures.

Rekker method

According to Rekker (g) the log Pow value is calculated as follows: log Pow=Σi aifi+Σj (interactious terms) where fi represents the different molecular fragment constants and ai the frequency of their occurrence in the molecule under investigation. The correction terms can be expressed as an integral multiple of one single constant Cm (so-called magic constant). The fragment constants fi and Cm were determined from a list of 1054 experimental Pow values (825 compounds) using multiple regression analysis (c)(h). The determination of the interaction terms is carried out according to set rules described in the literature (e)(h)(i).

Hansch-Leo method

According to Hansch and Leo (c), the log Pow value is calculated from: log Pow=Σi ai fi+Σj bj Fj where fi represents the different molecular fragment constants, Fj the correction terms and ai, bj the corresponding frequencies of occurrence. Derived from experimental Pow values, a list of atomic and group fragmental values and a list of correction terms Fj (so-called factors) were determined by trial and error. The correction terms have been ordered into several different classes (a)(c). It is relatively complicated and time consuming to take into account all the rules and correction terms. Software packages have been developed (b).

Combined method

The calculation of log Pow of complex molecules can be considerably improved, if the molecule is dissected into larger substructures for which reliable log Pow values are available, either from tables (b)(c) or from one's own measurements. Such fragments (e.g. heterocycles, anthraquinone, azobenzene) can then be combined with the Hansch π-values or with Rekker or Leo fragment constants.

Remarks

(i) The calculation methods can only be applied to partly or fully ionised compounds when it is possible to take the necessary correction factors into account;

(ii) if intramolecular hydrogen bonds can be assumed, the corresponding correction terms (approx. +0,6 to +1,0 log Pow units) have to be added (a). Indications for the presence of such bonds can be obtained from stereo models or spectroscopic data of the molecule;

(iii) If several tautomeric forms are possible, the most likely form should be used as the basis of the calculation;

(iv) the revisions of lists of fragment constants should be followed carefully.

Report

When using calculation/estimation methods, the test report shall, if possible, include the following information:

description of the substance (mixture, impurities, etc.),

indication of any possible intramolecular hydrogen bonding, dissociation, charge and any other unusual effects (e.g. tautomerism),

description of the calculation method,

identification or supply of database,

peculiarities in the choice of fragments,

comprehensive documentation of the calculation.

LITERATURE

(a) W.J. Lyman, W.F. Reehl and D.H. Rosenblatt (ed.), Handbook of Chemical Property Estimation Methods, McGraw-Hill, New York, 1983.

(b) Pomona College, Medicinal Chemistry Project, Claremont, California 91711, USA, Log P Database and Med. Chem. Software (Program CLOGP-3).

(c) C. Hansch, A.J. Leo, Substituent Constants for Correlation Analysis in Chemistry and Biology, John Wiley, New York, 1979.

(d) A. Leo, C. Hansch, D. Elkins, Chem. Rev., 1971, vol. 71, p. 525.

(e) R.F. Rekker, H.M. de Kort, Eur. J. Med. Chem. -Chill. Ther. 1979, vol. 14, p. 479.

(f) T. Fujita, J. Iwasa and C. Hansch, J. Amer. Chem. Soc., 1964, vol. 86, p. 5175.

(g) R.F. Rekker, The Hydrophobic Fragmental Constant, Pharmacochemistry Library, Elsevier, New York, 1977, vol. 1.

(h) C.V. Eadsforth, P. Moser, Chemosphere, 1983, vol. 12, p. 1459.

(i) R.A. Scherrer, ACS, American Chemical Society, Washington D.C., 1984, Symposium Series 255, p. 225.

Appendix 2 Recommended Reference Substances for the HLPC Method

No Reference Substance log Pow pKa 1 2-Butanone 0,3 2 4-Acetylpyridine 0,5 3 Aniline 0,9 4 Acetanilide 1,0 5 Benzylalcohol 1,1 6 p-Methoxyphenol 1,3 pKa = 10,26 7 Phenoxy acetic acid 1,4 pKa = 3,12 8 Phenol 1,5 pKa = 9,92 9 2,4-Dinitrophenol 1,5 pKa = 3,96 10 Benzonitrile 1,6 11 Phenylacetonitrile 1,6 12 4-Methylbenzyl alcohol 1,6 13 Acetophenone 1,7 14 2-Nitrophenol 1,8 pKa = 7,17 15 3-Nitrobenzoic acid 1,8 pKa = 3,47 16 4-Chloraniline 1,8 pKa = 4,15 17 Nitrobenzene 1,9 18 Cinnamic alcohol 1,9 19 Benzoic acid 1,9 pKa = 4,19 20 p-Cresol 1,9 pKa = 10,17 21 Cinnamic acid 2,1 pKa = 3,89 cis 4,44 trans 22 Anisole 2,1 23 Methylbenzoate 2,1 24 Benzene 2,1 25 3-Methylbenzoic acid 2,4 pKa = 4,27 26 4-Chlorophenol 2,4 pKa = 9,1 27 Trichloroethylene 2,4 28 Atrazine 2,6 29 Ethylbenzoate 2,6 30 2,6-Dichlorobenzonitrile 2,6 31 3-Chlorobenzoic acid 2,7 pKa = 3,82 32 Toluene 2,7 33 1-Naphthol 2,7 pKa = 9,34 34 2,3-Dichloroaniline 2,8 35 Chlorobenzene 2,8 36 Allyl-phenylether 2,9 37 Bromobenzene 3,0 38 Ethylbenzene 3,2 39 Benzophenone 3,2 40 4-Phenylphenol 3,2 pKa = 9,54 41 Thymol 3,3 42 1,4-Dichlorobenzene 3,4 43 Diphenylamine 3,4 pKa = 0,79 44 Naphthalene 3,6 45 Phenylbenzoate 3,6 46 Isopropylbenzene 3,7 47 2,4,6-Trichlorophenol 3,7 pKa = 6 48 Biphenyl 4,0 49 Benzylbenzoate 4,0 50 2,4-Dinitro-6 sec. butyophenol 4,1 51 1,2,4-Trichlorobenzene 4,2 52 Dodecanoic acid 4,2 53 Diphenylether 4,2 54 n-Butylbenzene 4,5 55 Phenanthrene 4,5 56 Fluoranthene 4,7 57 Dibenzyl 4,8 58 2,6-Diphenylpyridine 4,9 59 Triphenylamine 5,7 60 DDT 6,2 Other reference substances of low log Pow 1 Nicotinic acid -0,07

Appendix Figure Mould and accessories for the preparation of the pile

(All dimensions in millimetres) Length of the mould: 250 mm Material: aluminium

Appendix Figure Apparatus

Appendix Example of material specification for thermal sensitivity test (see DIN 1623) (1)(2)(3)

Tube: Material specification No 1.0336.505 g Orifice plate: Material specification No 1.4873 Threaded collar and nut: Material specification No 1.3817

Figure 1 Thermal sensitivity test apparatus

(all dimensions in millimetres) Fig. 1a Steel tube and accessories (1) tube (1a) outer flange (2) threaded collar; low-friction thread (3) orifice plate a = 2,0 or 6,0 mm diameter (4) nut b = 10 mm diameter (5) chamfered surface (6) 2 flat for spanner size 41 Fig. 1b Heating and protective device (7) 2 flat for spanner size 36 (8) splinter-proof box (9) 2 supporting rods for tube (10) assembled tube (11) position for rear burner; the other burners are visible (12) pilot jet

Figure 2 Thermal sensitivity test

(example of fragmentation) No explosion No explosion Explosion Explosion Explosion Explosion

Figure 3 Heating rate calibration for thermal sensitivity test

Temperature (°C) Time (s) Temperature/time curve obtained on heating dibutyl phtalate (27 cm3) in a closed (1,5 mm orifice plate) tube using a propane flow rate of 3,2 litre/minute. The temperature is measured with a 1 mm diameter stainless steel sheathed chromel/alumel thermocouple, placed centrally 43 mm below the rim of the tube. The heating rate between 135 oC and 285 oC should be between 185 and 215 K/minute.

Figure 4 Shock test apparatus

(all dimensions in millimetres) Fig. 4a Fall-hammer, front and side, general view (1) base, 450 x 450 x 60 (2) steel block, 230 x 250 x 200 (3) anvil, 100 diameter x 70 (4) column (5) median cross-member (6) 2 guides (7) toothed rack Fig. 4b Fall-hammer, lower part (8) graduated scale (9) fall-hammer (drop mass) (10) holding and releasing device (11) locating plate (12) intermediate anvil (interchangeable), 26 diameter x 26 (13) locating ring with orifices (14) impact device

Continued

Fig. 4c Shock device for substances in powdered or paste-like form Fig. 4d Shock device for liquid substances (1) steel cylinders (2) guide ring for steel cylinders (3) locating ring with orifices (a) vertical section (b) plan (4) rubber ring (5) liquid substance (40 mm3) (6) space free from liquid Fig. 4e Hammer (drop mass of 5 kg) (1) suspension spigot (2) height marker (3) positioning groove (4) cylindrical striking head (5) rebound catch

Figure 5 Friction sensitivity apparatus

Fig. 5a Friction apparatus; elevation and plan view (1) steel base (2) movable carriage (3) porcelain plate, 25 x 25 x 5 mm, held on carriage (4) fixed porcelain peg, 10 diameter x 15 mm (5) sample under test, approximately 10 mm3 Fig. 5b Starting position of peg on sample (6) peg-holder (7) loading arm (8) counterweight (9) switch (10) wheel for setting carriage at starting position (11) direction to electric drive motor

Appendix Figure Mould and accessories for the preparations of the pile

(All dimensions in millimetres)

Appendix Examples of other methods for determination of number average molecular weight (Mn) for polymers

Gel permeation chromatography (GPC) is the preferred method for determination of Mn, especially when a set of standards are available, whose structure are comparable with the polymer structure. However, where there are practical difficulties in using GPC or there is already an expectation that the substance will fail a regulatory Mn criterion (and which needs confirming), alternative methods are available, such as:

1.1.1.1.2.1.31.4

Use of colligative properties Ebullioscopy/Cryoscopy involves measurement of boiling point elevation (ebullioscopy) or freezing point depression (cryoscopy) of a solvent, when the polymer is added. The method relies on the fact that the effect of the dissolved polymer on the boiling/freezing point of the liquid is dependent on the molecular weight of the polymer (1) (2). Applicability, Mn < 20000. Lowering of vapour pressure involves the measurement of the vapour pressure of a chosen reference liquid before and after the addition of known quantities of polymer (1) (2). Applicability, Mn < 20000 (theoretically; in practice however of limited value). Membrane osmometry relies on the principle of osmosis, i.e. the natural tendency of solvent molecules to pass through a semi-permeable membrane from a dilute to a concentrated solution to achieve equilibrium. In the test, the dilute solution is at zero concentration, whereas the concentrated solution contains the polymer. The effect of drawing solvent through the membrane causes a pressure differential that is dependent on the concentration and the molecular weight of the polymer (1) (3) (4). Applicability, Mn between 20000 - 200000. Vapour phase osmometry involves comparison of the rate of evaporation of a pure solvent aerosol to at least three aerosols containing the polymer at different concentrations (1)(2)(4). Applicability, Mn < 20000.

2. End-group analysis To use this method, knowledge of both the overall structure of the polymer and the nature of the chain terminating end groups is needed (which must be distinguishable from the main skeleton by, e.g. NMR or titration/derivatisation). The determination of the molecular concentration of the end groups present on the polymer can lead to a value for the molecular weight (7) (8) (9). Applicability, Mn up to 50000 (with decreasing reliability).

3.(1)(2)(3)(4)(5)(6)(7)(8)(9)

References Billmeyer, F.W. Jr., (1984) Textbook of Polymer Science, 3rd Edn., John Wiley, New York. Glover, C.A., (1975) Absolute Colligative Property Methods. Chapter 4. In: Polymer Molecular Weights, Part I P.E. Slade, Jr. ed., Marcel Dekker, New York. ASTM D 3750-79, (1979) Standard Practice for Determination of Number-Average Molecular Weight of Polymers by Membrane Osmometry. American Society for Testing and Materials, Philadelphia, Pennsylvania. Coll, H. (1989) Membrane Osmometry. In: Determination of Molecular Weight, A.R. Cooper ed., J. Wiley and Sons, pp. 25-52. ASTM 3592-77, (1977) Standard Recommended Practice for Determination of Molecular Weight by Vapour Pressure, American Society for Testing and Materials, Philadelphia, Pennsylvania. Morris, C.E.M., (1989) Vapour Pressure Osmometry. In: Determinationn of Molecular Weight, A.R. Cooper ed., John Wiley and Sons. Schröder, E., Müller, G., and Arndt, K-F., (1989) Polymer Characterisation, Carl Hanser Verlag, Munich. Garmon, R.G., (1975) End-Group Determinations, Chapter 3 In: Polymer Molecular Weights, Part I, P.E. Slade, Jr. ed., Marcel Dekker, New York. Amiya, S., et al. (1990) Pure and Applied Chemistry, 62, 2139-2146.

Appendix Guidance for correcting low molecular content for the presence of insoluble polymer

When insoluble polymer is present in a sample, it results in mass loss during the GPC analysis. The insoluble polymer is irreversibly retained on the column or sample filter while the soluble portion of the sample passes through the column. In the case where the refractive index increment (dn/dc) of the polymer can be estimated or measured, one can estimate the sample mass lost on the column. In that case, one makes a correction using an external calibration with standard materials of known concentration and dn/dc to calibrate the response of the refractometer. In the example hereafter a poly(methyl methacrylate) (pMMA) standard is used. In the external calibration for analysis of acrylic polymers, a pMMA standard of known concentration in tetrahydrofuran, is analysed by GPC and the resulting data are used to find the refractometer constant according to the equation: K = R/(C × V × dn/dc) where: K the refractometer constant (in microvolt second/ml), R the response of the pMMA standard (in microvolt/second), C the concentration of the pMMA standard (in mg/ml), V the injection volume (in ml), and dn/dc the refractive index increment for pMMA in tetrahydrofuran (in ml/mg). The following data are typical for a pMMA standard: R 2937891 C 1,07 mg/ml V 0,1 ml dn/dc 9 × 10-5 ml/mg The resulting K value, 3,05 × 1011 is then used to calculate the theoretical detector response if 100 % of the polymer injected had eluted through the detector.

PART B METHODS FOR THE DETERMINATION OF TOXICITY AND OTHER HEALTH EFFECTS

GENERAL INTRODUCTION A.

CHARACTERISATION OF THE TEST SUBSTANCE The composition of the test substance, including major impurities, and its relevant physico-chemical properties including stability, should be known prior to the initiation of any toxicity study. The physico-chemical properties of the test substance provide important information for the selection of the route of administration, the design of each particular study and the handling and storage of the test substance. The development of an analytical method for qualitative and quantitative determination of the test substance (including major impurities when possible) in the dosing medium and the biological material should precede the initiation of the study. All information relating to the identification, the physico-chemical properties, the purity, and behaviour of the test substance should be included in the test report.

ANIMAL CARE Stringent control of environmental conditions and proper animal care techniques are essential in toxicity testing.

(i) Housing conditions The environmental conditions in the experimental animal rooms or enclosures should be appropriate to the test species. For rats, mice and guinea pigs, suitable conditions are a room temperature of 22 oC ± 3 oC with a relative humidity of 30 to 70 %; for rabbits the temperature should be 20 ± 3 oC with a relative humidity of 30 to 70 %. Some experimental techniques are particularly sensitive to temperature effects and, in these cases, details of appropriate conditions are included in the description of the test method. In all investigations of toxic effects, the temperature and humidity should be monitored, recorded, and included in the final report of the study. Lighting should be artificial, the sequence being 12 hours light, 12 hours dark. Details of the lighting pattern should be recorded and included in the final report of the study. Unless otherwise specified in the method, animals may be housed individually, or be caged in small groups of the same sex; for group caging, no more than five animals should be housed per cage. In reports of animal experiments, it is important to indicate the type of caging used and the number of animals housed in each cage both during exposure to the chemical and any subsequent observation period.

(ii) Feeding conditions Diets should meet all the nutritional requirements of the species under test. Where test substances are administered to animals in their diet the nutritional value may be reduced by interaction between the substance and a dietary constituent. The possibility of such a reaction should be considered when interpreting the results of tests. Conventional laboratory diets may be used with an unlimited supply of drinking water. The choice of the diet may be influenced by the need to ensure a suitable admixture of a test substance when administered by this method. Dietary contaminants which are known to influence the toxicity should not be present in interfering concentrations.

ALTERNATIVE TESTING The European Union is committed to promoting the development and validation of alternative techniques which can provide the same level of information as current animal tests, but which use fewer animals, cause less suffering or avoid the use of animals completely. Such methods, as they become available, must be considered wherever possible for hazard characterisation and consequent classification and labelling for intrinsic hazards and chemical safety assessment.

EVALUATION AND INTERPRETATION When tests are evaluated and interpreted, limitations in the extent to which the results of animal and in vitro studies can be extrapolated directly to man must be considered and therefore, evidence of adverse effects in humans, where available, may be used for confirmation of testing results.

LITERATURE REFERENCES Most of these methods are developed within the framework of the OECD programme for Testing Guidelines, and should be performed in conformity with the principles of Good Laboratory Practice, in order to ensure as wide as possible mutual acceptance of data. Additional information may be found in the references listed in the OECD guidelines and the relevant literature published elsewhere.

B.1 bis. ACUTE ORAL TOXICITY — FIXED DOSE PROCEDURE

B.1 bis.

1. METHOD This test method is equivalent to OECD TG 420 (2001)

1.

1.1. INTRODUCTION Traditional methods for assessing acute toxicity use death of animals as an endpoint. In 1984, a new approach to acute toxicity testing was suggested by the British Toxicology Society based on the administration at a series of fixed dose levels (1). The approach avoided using death of animals as an endpoint, and relied instead on the observation of clear signs of toxicity at one of a series of fixed dose levels. Following UK (2) and international (3) in vivo validation studies the procedure was adopted as a testing method in 1992. Subsequently, the statistical properties of the Fixed Dose Procedure have been evaluated using mathematical models in a series of studies (4)(5)(6). Together, the in vivo and modelling studies have demonstrated that the procedure is reproducible, uses fewer animals and causes less suffering than the traditional methods and is able to rank substances in a similar manner to the other acute toxicity testing methods. Guidance on the selection of the most appropriate test method for a given purpose can be found in the Guidance Document on Acute Oral Toxicity Testing (7). This guidance document also contains additional information on the conduct and interpretation of Testing Method B.1bis. It is a principle of the method that in the main study only moderately toxic doses are used, and that administration of doses that are expected to be lethal should be avoided. Also, doses that are known to cause marked pain and distress, due to corrosive or severely irritant actions, need not be administered. Moribund animals, or animals obviously in pain or showing signs of severe and enduring distress shall be humanely killed, and are considered in the interpretation of the test results in the same way as animals that died on test. Criteria for making the decision to kill moribund or severely suffering animals, and guidance on the recognition of predictable or impending death, are the subject of a separate Guidance Document (8). The method provides information on the hazardous properties and allows the substance to be ranked and classified according to the Globally Harmonised System (GHS) for the classification of chemicals which cause acute toxicity (9). The testing laboratory should consider all available information on the test substance prior to conducting the study. Such information will include the identity and chemical structure of the substance; its physico-chemical properties; the results of any other in vitro or in vivo toxicity tests on the substance; toxicological data on structurally related substances; and the anticipated use(s) of the substance. This information is necessary to satisfy all concerned that the test is relevant for the protection of human health, and will help in the selection of an appropriate starting dose.

1.2. DEFINITIONS Acute oral toxicity: refers to those adverse effects occurring following oral administration of a single dose of a substance or multiple doses given within 24 hours. Delayed death: means that an animal does not die or appear moribund within 48 hours but dies later during the 14-day observation period. Dose: is the amount of test substance administered. Dose is expressed as weight of test substance per unit weight of test animal (e.g. mg/kg). Evident toxicity: is a general term describing clear signs of toxicity following the administration of test substance (see (3) for examples) such that at the next highest fixed dose either severe pain and enduring signs of severe distress, moribund status (criteria are presented in the Humane Endpoints Guidance Document (8)), or probable mortality in most animals can be expected. GHS: Globally Harmonised Classification System for Chemical Substances and Mixtures. A joint activity of OECD (human health and the environment), UN Committee of Experts on Transport of Dangerous Goods (physical-chemical properties) and ILO (hazard communication) and coordinated by the Interorganisation Programme for the Sound Management of Chemicals (IOMC). Impending death: when moribund state or death is expected prior to the next planned time of observation. Signs indicative of this state in rodents could include convulsions, lateral position, recumbence and tremor. (See the Humane Endpoint Guidance Document (8) for more details). LD50(median lethal dose): is a statistically derived single dose of a substance that can be expected to cause death in 50 % of animals when administered by the oral route. The LD50 value is expressed in terms of weight of test substance per unit weight of test animal (mg/kg). Limit dose: refers to a dose at an upper limitation on testing (2000 or 5000 mg/kg). Moribund status: being in a state of dying or inability to survive, even if treated. (See the Humane Endpoint Guidance Document (8) for more details). Predictable death: presence of clinical signs indicative of death at a known time in the future before the planned end of the experiment, for example: inability to reach water or food. (See the Humane Endpoint Guidance Document (8) for more details).

1.3. PRINCIPLE OF THE TEST METHOD Groups of animals of a single sex are dosed in a stepwise procedure using the fixed doses of 5, 50, 300 and 2000 mg/kg (exceptionally an additional fixed dose of 5000 mg/kg may be considered, see Section 1.6.2). The initial dose level is selected on the basis of a sighting study as the dose expected to produce some signs of toxicity without causing severe toxic effects or mortality. Clinical signs and conditions associated with pain, suffering, and impending death, are described in detail in a separate OECD Guidance Document (8). Further groups of animals may be dosed at higher or lower fixed doses, depending on the presence or absence of signs of toxicity or mortality. This procedure continues until the dose causing evident toxicity or no more than one death is identified, or when no effects are seen at the highest dose or when deaths occur at the lowest dose.

1.4. DESCRIPTION OF THE TEST METHOD

1.4.

1.4.1. Selection of animal species The preferred rodent species is the rat, although other rodent species may be used. Normally females are used (7). This is because literature surveys of conventional LD50 tests show that usually there is little difference in sensitivity between the sexes, but in those cases where differences are observed, females are generally slightly more sensitive (10). However, if knowledge of the toxicological or toxicokinetic properties of structurally related chemicals indicates that males are likely to be more sensitive then this sex should be used. When the test is conducted in males, adequate justification should be provided. Healthy young adult animals of commonly used laboratory strains should be employed. Females should be nulliparous and non-pregnant. Each animal, at the commencement of its dosing, should be between eight and 12 weeks old and its weight should fall in an interval within ± 20 % of the mean weight of any previously dosed animals.

1.4.2. Housing and feeding conditions The temperature of the experimental animal room should be 22 oC (± 3 oC). Although the relative humidity should be at least 30 % and preferably not exceed 70 % other than during room cleaning the aim should be 50-60 %. Lighting should be artificial, the sequence being 12 hours light, 12 hours dark. For feeding, conventional laboratory diets may be used with an unlimited supply of drinking water. Animals may be group-caged by dose, but the number of animals per cage must not interfere with clear observations of each animal.

1.4.3. Preparation of animals The animals are randomly selected, marked to permit individual identification, and kept in their cages for at least five days prior to the start of dosing to allow for acclimatisation to the laboratory conditions.

1.4.4. Preparation of doses In general test substances should be administered in a constant volume over the range of doses to be tested by varying the concentration of the dosing preparation. Where a liquid end product or mixture is to be tested however, the use of the undiluted test substance, i.e. at a constant concentration, may be more relevant to the subsequent risk assessment of that substance, and is a requirement of some regulatory authorities. In either case, the maximum dose volume for administration must not be exceeded. The maximum volume of liquid that can be administered at one time depends on the size of the test animal. In rodents, the volume should not normally exceed 1ml /100 g of body weight: however in the case of aqueous solutions 2 ml/100 g body weight can be considered. With respect to the formulation of the dosing preparation, the use of an aqueous solution/suspension/emulsion is recommended wherever possible, followed in order of preference by a solution/suspension/emulsion in oil (e.g. corn oil) and then possibly solution in other vehicles. For vehicles other than water the toxicological characteristics of the vehicle should be known. Doses must be prepared shortly prior to administration unless the stability of the preparation over the period during which it will be used is known and shown to be acceptable.

1.5. PROCEDURE

1.5.

1.5.1. Administration of doses The test substance is administered in a single dose by gavage using a stomach tube or a suitable intubation canula. In the unusual circumstance that a single dose is not possible, the dose may be given in smaller fractions over a period not exceeding 24 hours. Animals should be fasted prior to dosing (e.g. with the rat, food but not water should be withheld over-night; with the mouse, food but not water should be withheld for three to four hours). Following the period of fasting, the animals should be weighed and the test substance administered. After the substance has been administered, food may be withheld for a further three to four hours in rats or one to two hours in mice. Where a dose is administered in fractions over a period of time, it may be necessary to provide the animals with food and water depending on the length of the period.

1.5.2. Sighting study The purpose of the sighting study is to allow selection of the appropriate starting dose for the main study. The test substance is administered to single animals in a sequential manner following the flowcharts in Appendix 1. The sighting study is completed when a decision on the starting dose for the main study can be made (or if a death is seen at the lowest fixed dose). The starting dose for the sighting study is selected from the fixed dose levels of 5, 50, 300 and 2000 mg/kg as a dose expected to produce evident toxicity based, when possible, on evidence from in vivo and in vitro data from the same chemical and from structurally related chemicals. In the absence of such information, the starting dose will be 300 mg/kg. A period of at least 24 hours will be allowed between the dosing of each animal. All animals should be observed for at least 14 days. Exceptionally, and only when justified by specific regulatory needs, the use of an additional upper fixed dose level of 5000 mg/kg may be considered (see Appendix 3). For reasons of animal welfare concern, testing of animals in GHS Category 5 ranges (2000-5000 mg/kg is discouraged and should only be considered when there is a strong likelihood that the results of such a test have a direct relevance for protecting human or animal health or the environment. In cases where an animal tested at the lowest fixed dose level (5 mg/kg) in the sighting study dies, the normal procedure is to terminate the study and assign the substance to GHS Category 1 (as shown in Appendix 1). However, if further confirmation of the classification is required, an optional supplementary procedure may be conducted, as follows. A second animal is dosed at 5 mg/kg. If this second animal dies, then GHS Category 1 will be confirmed and the study will be immediately terminated. If the second animal survives, then a maximum of three additional animals will be dosed at 5 mg/kg. Because there will be a high risk of mortality, these animals should be dosed in a sequential manner to protect animal welfare. The time interval between dosing each animal should be sufficient to establish that the previous animal is likely to survive. If a second death occurs, the dosing sequence will be immediately terminated and no further animals will be dosed. Because the occurrence of a second death (irrespective of the number of animals tested at the time of termination) falls into outcome A (two or more deaths), the classification rule of Appendix 2 at the 5 mg/kg fixed dose is followed (Category 1 if there are two or more deaths or Category 2 if there is no more than one death). In addition, Appendix 4 gives guidance on the classification in the EU system until the new GHS is implemented.

1.5.3. Main study

1.5.3.

1.5.3.1. Numbers of animals and dose levels The action to be taken following testing at the starting dose level is indicated by the flowcharts in Appendix 2. One of three actions will be required; either stop testing and assign the appropriate hazard classification class, test at a higher fixed dose or test at a lower fixed dose. However, to protect animals, a dose level that caused death in the sighting study will not be revisited in the main study (see Appendix 2). Experience has shown that the most likely outcome at the starting dose level will be that the substance can be classified and no further testing will be necessary. A total of five animals of one sex will normally be used for each dose level investigated. The five animals will be made up of one animal from the sighting study dosed at the selected dose level together with an additional four animals (except, unusually, if a dose level used on the main study was not included in the sighting study). The time interval between dosing at each level is determined by the onset, duration, and severity of toxic signs. Treatment of animals at the next dose should be delayed until one is confident of survival of the previously dosed animals. A period of three or four days between dosing at each dose level is recommended, if needed, to allow for the observation of delayed toxicity. The time interval may be adjusted as appropriate, e.g. in case of inconclusive response. When the use of an upper fixed dose of 5000 mg/kg is considered, the procedure outlined in Appendix 3 should be followed (see also section 1.6.2).

1.5.3.2. Limit test The limit test is primarily used in situations where the experimenter has information indicating that the test material is likely to be nontoxic, i.e., having toxicity only above regulatory limit doses. Information about the toxicity of the test material can be gained from knowledge about similar tested compounds or similar tested mixtures or products, taking into consideration the identity and percentage of components known to be of toxicological significance. In those situations where there is little or no information about its toxicity, or in which the test material is expected to be toxic, the main test should be performed. Using the normal procedure, a sighting study starting dose of 2000 mg/kg (or exceptionally 5000 mg/kg) followed by dosing of a further four animals at this level serves as a limit test for this guideline.

1.6. OBSERVATIONS Animals are observed individually after dosing at least once during the first 30 minutes, periodically during the first 24 hours, with special attention given during the first four hours, and daily thereafter, for a total of 14 days, except where they need to be removed from the study and humanely killed for animal welfare reasons or are found dead. However, the duration of observation should not be fixed rigidly. It should be determined by the toxic reactions, time of onset and length of recovery period, and may thus be extended when considered necessary. The times at which signs of toxicity appear and disappear are important, especially if there is a tendency for toxic signs to be delayed (11). All observations are systematically recorded, with individual records being maintained for each animal. Additional observations will be necessary if the animals continue to display signs of toxicity. Observations should include changes in skin and fur, eyes and mucous membranes, and also respiratory, circulatory, autonomic and central nervous systems, and somatomotor activity and behaviour pattern. Attention should be directed to observations of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma. The principles and criteria summarised in the Humane Endpoints Guidance Document should be taken into consideration (8). Animals found in a moribund condition and animals showing severe pain or enduring signs of severe distress should be humanely killed. When animals are killed for humane reasons or found dead, the time of death should be recorded as precisely as possible.

1.6.

1.6.1. Body weight Individual weights of animals should be determined shortly before the test substance is administered and at least weekly thereafter. Weight changes should be calculated and recorded. At the end of the test surviving animals are weighed and then humanely killed.

1.6.2. Pathology All test animals (including those that die during the test or are removed from the study for animal welfare reasons) should be subjected to gross necropsy. All gross pathological changes should be recorded for each animal. Microscopic examination of organs showing evidence of gross pathology in animals surviving 24 or more hours after the initial dosing may also be considered because it may yield useful information.

2. DATA Individual animal data should be provided. Additionally, all data should be summarised in tabular form, showing for each test group the number of animals used, the number of animals displaying signs of toxicity, the number of animals found dead during the test or killed for humane reasons, time of death of individual animals, a description and the time course of toxic effects and reversibility, and necropsy findings.

3. REPORTING

3.

3.1. TEST REPORT The test report must include the following information, as appropriate:

Test substance:

physical nature, purity, and, where relevant, physico-chemical properties (including isomerisation),

identification data, including CAS number.

Vehicle (if appropriate):

justification for choice of vehicle, if other than water.

Test animals:

species/strain used,

microbiological status of the animals, when known,

number, age and sex of animals (including, where appropriate, a rationale for use of males instead of females),

source, housing conditions, diet, etc.

Test conditions:

details of test substance formulation, including details of the physical form of the material administered,

details of the administration of the test substance including dosing volumes and time of dosing,

details of food and water quality (including diet type/source, water source),

the rationale for the selection of the starting dose.

Results:

tabulation of response data and dose level for each animal (i.e. animals showing signs of toxicity including mortality, nature, severity and duration of effects),

tabulation of body weight and body weight changes,

individual weights of animals at the day of dosing, in weekly intervals thereafter, and at time of death or sacrifice,

date and time of death if prior to scheduled sacrifice,

time course of onset of signs of toxicity and whether these were reversible for each animal,

necropsy findings and histopathological findings for each animal, if available.

Discussion and interpretation of results.

Conclusions.

4.(1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)

REFERENCES British Toxicology Society Working Party on Toxicity (1984) Special report: a new approach to the classification of substances and preparations on the basis of their acute toxicity. Human Toxicol., 3, p. 85-92. Van den Heuvel, M.J., Dayan, A.D. and Shillaker, R.O (1987) Evaluation of the BTS approach to the testing of substances and preparations for their acute toxicity. Human Toxicol.‚ 6, p. 279-291. Van den Heuvel, M.J., Clark, D.G., Fielder, R.J., Koundakjian, P.P., Oliver, G.J.A., Pelling, D., Tomlinson, N.J. and Walker, A.P (1990) The international validation of a fixed-dose procedure as an alternative to the classical LD50 test. Fd. Chem. Toxicol. 28, p. 469-482. Whitehead, A. and Curnow, R.N (1992) Statistical evaluation of the fixed-dose procedure. Fd. Chem. Toxicol., 30, p. 313-324. Stallard, N. and Whitehead, A (1995) Reducing numbers in the fixed-dose procedure. Human Exptl. Toxicol. 14, p. 315-323. Stallard, N., Whitehead, A and Ridgeway, P. (2002) Statistical evaluation of the revised fixed dose procedure. Hum. Exp. Toxicol., 21, p. 183-196. OECD (2001) Guidance Document on Acute Oral Toxicity Testing. Environmental Health and Safety Monograph Series on Testing and Assessment No 24. Paris OECD (2000) Guidance Document on the Recognition, Assessment and Use of Clinical Signs as Humane Endpoints for Experimental Animals Used in Safety Evaluation. Environmental Health and Safety Monograph Series on Testing and Assesment No 19. OECD (1998) Harmonised Integrated Hazard Classification for Human Health and Environmental Effects of Chemical Substances as endorsed by the 28th Joint Meeting of the Chemicals Committee and the Working Party on Chemicals in November 1998, Part 2, p. 11 [http://webnet1.oecd.org/oecd/pages/home/displaygeneral/0,3380, EN-documents-521-14-no-24-no-0,FF.html]. Lipnick, R.L., Cotruvo, J.A., Hill, R.N., Bruce, R.D., Stitzel, K.A., Walker, A.P., Chu, I., Goddard, M., Segal, L., Springer, J.A. and Myers, R.C (1995) Comparison of the Up-and-Down, Conventional LD50, and Fixed-Dose Acute Toxicity Procedures. Fd. Chem. Toxicol. 33, p. 223-231. Chan P.K and A. W Hayes (1994) Chapter 16 Acute Toxicity and Eye Irritation. In: Principles and Methods of Toxicology. 3rd Edition. A.W. Hayes, Editor. Raven Press Ltd. New York, USA.

B.1 tris. ACUTE ORAL TOXICITY — ACUTE TOXIC CLASS METHOD

B.1 tris.

1. METHOD This test method is equivalent to OECD TG 423 (2001)

1.

1.1. INTRODUCTION The acute toxic class method (1) set out in this test is a stepwise procedure with the use of three animals of a single sex per step. Depending on the mortality and/or the moribund status of the animals, on average two to four steps may be necessary to allow judgement on the acute toxicity of the test substance. This procedure is reproducible, uses very few animals and is able to rank substances in a similar manner to the other acute toxicity testing methods. The acute toxic class method is based on biometric evaluations (2)(3)(4)(5) with fixed doses, adequately separated to enable a substance to be ranked for classification purposes and hazard assessment. The method as adopted in 1996 was extensively validated in vivo against LD50 data obtained from the literature, both nationally (6) and internationally (7). Guidance on the selection of the most appropriate test method for a given purpose can be found in the Guidance Document on Acute Oral Toxicity Testing (8). This Guidance Document also contains additional information on the conduct and interpretation of testing method B.1tris. Test substances, at doses that are known to cause marked pain and distress due to corrosive or severely irritant actions, need not be administered. Moribund animals, or animals obviously in pain or showing signs of severe and enduring distress shall be humanely killed, and are considered in the interpretation of the test results in the same way as animals that died on test. Criteria for making the decision to kill moribund or severely suffering animals, and guidance on the recognition of predictable or impending death, are the subject of a separate Guidance Document (9). The method uses pre-defined doses and the results allow a substance to be ranked and classified according to the Globally Harmonised System for the classification of chemicals which cause acute toxicity (10). In principle, the method is not intended to allow the calculation of a precise LD50, but does allow for the determination of defined exposure ranges where lethality is expected since death of a proportion of the animals is still the major endpoint of this test. The method allows for the determination of an LD50 value only when at least two doses result in mortality higher than 0 % and lower than 100 %. The use of a selection of pre-defined doses, regardless of test substance, with classification explicitly tied to number of animals observed in different states improves the opportunity for laboratory to laboratory reporting consistency and repeatability. The testing laboratory should consider all available information on the test substance prior to conducting the study. Such information will include the identity and chemical structure of the substance; its physico-chemical properties; the result of any other in vivo or in vitro toxicity tests on the substance; toxicological data on the structurally related substances; and the anticipated use(s) of the substance. This information is necessary to satisfy all concerned that the test is relevant for the protection of human health and will help in the selection of the most appropriate starting dose.

1.2. DEFINITIONS Acute oral toxicity: refers to those adverse effects occurring following oral administration of a single dose of a substance or multiple doses given within 24 hours. Delayed death: means that an animal does not die or appear moribund within 48 hours but dies later during the 14-day observation period. Dose: is the amount of test substance administered. Dose is expressed as weight of test substance per unit weight of test animal (e.g. mg/kg). GHS: Globally Harmonised Classification System for Chemical Substances and Mixtures. A joint activity of OECD (human health and the environment), UN Committee of Experts on Transport of Dangerous Goods (physical-chemical properties) and ILO (hazard communication) and coordinated by the Interorganisation Programme for the Sound Management of Chemicals (IOMC). Impending death: when moribund state or death is expected prior to the next planned time of observation. Signs indicative of this state in rodents could include convulsions, lateral position, recumbence and tremor (See the Humane Endpoint Guidance Document (9) for more details). LD50(median lethal oral dose): is a statistically derived single dose of a substance that can be expected to cause death in 50 % of animals when administered by the oral route. The LD50 value is expressed in terms of weight of test substance per unit weight of test animal (mg/kg). Limit dose: refers to a dose at an upper limitation on testing (2000 or 5000 mg/kg). Moribund status: being in a state of dying or inability to survive, even if treated (See the Humane Endpoint Guidance Document (9) for more details). Predictable death: presence of clinical signs indicative of death at a known time in the future before the planned end of the experiment; for example: inability to reach water or food. (See the Humane Endpoint Guidance Document (9) for more details).

1.3. PRINCIPLE OF THE TEST It is the principle of the test that, based on a stepwise procedure with the use of a minimum number of animals per step, sufficient information is obtained on the acute toxicity of the test substance to enable its classification. The substance is administered orally to a group of experimental animals at one of the defined doses. The substance is tested using a stepwise procedure, each step using three animals of a single sex (normally females). Absence or presence of compound-related mortality of the animals dosed at one step will determine the next step, i.e.; Details of the test procedure are described in Appendix 1. The method will enable a judgement with respect to classifying the test substance to one of a series of toxicity classes defined by fixed LD50 cut-off values.

no further testing is needed,

dosing of three additional animals, with the same dose,

dosing of three additional animals at the next higher or the next lower dose level.

1.4. DESCRIPTION OF THE METHOD

1.4.

1.4.1. Selection of animal species The preferred rodent species is the rat, although other rodent species may be used. Normally females are used (9). This is because literature surveys of conventional LD50 tests show that, although there is little difference in sensitivity between the sexes, in those cases where differences are observed females are generally slightly more sensitive (11). However if knowledge of the toxicological or toxicokinetic properties of structurally related chemicals indicates that males are likely to be more sensitive, then this sex should be used. When the test is conducted in males, adequate justification should be provided. Healthy young adult animals of commonly used laboratory strains should be employed. Females should be nulliparous and non-pregnant. Each animal, at the commencement of its dosing, should be between eight and 12 weeks old and its weight should fall in an interval within ± 20 % of the mean weight of any previously dosed animals.

1.4.2. Housing and feeding conditions The temperature in the experimental animal room should be 22 oC (± 3 oC). Although the relative humidity should be at least 30 % and preferably not exceed 70 % other than during room cleaning the aim should be 50-60 %. Lighting should be artificial, the sequence being 12 hours light, 12 hours dark. For feeding, conventional laboratory diets may be used with an unlimited supply of drinking water. Animals may be group-caged by dose, but the number of animals per cage must not interfere with clear observations of each animal.

1.4.3. Preparation of animals The animals are randomly selected, marked to permit individual identification, and kept in their cages for at least five days prior to dosing to allow for acclimatisation to the laboratory conditions.

1.4.4. Preparation of doses In general, test substances should be administered in a constant volume over the range of doses to be tested by varying the concentration of the dosing preparation. Where a liquid end product or mixture is to be tested however, the use of the undiluted test substance, i.e. at a constant concentration, may be more relevant to the subsequent risk assessment of that substance, and is a requirement of some regulatory authorities. In either case, the maximum dose volume for administration must not be exceeded. The maximum volume of liquid that can be administered at one time depends on the size of the test animal. In rodents, the volume should not normally exceed 1 ml/100 g of body weight: however in the case of aqueous solutions 2 ml/100 g body weight can be considered. With respect to the formulation of the dosing preparation, the use of an aqueous solution/suspension/emulsion is recommended wherever possible, followed in order of preference by a solution/suspension/emulsion in oil (e.g. corn oil) and then possibly solution in other vehicles. For vehicles other than water the toxicological characteristics of the vehicle should be known. Doses must be prepared shortly prior to administration unless the stability of the preparation over the period during which it will be used is known and shown to be acceptable.

1.5. PROCEDURE

1.5.

1.5.1. Administration of doses The test substance is administered in a single dose by gavage using a stomach tube or a suitable intubation canula. In the unusual circumstance that a single dose is not possible, the dose may be given in smaller fractions over a period not exceeding 24 hours. Animals should be fasted prior to dosing (e.g. with the rat, food but not water should be withheld overnight, with the mouse, food but not water should be withheld for three or four hours). Following the period of fasting, the animals should be weighed and the test substance administered. After the substance has been administered, food may be withheld for a further three or fours hours in rats or one or two hours in mice. Where a dose is administered in fractions over a period it may be necessary to provide the animals with food and water depending on the length of the period.

1.5.2. Number of animals and dose levels Three animals are used for each step. The dose level to be used as the starting dose is selected from one of four fixed levels, 5, 50, 300 and 2000 mg/kg body weight. The starting dose level should be that which is most likely to produce mortality in some of the dosed animals. The flowcharts of Appendix 1 describe the procedure that should be followed for each of the starting doses. In addition, Appendix 4 gives guidance on the classification in the EU system until the new GHS is implemented. When available information suggests that mortality is unlikely at the highest starting dose level (2000 mg/kg body weight), then a limit test should be conducted. When there is no information on a substance to be tested, for animal welfare reasons it is recommended to use the starting dose of 300 mg/kg body weight. The time interval between treatment groups is determined by the onset, duration, and severity of toxic signs. Treatment of animals at the next dose should be delayed until one is confident of survival of the previously dosed animals. Exceptionally, and only when justified by specific regulatory needs, the use of additional upper dose level of 5000 mg/kg body weight may be considered (see Appendix 2). For reasons of animal welfare concern, testing of animals in GHS Category 5 ranges (2000-5000 mg/kg) is discouraged and should only be considered when there is a strong likelihood that the results of such a test would have a direct relevance for protecting human or animal health or the environment.

1.5.3. Limit test The limit test is primarily used in situations where the experimenter has information indicating that the test material is likely to be non-toxic, i.e., having toxicity only above regulatory limit doses. Information about the toxicity of the test material can be gained from knowledge about similar tested compounds or similar tested mixtures or products, taking into consideration the identity and percentage of components known to be of toxicological significance. In those situations where there is little or no information about its toxicity, or in which the test material is expected to be toxic, the main test should be performed. A limit test at one dose level of 2000 mg/kg body weight may be carried out with six animals (three animals per step). Exceptionally a limit test at one dose level of 5000 mg/kg may be carried out with three animals (see Appendix 2). If test substance-related mortality is produced, further testing at the next lower level may need to be carried out.

1.6. OBSERVATIONS Animals are observed individually after dosing at least once during the first 30 minutes, periodically during the first 24 hours, with special attention given during the first four hours, and daily thereafter, for a total of 14 days, except where they need to be removed from the study and humanely killed for animal welfare reasons or are found dead. However, the duration of observation should not be fixed rigidly. It should be determined by the toxic reactions, time of onset and length of recovery period, and may thus be extended when considered necessary. The times at which signs of toxicity appear and disappear are important, especially if there is a tendency for toxic signs to be delayed (12). All observations are systematically recorded with individual records being maintained for each animal. Additional observations will be necessary if the animals continue to display signs of toxicity. Observations should include changes in skin and fur, eyes and mucous membranes, and also respiratory, circulatory, autonomic and central nervous systems, and somatomotor activity and behaviour pattern. Attention should be directed to observations of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma. The principles and criteria summarised in the Humane Endpoints Guidance Document (9) should be taken into consideration. Animals found in a moribund condition and animals showing severe pain or enduring signs of severe distress should be humanely killed. When animals are killed for humane reasons or found dead, the time of death should be recorded as precisely as possible.

1.6.

1.6.1. Body weight Individual weights of animals should be determined shortly before the test substance is administered, and at least weekly thereafter. Weight changes should be calculated and recorded. At the end of the test surviving animals are weighed and humanely killed.

1.6.2. Pathology All test animals (including those that die during the test or are removed from the study for animal welfare reasons) should be subjected to gross necropsy. All gross pathological changes should be recorded for each animal. Microscopic examination of organs showing evidence of gross pathology in animals surviving 24 or more hours may also be considered because it may yield useful information.

3. REPORTING

3.

3.1. Test report The test report must include the following information, as appropriate:

Test substance:

physical nature, purity, and, where relevant, physico-chemical properties (including isomerisation),

identification data, including CAS number.

Vehicle (if appropriate):

justification for choice of vehicle, if other than water.

Test animals:

species/strain used,

microbiological status of the animals, when known,

number, age, and sex of animals (including, where appropriate, a rationale for the use of males instead of females),

source, housing conditions, diet, etc.

Test conditions:

details of test substance formulation including details of the physical form of the material administered,

details of the administration of the test substance including dosing volumes and time of dosing,

details of food and water quality (including diet type/source, water source),

the rationale for the selection of the starting dose.

Results:

tabulation of response data and dose level for each animal (i.e. animals showing signs of toxicity including mortality; nature, severity, and duration of effects),

tabulation of body weight and body weight changes,

individual weights of animals at the day of dosing, in weekly intervals thereafter, and at the time of death or sacrifice,

date and time of death if prior to scheduled sacrifice,

time course of onset of signs of toxicity, and whether these were reversible for each animal,

necropsy findings and histopathological findings for each animal, if available.

Discussion and interpretation of results.

Conclusions.

4.(1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)

REFERENCES Roll R., Höfer-Bosse Th. And Kayser D (1986) New Perspectives in Acute Toxicity Testing of Chemicals. Toxicol. Lett., Suppl. 31, p. 86. Roll R., Riebschläger M., Mischke U. and Kayser D (1989) Neue Wege zur Bestimmung der akuten Toxizität von Chemikalien. Bundesgesundheitsblatt 32, p. 336-341. Diener W., Sichha L., Mischke U., Kayser D. and Schlede E (1994) The Biometric Evaluation of the Acute-Toxic-Class Method (Oral). Arch. Toxicol. 68, p. 559-610. Diener W., Mischke U., Kayser D. and Schlede E., (1995) The Biometric Evaluation of the OECD Modified Version of the Acute-Toxic-Class Method (Oral). Arch. Toxicol. 69, p. 729-734. Diener W., and Schlede E., (1999) Acute Toxicity Class Methods: Alterations to LD/LC50 Tests. ALTEX 16, p. 129-134. Schlede E., Mischke U., Roll R. and Kayser D., (1992). A National Validation Study of the Acute-Toxic- Class Method — An Alternative to the LD50 Test. Arch. Toxicol. 66, 455-470. Schlede E., Mischke U., Diener W. and Kayser D., (1994) The International Validation Study of the Acute-Toxic-Class Method (Oral). Arch. Toxicol. 69, p. 659-670. OECD, (2001) Guidance Document on Acute Oral Toxicity Testing. Environmental Health and Safety Monograph Series on Testing and Assessment No 24. Paris. OECD, (2000) Guidance Document on the Recognition, Assessment and Use of Clinical Signs as Humane Endpoints for Experimental Animals Used in Safety Evaluation. Environmental Health and Safety Monograph Series on Testing and Assessment No 19. OECD, (1998) Harmonised Integrated Hazard Classification System For Human Health And Environmental Effects Of Chemical Substances as endorsed by the 28th Joint Meeting of the Chemicals Committee and the Working Party on Chemicals in November 1998, Part 2, p. 11 [http://webnet1.oecd.org/oecd/pages/home/displaygeneral/0,3380, EN-documents-521-14-no-24-no-0,FF.html]. Lipnick R. L., Cotruvo, J.A., Hill R. N., Bruce R. D., Stitzel K. A., Walker A. P., Chu I.; Goddard M., Segal L., Springer J. A. and Myers R. C. (1995) Comparison of the Up-and Down, Conventional LD50, and Fixed Dose Acute Toxicity Procedures. Fd. Chem. Toxicol 33, p. 223-231. Chan P.K. and A.W. Hayes. (1994). Chap. 16. Acute Toxicity and Eye Irritancy. Principles and Methods of Toxicology. Third Edition. A.W. Hayes, Editor. Raven Press, Ltd., New York, USA.

B.2. ACUTE TOXICITY (INHALATION)

B.2.

1. METHOD

1.

1.1. INTRODUCTION It is useful to have preliminary information on the particle size distribution, the vapour pressure, the melting point, the boiling point, the flash point and explosivity (if applicable) of the substance. See also General introduction Part B (A).

1.2. DEFINITIONS See General introduction Part B (B).

1.3. REFERENCE SUBSTANCES None.

1.4. PRINCIPLE OF THE TEST METHOD Several groups of experimental animals are exposed for a defined period to the test substance in graduated concentrations, one concentration being used per group. Subsequently observations of effects and deaths are made. Animals, which die during the test are necropsied and at the conclusion of the test surviving animals are necropsied. Animals showing severe and enduring signs of distress and pain may need to be humanely killed. Dosing test substances in a way known to cause marked pain and distress due to corrosive or severe irritating properties need not be carried out.

1.5. QUALITY CRITERIA None.

1.6. DESCRIPTION OF THE TEST METHOD

1.6.

1.6.1. Preparations The animals are kept under the experimental housing and feeding conditions for at least five days prior to the experiment. Before the test healthy young animals are randomiseds and assigned to the required number of groups. They need not be subjected to simulated exposure unless this is indicated by the type of exposure apparatus being used. Solid test substances may need to be micronised in order to achieve particles of an appropriate size. Where necessary a suitable vehicle may be added to the test substance to help generate an appropriate concentration of the test substance in the atmosphere and a vehicle control group should then be used. If a vehicle or other additives are used to facilitate dosing, they should be known not to produce toxic effects. Historical data can be used if appropriate.

1.6.2. Test conditions

1.6.2.

1.6.2.1. Experimental animals Unless there are contra-indications the rat is the preferred species. Commonly used laboratory strains should be employed. For each sex, at the start of the test the range of weight variation in the animals used should not exceed ± 20 % of the appropriate mean value.

1.6.2.2. Number and sex At least 10 rodents (five female and five male) are used at each concentration level. The females should be nulliparous and non-pregnant. Note: in acute toxicity tests with animals of a higher order than rodents, the use of smaller numbers should be considered. Doses should be carefully selected, and every effort should be made not to exceed moderately toxic doses. In such tests administration of lethal doses of the test substance should be avoided.

1.6.2.3. Exposure concentrations These should be sufficient in number, at least three, and spaced appropriately to produce test groups with a range of toxic effects and mortality rates. The data should be sufficient to produce a concentration mortality curve and, where possible, permit an acceptable determination of an LC50.1.6.2.4. Limit test

1.6.2.4. Limit test If an exposure of five male and five female test animals to 20 mg per litre of a gas or 5 rug per litre of an aerosol or a particulate for four hours (or where this is not possible due to the physical or chemical, including explosive, properties of the test substance, the maximum attainable concentration) produces no compound related mortality within 14 days further testing may not be considered necessary (18th ATP, dir. 93/21/EEC, Ll10/93)

1.6.2.5. Exposure time The period of exposure should be four hours.

1.6.2.6. Equipment The animals should be tested with inhalation equipment designed to sustain a dynamic airflow of at least 12 air changes per hour, to ensure an adequate oxygen content and an evenly distributed exposure atmosphere. Where a chamber is used its design should minimise crowding of the test animals and maximise their exposure by inhalation to the test substance. As a general rule to ensure stability of a chamber atmosphere the total volume of the test animals should not exceed 5 % of the volume of the test chamber. Oro-nasal, head only, or whole body individual chamber exposure may be used; the first two will help to minimise the uptake of the test substance by other routes.

1.6.2.7. Observation period The observation period should be at least 14 days. However, the duration of observations should not be rigidly fixed. It should be determined by the toxic reactions, their rate of onset and the length of the recovery period; it may thus be extended when considered necessary. The time at which signs of toxicity appear and disappear and the time of death are important, especially if there is a tendency for deaths to be delayed.

1.6.3. Procedure Shortly before exposure, the animals are weighed, and then exposed to the test concentration in the designated apparatus for a period of four hours, after equilibration of the chamber concentration. Time for equilibration should be short. The temperature at which the test is performed should be maintained at 22 ± 3 oC. Ideally the relative humidity should be maintained between 30 % and 70 %, but in certain instances (e.g. tests of some aerosols) this may not be practicable. Maintenance of a slight negative pressure inside the chamber (≥ 5 mm of water) will prevent leakage of the test substance into the surrounding area. Food and water should be withheld during exposure. Suitable systems for the generation and monitoring of the test atmosphere should be used. The system should ensure that stable exposure conditions are achieved as rapidly as possible. The chamber should be designed and operated in such a way that a homogeneous distribution of the test atmosphere within the chamber is maintained. Measurements or monitoring should be made: During and following exposure, observations are made and recorded systematically; individual records should be maintained for each animal. Observations should be made frequently during the first day. A careful clinical examination should be made at least once each working day, other observations should be made daily with appropriate actions taken to minimise loss of animals from the study, e.g. necropsy or refrigeration of those animals found dead and isolation or sacrifice of weak or moribund animals. Observations should include changes in the skin and fur, eyes, mucous membranes, respiratory, circulatory, autonomic and central nervous systems, and somatomotor activity and behaviour pattern. Particular attention should be directed to observation of respiratory behaviour, tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma. The time of death should be recorded as precisely as possible. Individual weights of animals should be determined weekly after exposure, and at death. Animals that die during the test and those surviving at the termination of the test are subjected to necropsy with particular reference to any changes in the upper and lower respiratory tract. All gross pathological changes should be recorded. Where indicated, tissues should be taken for histopathological examination.

1.6.3.

(a) of the rate of air flow (continuously);

(b) of the actual concentration of the test substance measured in the breathing zone at least three times during exposure (some atmospheres, e.g. aerosols at high concentrations, may need more frequent monitoring). During the exposure period the concentration should not vary by more than ± 15 % of the mean value. However in the case of some aerosols, this level of control may not be achievable and a wider range would then be acceptable. For aerosols, particle size analysis should be performed as often as necessary (at least once per test group);

2. DATA Data should be summarised in tabular form showing for each test group the number of animals at the start of the test, time of death of individual animals, number of animals displaying other signs of toxicity, description of toxic effects and necropsy findings. Changes in weight must be calculated and recorded when survival exceeds one day. Animals, which are humanely killed due to compound-related distress and pain are recorded as compound-related deaths. The LC50 should be determined by a recognised method. Data evaluation should include the relationship, if any, between the animal's exposure to the test substance and the incidence and severity of all abnormalities, including behavioural and clinical abnormalities, gross lesions, body weight changes, mortality and any other toxic effects.

3. REPORTING

3.

3.1. TEST REPORT The test report shall, if possible, include the following information: Exposure data These should be tabulated and presented with mean values and a measure of variability (e.g. standard deviation) and shall, if possible, include:

species, strain, source, environmental conditions, diet, etc.,

test conditions: description of exposure apparatus, including design, type, dimensions, source of air, system for generating aerosols, method of conditioning air and the method of housing animals in a test chamber when this is used. The equipment for measuring temperature, humidity, and aerosol concentrations and particle size distribution should be described.

3.1.

(a) airflow rates through the inhalation equipment;

(b) temperature and humidity of the air;

(d) nature of vehicle, if used;

(e) actual concentrations in test breathing zone;

(f) The mass median aerodynamic diameter (Mmad) and the geometric standard deviation (GSD);

(g) equilibration period;

(h) exposure period;

tabulation of response data by sex and exposure level (i.e. number of animals that died or were killed during the test, number of animals showing signs of toxicity, number of animals exposed),

time of death during or following exposure, reasons and criteria used for humane killing of animals,

all observations,

LC50 value for each sex determined at the end of the observation period (with method of calculation specified),

95 % confidence interval for the LC50 (where this can be provided),

dose/mortality curve and slope (where permitted by the method of determination),

necropsy findings,

any histopathological findings,

discussions of the results (particular attention should be given to the effect that humane killing of animals during the test may have on the calculated LC50 value),

interpretation of the results.

3.2. EVALUATION AND INTERPRETATION See General introduction Part B (D).

4. REFERENCES See General introduction Part B (E).

B.3. ACUTE TOXICITY (DERMAL)

B.3.

1. METHOD

1.

1.1. INTRODUCTION See General introduction Part B (A).

1.2. DEFINITION See General introduction Part B (B).

1.3. REFERENCE SUBSTANCES None.

1.4. PRINCIPLE OF THE TEST METHOD The test substance is applied to the skin in graduated doses to several groups of experimental animals, one dose being used per group. Subsequently, observations of effects and deaths are made. Animals, which die during the test are necropsied and at the conclusion of the test surviving animals are necropsied. Animals showing severe and enduring signs of distress and pain may need to be humanely killed. Dosing test substances in a way known to cause marked pain and distress due to corrosive or irritating properties need not be carried out.

1.5. QUALITY CRITERIA None.

1.6. DESCRIPTION OF THE TEST METHOD

1.6.

1.6.1. Preparations The animals are kept in their experimental cages under the experimental housing and feeding conditions for at least five days prior to the experiment. Before the test, healthy young adult animals are randomised and assigned to the treatment groups. Approximately 24 hours before the test, fur should be removed by clipping or shaving from the dorsal area of the trunk of the animals. When clipping or shaving the fur, care must be taken to avoid abrading the skin which could alter its permeability. Not less than 10 % of the body surface should be clear for the application of the test substance. When testing solids, which may be pulverised if appropriate, the test substance should be moistened sufficiently with water or, where necessary, a suitable vehicle to ensure good contact with the skin. When a vehicle is used, the influence of the vehicle on penetration of skin by the test substance should be taken into account. Liquid test substances are generally used undiluted.

1.6.2. Test conditions

1.6.2.

1.6.2.1. Experimental animals The adult rat or rabbit may be used. Other species may be used but their use would require justification. Commonly used laboratory strains should be employed. For each sex, at the start of the test the range of weight variation in the animals used should not exceed ± 20 % of the appropriate mean value.

1.6.2.2. Number and sex At least five animals are used at each dose level. They should all be of the same sex. If females are used, they should be nulliparous and non-pregnant. Where information is available demonstrating that a sex is markedly more sensitive, animals of this sex should be dosed. Note: in acute toxicity tests with animals of a higher order than rodents, the use of smaller numbers should be considered. Doses should be carefully selected, and every effort should be made not to exceed moderately toxic doses. In such tests, administration of lethal doses of the test substance should be avoided.

1.6.2.3. Dose levels These should be sufficient in number, at least three, and spaced appropriately to produce test groups with a range of toxic effects and mortality rates. Any irritant or corrosive effects should be taken into account when deciding on dose levels. The data should be sufficient to produce a dose/response curve and, where possible, permit an acceptable determination of the LD50.

1.6.2.4. Limit test A limit test at one dose level of at least 2000 mg/kg bodyweight may be carried out in a group of five male and five female animals, using the procedures described above. If compound-related mortality is produced, a full study may need to be considered.

1.6.2.5. Observation period The observation period should be at least 14 days. However, the duration of observation should not be rigidly fixed. It should be determined by the toxic reactions, their rate of onset and the length of the recovery period; it may thus be extended when considered necessary. The time at which signs of toxicity appear and disappear, their duration and the time of death are important, especially if there is a tendency for deaths to be delayed.

1.6.3. Procedure Animals should be caged individually. The test substance should be applied uniformly over an area, which is approximately 10 % of the total body surface area. With highly toxic substances the surface area covered may be less but as much of the area should be covered with a layer as thin and uniform as possible. Test substances should be held in contact with the skin with a porous gauze dressing and non-irritating tape throughout a 24-hour exposure period. The test site should be further covered in a suitable manner to retain the gauze dressing and test substance and ensure that the animals cannot ingest the test substance. Restrainers may be used to prevent the ingestion of the test substance but complete immobilisation is not a recommended method. At the end of the exposure period, residual test substance should be removed, where practicable, using water or some other appropriate method of cleansing the skin. Observations should be recorded systematically as they are made. Individual records should be maintained for each animal. Observations should be made frequently during the first day. A careful clinical examination should be made at least once each working day, other observations should be made daily with appropriate actions taken to minimise loss of animals to the study, e.g. necropsy or refrigeration of those animals found dead and isolation or sacrifice of weak or moribund animals. Observations should include changes in fur, treated skin, eyes and mucous membranes, and also respiratory, circulatory, autonomic and central nervous systems, and somatomotor activity and behaviour pattern. Particular attention should be directed to observations of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma. The time of death must be recorded as precisely as possible. Animals that die during the test and those surviving at the termination of the test are subjected to necropsy. All gross pathological changes should be recorded. Where indicated, tissues should be taken for histopathological examination.

1.6.3. Assessment of toxicity in the other sex

After completion of the study in one sex, at least one group of five animals of the other sex is dosed to establish that animals of this sex are not markedly more sensitive to the test substance. The use of fewer animals may be justified in individual circumstances. Where adequate information is available to demonstrate that animals of the sex tested are markedly more sensitive, testing in animals of the other sex may be dispensed with.

2. DATA Data should be summarised in tabular form, showing for each test group the number of animals at the start of the test, time of death of individual animals, number of animals displaying other signs of toxicity, description of toxic effects and necropsy findings. Individual weights of animals should be determined and recorded shortly before the test substance is applied, weekly thereafter, and at death; changes in weight should be calculated and recorded when survival exceeds one day. Animals, which are humanely killed due to compound-related distress and pain are recorded as compound-related deaths. The LD50 should be determined by a recognised method. Data evaluation should include an evaluation of relationships, if any, between the animal's exposure to the test substance and the incidence and severity of all abnormalities, including behavioural and clinical abnormalities, gross lesions, body weight changes, mortality, and any other toxicological effects.

3. REPORTING

3.

3.1. TEST REPORT The test report shall, if possible, include the following information:

species, strain, source, environmental conditions, diet, etc.,

test conditions (including method of skin cleansing and type of dressing: occlusive or not occlusive),

dose levels (with vehicle, if used, and concentrations),

sex of animals dosed,

tabulation of response data by sex and dose level (i.e. number of animals that died or were killed during the test, number of animals showing signs of toxicity, number of animals exposed),

time of death after dosing, reasons and criteria used for humane killing of animals,

all observations,

LD50 value for the sex subjected to a full study, determined at 14 days with the method of determination specified,

95 % confidence interval for the LD50 (where this can be provided),

dose/mortality curve and slope where permitted by the method of determination,

necropsy findings,

any histopathological findings,

results of any test on the other sex,

discussion of results (particular attention should be given to the effect that humane killing of animals during the test may have on the calculated LD50 value),

interpretation of the results.

3.2. EVALUATION AND INTERPRETATION See General introduction Part B (D).

4. REFERENCES See General introduction Part B (E).

B.4. ACUTE TOXICITY: DERMAL IRRITATION/CORROSION

B.4.

1. METHOD This method is equivalent to the OECD TG 404 (2002).

1.

1.1. INTRODUCTION In the preparation of this updated method special attention was given to possible improvements in relation to animal welfare concerns and to the evaluation of all existing information on the test substance in order to avoid unnecessary testing in laboratory animals. This method includes the recommendation that prior to undertaking the described in vivo test for corrosion/irritation of the substance, a weight-of-the-evidence analysis be performed on the existing relevant data. Where insufficient data are available, they can be developed through application of sequential testing (1). The testing strategy includes the performance of validated and accepted in vitro tests and is provided as an Appendix to this method. In addition, where appropriate, the successive, instead of simultaneous, application of the three test patches to the animal in the initial in vivo test is recommended. In the interest of both sound science and animal welfare, in vivo testing should not be undertaken until all available data relevant to the potential dermal corrosivity/irritation of the substance have been evaluated in a weight-of-the-evidence analysis. Such data will include evidence from existing studies in humans and/or laboratory animals, evidence of corrosivity/irritation of one or more structurally related substances or mixtures of such substances, data demonstrating strong acidity or alkalinity of the substance (2)(3), and results from validated and accepted in vitro or ex vivo tests (4)(5)(5a). This analysis should decrease the need for in vivo testing for dermal corrosivity/irritation of substances for which sufficient evidence already exists from other studies as to those two endpoints. A preferred sequential testing strategy, which includes the performance of validated and accepted in vitro or ex vivo tests for corrosion/irritation, is included as an Appendix to this Method. The strategy was developed at, and unanimously recommended by the participants of, an OECD workshop (6), and has been adopted as the recommended testing strategy in the Globally Harmonised System for the Classification of Chemical Substances (GHS) (7). It is recommended that this testing strategy be followed prior to undertaking in vivo testing. For new substances it is the recommended a stepwise testing approach for developing scientifically sound data on the corrosivity/irritation of the substance. For existing substances with insufficient data on dermal corrosion/irritation, the strategy should be used to fill missing data gaps. The use of a different testing strategy or procedure, or a decision not to use a stepwise testing approach, should be justified. If a determination of corrosivity or irritation cannot be made using a weight-of-the-evidence analysis, consistent with the sequential testing strategy, an in vivo test should be considered (see Appendix).

1.2. DEFINITIONS Dermal irritation: is the production of reversible damage of the skin following the application of a test substance for up to four hours. Dermal corrosion: is the production of irreversible damage of the skin; namely, visible necrosis through the epidermis and into the dermis, following the application of a test substance for up to four hours. Corrosive reactions are typified by ulcers, bleeding, bloody scabs, and, by the end of observation at 14 days, by discoloration due to blanching of the skin, complete areas of alopecia, and scars. Histopathology should be considered to evaluate questionable lesions.

1.3. PRINCIPLE OF THE TEST METHOD The substance to be tested is applied in a single dose to the skin of an experimental animal; untreated skin areas of the test animal serve as the control. The degree of irritation/corrosion is read and scored at specified intervals and is further described in order to provide a complete evaluation of the effects. The duration of the study should be sufficient to evaluate the reversibility or irreversibility of the effects observed. Animals showing continuing signs of severe distress and/or pain at any stage of the test should be humanely killed, and the substance assessed accordingly. Criteria for making the decision to humanely kill moribund and severely suffering animals can be found in reference (8).

1.4. DESCRIPTION OF THE TEST METHOD

1.4.

1.4.1. Preparation for the in vivo test

1.4.1.

1.4.1.1. Selection of animal species The albino rabbit is the preferable laboratory animal and healthy young adult rabbits are used. A rationale for using other species should be provided.

1.4.1.2. Preparation of the animals Approximately 24 hours before the test, fur should be removed by closely clipping the dorsal area of the trunk of the animals. Care should be taken to avoid abrading the skin, and only animals with healthy, intact skin should be used. Some strains of rabbit have dense patches of hair that are more prominent at certain times of the year. Such areas of dense hair growth should not be used as test sites.

1.4.1.3. Housing and feeding conditions Animals should be individually housed. The temperature of the experimental animal room should be 20 oC (± 3 oC) for rabbits. Although the relative humidity should be at least 30 % and preferably not exceed 70 %, other than during room cleaning, the aim should be 50-60 %. Lighting should be artificial, the sequence being 12 hours light, 12 hours dark. For feeding, conventional laboratory diets may be used with an unrestricted supply of drinking water.

1.4.2. Test procedure

1.4.2.

1.4.2.1. Application of the test substance The test substance should be applied to a small area (approximately 6 cm2) of skin and covered with a gauze patch, which is held in place with non-irritating tape. In cases in which direct application is not possible (e.g. liquids or some pastes), the test substance should first be applied to the gauze patch, which is then applied to the skin. The patch should be loosely held in contact with the skin by means of a suitable semi-occlusive dressing for the duration of the exposure period. If the test substance is applied to the patch, it should be attached to the skin in such a manner that there is good contact and uniform distribution of the substance on the skin. Access by the animal to the patch and ingestion or inhalation of the test substance should be prevented. Liquid test substances are generally used undiluted. When testing solids (which may be pulverised, if considered necessary), the test substance should be moistened with the smallest amount of water (or, where necessary, of another suitable vehicle) sufficient to ensure good skin contact. When vehicles other than water are used, the potential influence of the vehicle on irritation of the skin by the test substance should be minimal, if any. At the end of the exposure period, which is normally four hours, residual test substance should be removed, where practicable, using water or an appropriate solvent without altering the existing response or the integrity of the epidermis.

1.4.2.2. Dose level A dose of 0,5 ml. of liquid or 0,5 g of solid or paste is applied to the test site.

1.4.2.3. Initial test (in vivo dermal irritation/corrosion test using one animal) It is strongly recommended that the in vivo test be performed initially using one animal, especially when the substance is suspected to have corrosion potential. This is in accordance with the sequential testing strategy (see Appendix 1). When a substance has been judged to be corrosive on the basis of a weight-of-the-evidence analysis, no further animal testing is needed. For most substances suspected of being corrosive, further in vivo testing is normally not necessary. However, in those cases where additional data are felt warranted because of insufficient evidence, limited animal testing may be carried out using the following approach: up to three tests patches are applied sequentially to the animal. The first patch is removed after three minutes. If no serious skin reaction is observed, a second patch is applied and removed after one hour. If the observations at this stage indicate that exposure can humanely be allowed to extend to four hours, a third patch is applied and removed after four hours, and the response is graded. If a corrosive effect is observed after any of the three sequential exposures, the test is immediately terminated. If a corrosive effect is not observed after the last patch is removed, the animal is observed for 14 days, unless corrosion develops at an earlier time point. In those cases in which the test substance is not expected to produce corrosion but may be irritating, a single patch should be applied to one animal for four hours.

1.4.2.4. Confirmatory test (in vivo dermal irritation test with additional animals) If a corrosive effect is not observed in the initial test, the irritant or negative response should be confirmed using up to two additional animals, each with one patch, for an exposure period of four hours. If an irritant effect is observed in the initial test, the confirmatory test may be conducted in a sequential manner, or by exposing two additional animals simultaneously. In the exceptional case, in which the initial test is not conducted, two or three animals may be treated with a single patch, which is removed after four hours. When two animals are used, if both exhibit the same response, no further testing is needed. Otherwise, the third animal is also tested. Equivocal responses may need to be evaluated using additional animals.

1.4.2.5. Observation period The duration of the observation period should be sufficient to evaluate fully the reversibility of the effects observed. However, the experiment should be terminated at any time that the animal shows continuing signs of severe pain or distress. To determine the reversibility of effects, the animals should be observed up to 14 days after removal of the patches. If reversibility is seen before 14 days, the experiment should be terminated at that time.

1.4.2.6. Clinical observations and grading of skin reactions All animals should be examined for signs of erythema and oedema, and the responses scored at 60 minutes, and then at 24, 48 and 72 hours after patch removal. For the initial test in one animal, the test site is also examined immediately after the patch has been removed. Dermal reactions are graded and recorded according to the grades in the Table below. If there is damage to skin which cannot be identified as irritation or corrosion at 72 hours, observations may be needed until day 14 to determine the reversibility of the effects. In addition to the observation of irritation, all local toxic effects, such as defatting of the skin, and any systemic adverse effects (e.g. effects on clinical signs of toxicity and body weight), should be fully described and recorded. Histopathological examination should be considered to clarify equivocal responses. The grading of skin responses is necessarily subjective. To promote harmonisation in grading of skin response and to assist testing laboratories and those involved in making and interpreting the observations, the personnel performing the observations need to be adequately trained in the scoring system used (see Table below). An illustrated guide for grading skin irritation and other lesions could be helpful (9).

2. DATA

2.

2.1. PRESENTATION OF RESULTS Study results should be summarised in tabular form in the final test report and should cover all items listed in section 3.1.

2.2. EVALUATION OF RESULTS The dermal irritation scores should be evaluated in conjunction with the nature and severity of lesions, and their reversibility or lack of reversibility. The individual scores do not represent an absolute standard for the irritant properties of a material, as other effects of the test material are also evaluated. Instead, individual scores should be viewed as reference values, which need to be evaluated in combination with all other observations from the study. Reversibility of dermal lesions should be considered in evaluating irritant responses. When responses such as alopecia (limited area), hyperkeratosis, hyperplasia and scaling, persist to the end of the 14-day observation period, the test substance should be considered an irritant.

3. REPORTING

3.

3.1. TEST REPORT The test report must include the following information:

Rationale for in vivo testing: weight-of-evidence analysis of pre-existing test data, including results from sequential testing strategy:

description of relevant data available from prior testing,

data derived at each stage of testing strategy,

description of in vitro tests performed, including details of procedures, results obtained with test/reference substances,

weight-of-the-evidence analysis for performing in vivo study.

Test substance:

identification data (e.g. CAS number, source, purity, known impurities, lot number),

physical nature and physicochemical properties (e.g. pH, volatility, solubility, stability),

if mixture, composition and relative percentages of components.

Vehicle:

identification, concentration (where appropriate), volume used,

justification for choice of vehicle.

Test animals:

species/strain used, rationale for using animals other than albino rabbit,

number of animals of each sex,

individual animal weights at start and conclusion of test,

age at start of study,

source of animals, housing conditions, diet, etc.

Test conditions:

technique of patch site preparation,

details of patch materials used and patching technique,

details of test substance preparation, application, and removal.

Results:

tabulation of irritation/corrosion response scores for each animal at all time points measured,

descriptions of all lesions observed,

narrative description of nature and degree of irritation or corrosion observed, and any histopathological findings,

description of other adverse local (e.g. defatting of skin) and systemic effects in addition to dermal irritation or corrosion.

Discussion of results

4.(1)(2)(3)(4)(5)(5a)(6)(7)(8)(9)

REFERENCES Barratt, M.D., Castell, J.V., Chamberlain, M., Combes, R.D., Dearden, J.C., Fentem, J.H., Gerner, I., Giuliani, A., Gray, T.J.B., Livingston, D.J., Provan, W.M., Rutten, F.A.J.J.L., Verhaar, H.J.M., Zbinden, P. (1995) The Integrated Use of Alternative Approaches for Predicting Toxic Hazard. ECVAM Workshop Report 8. ATLA 23, p. 410-429. Young, J.R., How, M.J., Walker, A.P., Worth W.M.H. (1988) Classification as Corrosive or Irritant to Skin of Preparations Containing Acidic or Alkaline Substance Without Testing on Animals. Toxicollogy In Vitro, 2, p. 19-26. Worth, A.P., Fentem, J.H., Balls, M., Botham, P.A., Curren, R.D., Earl, L.K., Esdaile, D.J., Liebsch, M. (1998) Evaluation of the proposed OECD Testing Strategy for skin corrosion. ATLA 26, p. 709-720. ECETOC (1990) Monograph No 15, Skin Irritation, European Chemical Industry, Ecology and Toxicology Centre, Brussels. Fentem, J.H., Archer, G.E.B., Balls, M., Botham, P.A., Curren, R.D., Earl, L.K., Edsail, D.J., Holzhutter, H.G. and Liebsch, M. (1998) The ECVAM international validation study on in vitro tests for skin corrosivity. 2. Results and evaluation by the Management Team. Toxicology In Vitro 12, p. 483-524. Testing Method B.40 Skin Corrosion. OECD (1996) OECD Test Guidelines Programme: Final Report of the OECD Workshop on Harmonisation of Validation and Acceptance Criteria for Alternative Toxicological Test Methods. Held in Solna, Sweden, 22-24 January 1996 (http://www.oecd1.org/ehs/test/background.htm). OECD (1998) Harmonised Integrated Hazard Classification System for Human Health and Environmental Effects of Chemical Substances, as endorsed by the 28th Joint Meeting of the Chemicals Committee and the Working Party on Chemicals, November 1998 (http://www.oecd1.org/ehs/Class/HCL6.htm). OECD (2000). Guidance Document on the Recognition, Assessment and Use of Clinical Signs as Humane Endpoints for Experimental Animals Used in Safety Evaluation. OECD Environmental Health and Safety Publications. Series on Testing and Assessment No 19 (http://www.oecd1.org/ehs/test/monos.htm). EPA (1990). Atlas of Dermal Lesions, (20T-2004). United States Environmental Protection Agency, Office of Pesticides and Toxic Substances, Washington, DC, August 1990. [Available from OECD Secretariat upon request].

Table I Erythema and Eschar formation Oedema formation GRADING OF SKIN REACTIONS

No erythema … 0 Very slight erythema (barely perceptible) … 1 Well defined erythema … 2 Moderate to severe erythema … 3 Severe erythema (beef redness) to eschar formation preventing grading of erythema … 4 Maximum possible: 4 No oedema … 0 Very slight oedema (barely perceptible) … 1 Slight oedema (edges of area well defined by definite raising) … 2 Moderate oedema (raised approximately 1 mm) … 3 Severe oedema (raised more than 1 mm and extending beyond area of exposure) … 4 Maximum possible: 4 Histopathological examination may be carried out to clarify equivocal responses.

B.5. ACUTE TOXICITY: EYE IRRITATION/CORROSION

B.5.

1. METHOD This method is equivalent to the OECD TG 405 (2002)

1.

1.1. INTRODUCTION In the preparation of this updated method special attention was given to possible improvements through the evaluation of all existing information on the test substance in order to avoid unnecessary testing in laboratory animals and thereby address animal welfare concerns. This method includes the recommendation that prior to undertaking the described in vivo test for acute eye irritation/corrosion, a weight-of-the-evidence analysis be performed (1) on the existing relevant data. Where insufficient data are available, it is recommended that they be developed through application of sequential testing (2)(3). The testing strategy includes the performance of validated and accepted in vitro tests and is provided as an Appendix to the testing method. In addition, the use of an in vivo dermal irritation/corrosion test to predict eye corrosion prior to consideration of an in vivo eye test is recommended. In the interest of both sound science and animal welfare, in vivo testing should not be considered until all available data relevant to the potential eye corrosivity/irritation of the substance has been evaluated in a weight-of-the-evidence analysis. Such data will include evidence from existing studies in humans and/or laboratory animals, evidence of corrosivity/irritation of one or more structurally related substances or mixtures of such substances, data demonstrating high acidity or alkalinity of the substance (4)(5), and results from validated and accepted in vitro or ex vivo tests for skin corrosion and irritation (6)(6a). The studies may have been conducted prior to, or as a result of, a weight-of-the-evidence analysis. For certain substances, such an analysis may indicate the need for in vivo studies of the ocular corrosion/irritation potential of the substance. In all such cases, before considering the use of the in vivo eye test, preferably a study of the in vivo dermal effects of the substance should be conducted first and evaluated in accordance with testing method B.4 (7). The application of a weight-of-the-evidence analysis and the sequential testing strategy should decrease the need for in vivo testing for eye corrosivity/irritation of substances for which sufficient evidence already exists from other studies. If a determination of eye corrosion or irritation potential cannot be made using the sequential testing strategy, even after the performance of an in vivo study of dermal corrosion and irritation, an in vivo eye corrosion/irritation test may be performed. A preferred sequential testing strategy, which includes the performance of validated in vitro or ex vivo tests for corrosion/irritation, is included in the Appendix to this testing method. The strategy was developed at, and unanimously recommended by the participants of, an OECD workshop (8), and has been adopted as the recommended testing strategy in the Globally Harmonised System for the Classification of Chemical Substances (GHS) (9). It is recommended that this testing strategy be followed prior to undertaking in vivo testing. For new substances it is the recommended stepwise testing approach for developing scientifically sound data on the corrosivity/irritation of the substance. For existing substances with insufficient data on skin and eye corrosion/irritation, the strategy should be used to fill missing data gaps. The use of a different testing strategy or procedure, or the decision not to use a stepwise testing approach, should be justified.

1.2. DEFINITIONS Eye irritation: is the production of changes in the eye following the application of a test substance to the anterior surface of the eye, which are fully reversible within 21 days of application. Eye corrosion: is the production of tissue damage in the eye, or serious physical decay of vision, following application of a test substance to the anterior surface of the eye, which is not fully reversible within 21 days of application.

1.3. PRINCIPLE OF THE TEST METHOD The substance to be tested is applied in a single dose to one of the eyes of the experimental animal; the untreated eye serves as the control. The degree of eye irritation/corrosion is evaluated by scoring lesions of conjunctiva, cornea, and iris, at specific intervals. Other effects in the eye and adverse systemic effects are also described to provide a complete evaluation of the effects. The duration of the study should be sufficient to evaluate the reversibility or irreversibility of the effects. Animals showing continuing signs of severe distress and/or pain at any stage of the test should be humanely killed, and the substance assessed accordingly. Criteria for making the decision to humanely kill moribund and severely suffering animals can be found in reference (10).

1.4. DESCRIPTION OF THE TEST METHOD

1.4.

1.4.1. Preparation for the in vivo test

1.4.1.

1.4.1.1. Selection of species The albino rabbit is the preferable laboratory animal, and healthy young adult animals are used. A rationale for using other strains or species should be provided.

1.4.1.2. Preparation of animals Both eyes of each experimental animal provisionally selected for testing should be examined within 24 hours before testing starts. Animals showing eye irritation, ocular defects, or pre-existing corneal injury should not be used.

1.4.2. Test procedure

1.4.2.

1.4.2.1. Application of the test substance The test substance should be placed in the conjunctival sac of one eye of each animal after gently pulling the lower lid away from the eyeball. The lids are then gently held together for about one second in order to prevent loss of the material. The other eye, which remains untreated, serves as a control.

1.4.2.2. Irrigation The eyes of the test animals should not be washed for at least 24 hours following instillation of the test substance, except for solids (see Section 1.4.2.3.2), and in case of immediate corrosive or irritating effects. At 24 hours a washout may be used if considered appropriate. Use of a satellite group of animals to investigate the influence of washing is not recommended unless it is scientifically justified. If a satellite group is needed, two rabbits should be used. Conditions of washing should be carefully documented, e.g. time of washing; composition and temperature of wash solution; duration, volume, and velocity of application.

1.4.2.3. Dose level

1.4.2.3.

1.4.2.3.1. Testing of liquids For testing liquids, a dose of 0,1 ml is used. Pump sprays should not be used for instilling the substance directly into the eye. The liquid spray should be expelled and collected in a container prior to instilling 0,1 ml into the eye.

1.4.2.3.2. Testing of solids When testing solids, pastes, and particulate substances, the amount used should have a volume of 0,1 ml or a weight of not more than 100 mg. The test material should be ground to a fine dust. The volume of solid material should be measured after gently compacting it, e.g. by tapping the measuring container. If the solid test substance has not been removed from the eye of the test animal by physiological mechanisms at the first observation time point of one hour after treatment, the eye may be rinsed with saline or distilled water.

1.4.2.3.3. Testing of aerosols It is recommended that all pump sprays and aerosols be collected prior to instillation into the eye. The one exception is for substances in pressurised aerosol containers, which cannot be collected due to vaporisation. In such cases, the eye should be held open, and the test substance administered to the eye in a simple burst of about one second, from a distance of 10 cm directly in front of the eye. This distance may vary depending on the pressure of the spray and its contents. Care should be taken not to damage the eye from the pressure of the spray. In appropriate cases, there may be a need to evaluate the potential for mechanical damage to the eye from the force of the spray. An estimate of the dose from an aerosol can be made by simulating the test as follows: the substance is sprayed on to weighing paper through an opening the size of a rabbit eye placed directly before the paper. The weight increase of the paper is used to approximate the amount sprayed into the eye. For volatile substances, the dose may be estimated by weighing a receiving container before and after removal of the test material.

1.4.2.4. Initial test (in vivo eye irritation/corrosion test using one animal) As articulated in the sequential testing strategy (see Appendix 1), it is strongly recommended that the in vivo test be performed initially using one animal. If the results of this test indicate the substance to be corrosive or a severe irritant to the eye using the procedure described, further testing for ocular irritancy should not be performed.

1.4.2.5. Local anaesthetics Local anaesthetics may be used on a case-by-case basis. If the weight-of-the-evidence analysis indicates that the substance has the potential to cause pain, or initial testing shows that a painful reaction will occur, a local anaesthetic may be used prior to instillation of the test substance. The type, concentration, and dose of the local anaesthetic should be carefully selected to ensure that differences in reaction to the test substance will not result from its use. The control eye should be similarly anaesthetised.

1.4.2.6. Confirmatory test (in vivo eye irritation test with additional animals) If a corrosive effect is not observed in the initial test, the irritant or negative response should be confirmed using up to two additional animals. If a severe irritant effect is observed in the initial test indicating a possible strong (irreversible) effect in the confirmatory testing, it is recommended that the confirmatory test be conducted in a sequential manner in one animal at a time, rather than exposing the two additional animals simultaneously. If the second animal reveals corrosive or severe irritant effects, the test is not continued. Additional animals may be needed to confirm weak or moderate irritant responses.

1.4.2.7. Observation period The duration of the observation period should be sufficient to evaluate fully the magnitude and reversibility of the effects observed. However, the experiment should be terminated at any time that the animal shows continuing signs of severe pain or distress (9). To determine reversibility of effects, the animals should be observed normally for 21 days post administration of the test substance. If reversibility is seen before 21 days, the experiment should be terminated at that time.

1.4.2.7.

1.4.2.7.1. Clinical observations and grading of eye reactions The eyes should be examined at one, 24, 48, and 72 hours after test substance application. Animals should be kept on test no longer than necessary once definitive information has been obtained. Animals showing continuing severe pain or distress should be humanely killed without delay, and the substance assessed accordingly. Animals with the following eye lesions post-instillation should be humanely killed: corneal perforation or significant corneal ulceration including staphyloma; blood in the anterior chamber of the eye; grade 4 corneal opacity which persists for 48 hours; absence of a light reflex (iridial response grade 2) which persists for 72 hours; ulceration of the conjunctival membrane; necrosis of the conjuctivae or nictitating membrane; or sloughing. This is because such lesions generally are not reversible Animals that do not develop ocular lesions may be terminated not earlier than three days post instillation. Animals with mild to moderate lesions should be observed until the lesions clear, or for 21 days, at which time the study is terminated. Observations should be performed at seven, 14, and 21 days in order to determine the status of the lesions, and their reversibility or irreversibility. The grades of ocular reaction (conjunctivae, cornea and iris) should be recorded at each examination (Table I). Any other lesions in the eye (e.g. pannus, staining) or adverse systemic effects should also be reported. Examination of reactions can be facilitated by use of a binocular loupe, hand slit-lamp, biomicroscope, or other suitable device. After recording the observations at 24 hours, the eyes may be further examined with the aid of fluorescein. The grading of ocular responses is necessarily subjective. To promote harmonisation of grading of ocular response and to assist testing laboratories and those involved in making and interpreting the observations, the personnel performing the observations need to be adequately trained in the scoring system used.

2. DATA

2.

2.2. EVALUATION OF RESULTS The ocular irritation scores should be evaluated in conjunction with the nature and severity of lesions, and their reversibility or lack of reversibility. The individual scores do not represent an absolute standard for the irritant properties of a material, as other effects of the test material are also evaluated. Instead, individual scores should be viewed as reference values and are only meaningful when supported by a full description and evaluation of all observations.

3. REPORTING

3.

3.1. TEST REPORT The test report must include the following information:

Rationale for in vivo testing: weight-of-the-evidence analysis of pre-existing test data, including results from sequential testing strategy

description of relevant data available from prior testing,

data derived in each step of testing strategy,

description of in vitro tests performed, including details of procedures, results obtained with test/reference substances,

description of in vivo dermal irritation/corrosion study performed, including results obtained,

weight-of-the-evidence analysis for performing in vivo study.

Test substance:

identification data (e.g. CAS number, source, purity, known impurities, lot number),

physical nature and physicochemical properties (e.g. pH, volatility, solubility, stability, reactivity with water),

in case of a mixture, composition and relative percentages of components,

if local anaesthetic is used, identification, purity, type, dose, and potential interaction with test substance.

Vehicle:

identification, concentration (where appropriate), volume used,

justification for choice of vehicle.

Test animals:

species/strain used, rationale for using animals other than albino rabbit,

age of each animal at start of study,

number of animals of each sex in test and control groups (if required),

individual animal weights at start and conclusion of test,

source, housing conditions, diet, etc.

Results:

description of method used to score irritation at each observation time (e.g. hand slitlamp, biomicroscope, fluorescein),

tabulation of irritant/corrosive response data for each animal at each observation time up to removal of each animal from the test,

narrative description of the degree and nature of irritation or corrosion observed,

description of any other lesions observed in the eye (e.g. vascularisation, pannus formation, adhesions, staining),

description of non-ocular local and systemic adverse effects, and histopathological findings, if any.

Discussion of results.

3.2. INTERPRETATION OF THE RESULTS Extrapolation of the results of eye irritation studies in laboratory animals to humans is valid only to a limited degree. In many cases the albino rabbit is more sensitive than humans to ocular irritants or corrosives. Care should be taken in the interpretation of data to exclude irritation resulting from secondary infection.

4.(1)(2)(3)(4)(5)(6)(6a)(7)(8)(9)(10)

REFERENCES Barratt, M.D., Castell, J.V., Chamberlain, M., Combes, R.D., Dearden, J.C., Fentem, J.H., Gerner, I., Giuliani, A., Gray, T.J.B., Livingston, D.J., Provan, W.M., Rutten, F.A.J.J.L., Verhaar, H.J.M., Zbinden, P. (1995) The Integrated Use of Alternative Approaches for Predicting Toxic Hazard. ECVAM Workshop Report 8. ATLA 23, p. 410-429. de Silva, O., Cottin, M., Dami, N., Roguet, R., Catroux, P., Toufic, A., Sicard, C., Dossou, K.G., Gerner, I., Schlede, E., Spielmann, H., Gupta, K.C., Hill, R.N., (1997) Evaluation of Eye Irritation Potential: Statistical Analysis and Tier Testing Strategies. Food Chem. Toxicol 35, p. 159-164. Worth A.P. and Fentem J.H., (1999) A general approach for evaluating stepwise testing strategies ATLA 27, p. 161-177 Young, J.R., How, M.J., Walker, A.P., Worth W.M.H., (1988) Classification as Corrosive or Irritant to Skin of Preparations Containing Acidic or Alkaline Substance Without Testing on Animals. Toxicollogy In Vitro, 2, p. 19-26. Neun, D.J. (1993) Effects of Alkalinity on the Eye Irritation Potential of Solutions Prepared at a Single pH. J. Toxicol. Cut. Ocular Toxicol. 12, p. 227-231. Fentem, J.H., Archer, G.E.B., Balls, M., Botham, P.A., Curren, R.D., Earl, L.K., Edsaile, D.J., Holzhutter, H.G. and Liebsch, M. (1998) The ECVAM international validation study on in vitro tests for skin corrosivity. 2. Results and evaluation by the Management Team. Toxicology In Vitro 12, p. 483-524. Testing Method B.40 Skin Corrosion. Testing method B.4. Acute toxicity: dermal irritation/corrosion. OECD, (1996) OECD Test Guidelines Programme: Final Report of the OECD Workshop on Harmonisation of Validation and Acceptance Criteria for Alternative Toxicological Test Methods. Held in Solna, Sweden, 22-24 January 1996 (http://www.oecd.org/ehs/test/background.htm). OECD, (1998) Harmonised Integrated Hazard Classification System for Human Health and Environmental Effects of Chemical Substances, as endorsed by the 28th Joint Meeting of the Chemicals Committee and the Working Party on Chemicals, November 1998 (http://www.oecd.org/ehs/Class/HCL6.htm). OECD, (2000) Guidance Document on the Recognition, Assessment and Use of Clinical Signs as Humane Endpoints for Experimental Animals Used in Safety Evaluation. OECD Environmental Health and Safety Publications. Series on Testing and Assessment No 19 (http://www.oecd.org/ehs/test/monos.htm).

Table I GRADING OF OCULAR LESIONS

Cornea

Opacity: degree of density (readings should be taken from most dense area) No ulceration or opacity … 0 Scattered or diffuse areas of opacity (other than slight dulling of normal lustre); details of iris clearly visible … 1 Easily discernible translucent area; details of iris slightly obscured … 2 Nacrous area; no details of iris visible; size of pupil barely discernible … 3 Opaque cornea; iris not discernible through the opacity … 4 Maximum possible: 4 NOTES

Iris

Normal … 0 Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperaemia; or injection; iris reactive to light (a sluggish reaction is considered to be an effect) … 1 Hemorrhage, gross destruction, or no reaction to light … 2 Maximum possible: 2

Conjunctivae

Redness (refers to palpebral and bulbar conjunctivae; excluding cornea and iris) Normal … 0 Some blood vessels hyperaemic (injected) … 1 Diffuse, crimson colour; individual vessels not easily discernible … 2 Diffuse beefy red … 3 Maximum possible: 3

Chemosis

Swelling (refers to lids and/or nictating membranes) Normal … 0 Some swelling above normal … 1 Obvious swelling, with partial eversion of lids … 2 Swelling, with lids about half closed … 3 Swelling, with lids more than half closed … 4 Maximum possible: 4

B.6. SKIN SENSITISATION

B.6.

1. METHOD

1.

1.1. INTRODUCTION Remarks: The sensitivity and ability of tests to detect potential human skin sensitisers are considered important in a classification system for toxicity relevant to public health. There is no single test method which will adequately identify all substances with a potential for sensitising human skin and which is relevant for all substances. Factors such as the physical characteristics of a substance, including its ability to penetrate the skin, must be considered in the selection of a test. Two types of tests using guinea pigs have been developed: the adjuvant-type tests, in which an allergic state is potentiated by dissolving or suspending the test substance in Freunds Complete Adjuvant (FCA), and the non-adjuvant tests. Adjuvant-type tests are likely to be more accurate in predicting a probable skin sensitising effect of a substance in humans than those methods not employing Freunds Complete Adjuvant and are thus the preferred methods. The Guinea-Pig Maximisation Test (Gpmt) is a widely used adjuvant-type test. Although several other methods can be used to detect the potential of a substance to provoke skin sensitisation reaction, the Gpmt is considered to be the preferred adjuvant technique. With many chemical classes, non-adjuvant tests (the preferred one being the Buehler test) are considered to be less sensitive. In certain cases there may be good reasons for choosing the Buehler test involving topical application rather than the intradermal injection used in the Guinea-Pig Maximisation Test. Scientific justification should be given when the Buehler test is used. The Guinea-Pig Maximisation Test (Gpmt) and the Buehler test are described in this method. Other methods may be used provided that they are well-validated and scientific justification is given. If a positive result is seen in a recognised screening test, a test substance may be designated as a potential sensitiser, and it may not be necessary to conduct a further guinea pig test. However, if a negative result is seen in such a test, the guinea pig test must be conducted using the procedure described in this tes method. See also General introduction Part B.

1.2. DEFINITIONS Skin sensitisation: (allergic contact dermatitis) is an immunologically mediated cutaneous reaction to a substance. In the human, the responses may be characterised by pruritis, erythema, oedema, papules, vesicles, bullae or a combination of these. In other species the reactions may differ and only erythema and oedema may be seen. Induction exposure: an experimental exposure of a subject to a test substance with the intention of inducing a hypersensitive state. Induction period: a period of at least one week following an induction exposure during which a hypersensitive state may be developed. Challenge exposure: an experimental exposure of a previously treated subject to a test substance following an induction period, to determine if the subject reacts in a hypersensitive manner.

1.3. REFERENCE SUBSTANCES The sensitivity and reliability of the experimental technique used should be assessed every six months by use of substances, which are known to have mild-to-moderate skin sensitisation properties. In a properly conducted test, a response of at least 30 % in an adjuvant test and at least 15 % in a non-adjuvant test should be expected for mild/moderate sensitisers. The following substances are preferred. CAS numbers EINECS numbers EINECS names Common names 101-86-0 202-983-3 α-hexylcinnamaldehyde α-hexylcinnamaldehyde 149-30-4 205-736-8 Benzothiazole-2-thiol (mercaptobenzothiazole) kaptax 94-09-7 202-303-5 Benzocaine norcaine There may be circumstances where, given adequate justification other control substances meeting the above criteria may be used.

1.4. PRINCIPLE OF THE TEST METHOD The test animals are initially exposed to the test substance by intradermal injections and/or epidermal application (induction exposure). Following a rest period of 10 to 14 days (induction period), during which an immune response may develop, the animals are exposed to a challenge dose. The extent and degree of skin reaction to the challenge exposure in the test animals is compared with that demonstrated by control animals which undergo sham treatment during induction and receive the challenge exposure.

1.5. DESCRIPTION OF THE TEST METHODS If removal of the test substance is considered necessary, this should be achieved using water or an appropriate solvent without altering the existing response or the integrity of the epidermis.

1.5.

1.5.1. Guinea-Pig Maximisation Test (GPMT)

1.5.1.

1.5.1.1. Preparations Healthy young adult albino guinea pigs are acclimatised to the laboratory conditions for at least five days prior to the test. Before the test, animals are randomised and assigned to the treatment groups. Removal of hair is by clipping, shaving or possibly by chemical depilation, depending on the test method used. Care should be taken to avoid abrading the skin. The animals are weighed before the test commences and at the end of the test.

1.5.1.2. Test conditions

1.5.1.2.

1.5.1.2.1. Test animals Commonly used laboratory strains of albino guinea-pigs are used.

1.5.1.2.2. Number and sex Male and/or female animals can be used. If females are used, they should be nulliparous and non-pregnant. A minimum of 10 animals is used in the treatment group and at least five animals in the control group. When fewer than 20 test and 10 control guinea pigs have been used, and it is not possible to conclude that the test substance is a sensitiser, testing in additional animals to give a total of at least 20 test and 10 control animals is strongly recommended.

1.5.1.2.3. Dose levels The concentration of the test substance used for each induction exposure should be well-tolerated systemically and should be the highest to cause mild-to-moderate skin irritation. The concentration used for the challenge exposure should be the highest non-irritant dose. The appropriate concentrations should be determined from a pilot study using two or three animals, if other information is not available. Consideration should be given to the use of FCA-treated animals for this purpose.

1.5.1.3. Procedure

1.5.1.3.

1.5.1.3.1. Induction Day 0-treated group Three pairs of intradermal injections of 0,1 ml volume are given in the shoulder region which is cleared of hair so that one of each pair lies on each side of the midline. Injection 1: a 1:1 mixture (v/v) FCA/water or physiological saline. Injection 2: the test substance in an appropriate vehicle at the selected concentration. Injection 3: the test substance at the selected concentration formulated in a 1:1 mixture (v/v) FCA/water or physiological saline. In injection 3, water soluble substances are dissolved in the aqueous phase prior to mixing with FCA. Liposoluble or insoluble substances are suspended in FCA prior to combining with the aqueous phase. The final concentration of test substance shall be equal to that used in injection 2. Injections 1 and 2 are given close to each other and nearest the head, while 3 is given towards the caudal part of the test area. Day 0-control group Three pairs of intradermal injections of 0,1 ml volume are given in the same sites as in the treated animals. Injection 1: a 1:1 mixture (v/v) FCA/water or physiological saline. Injection 2: the undiluted vehicle. Injection 3: a 50 % w/v formulation of the vehicle in a 1:1 mixture (v/v) FCA/water or physiological saline. Day 5-7-treated and control groups Approximately 24 hours before the topical induction application, if the substance is not a skin irritant, the test area, after close-clipping and/or shaving is treated with 0,5 ml of 10 % sodium lauryl sulphate in vaseline, in order to create a local irritation. Day 6-8-treated group The test area is again cleared of hair. A filter paper (2 × 4 cm) is fully-loaded with test substance in a suitable vehicle and applied to the test area and held in contact by an occlusive dressing for 48 hours. The choice of the vehicle should be justified. Solids are finely pulverised and incorporated in a suitable vehicle. Liquids can be applied undiluted, if appropriate. Day 6-8-control group The test area is again cleared of hair. The vehicle only is applied in a similar manner to the test area and held in contact by an occlusive dressing for 48 hours.

1.5.1.3.2. Challenge Day 20-22-treated and control groups The flanks of treated and control animals are cleared of hair. A patch or chamber loaded with the test substance is applied to one flank of the animals and, when relevant, a patch or chamber loaded with the vehicle only may also be applied to the other flank. The patches are held in contact by an occlusive dressing for 24 hours.

1.5.1.3.3. Observation and Grading: treated and control groups Blind reading of test and control animals is encouraged. If it is necessary to clarify the results obtained in the first challenge, a second challenge (i.e. a rechallenge), where appropriate with a new control group, should be considered approximately one week after the first one. A rechallenge may also be performed on the original control group. All skin reactions and any unusual findings, including systemic reactions, resulting from induction and challenge procedures should be observed and recorded according to the grading scale of Magnusson/Kligman (See Appendix). Other procedures, e.g. histopathological examination, the measurement of skin fold thickness, may be carried out to clarify doubtful reactions.

approximately 21 hours after removing the patch the challenge area is cleaned and closely-clipped and/or shaved and depilated if necessary;

approximately three hours later (approximately 48 hours from the start of the challenge application) the skin reaction is observed and recorded according to the grades shown in the Appendix;

approximately 24 hours after this observation a second observation (72 hours) is made and once again recorded.

1.5.2. Buehler test

1.5.2.

1.5.2.1. Preparations Healthy young adult albino guinea-pigs are acclimatised to the laboratory conditions for at least five days prior to the test. Before the test, animals are randomised and assigned to the treatment groups. Removal of hair is by clipping, shaving or possibly by chemical depilation, depending on the test method used. Care should be taken to avoid abrading the skin. The animals are weighed before the test commences and at the end of the test.

1.5.2.2. Test conditions

1.5.2.2.

1.5.2.2.1. Test animals Commonly used laboratory strains of albino guinea-pigs are used.

1.5.2.2.2. Number and sex Male and/or female animals can be used. If females are used, they should be nulliparous and non-pregnant. A minimum of 20 animals is used in the treatment group and at least 10 animals in the control group.

1.5.2.2.3. Dose levels The concentration of test substance used for each induction exposure should be the highest possible to produce a mild but not excessive irritation. The concentration used for the challenge exposure should be the highest non-irritating dose. If necessary, the appropriate concentration can be determined from a pilot study using two or three animals. For water soluble test materials, it is appropriate to use water or a dilute non-irritating solution of surfactant as the vehicle. For other test materials 80 % ethanol/water is preferred for induction and acetone for challenge.

1.5.2.3. Procedure

1.5.2.3.

1.5.2.3.1. Induction Day 0-treated group One flank is cleared of hair (closely-clipped). The test patch system should be fully loaded with test substance in a suitable vehicle (the choice of the vehicle should be justified; liquid test substances can be applied undiluted, if appropriate). The test patch system is applied to the test area and held in contact with the skin by an occlusive patch or chamber and a suitable dressing for six hours. The test patch system must be occlusive. A cotton pad is appropriate and can be circular or square, but should approximate 4-6 cm2. Restraint using an appropriate restrainer is preferred to assure occlusion. If wrapping is used, additional exposures may be required. Day 0-control group One flank is cleared of hair (closely-clipped). The vehicle only is applied in a similar manner to that used for the treated group. The test patch system is held in contact with the skin by an occlusive patch or chamber and a suitable dressing for six hours. If it can be demonstrated that a sham control group is not necessary, a naive control group may be used. Days 6-8 and 13-15-treated and control group The same application as on day 0 is carried out on the same test area (cleared of hair if necessary) of the same flank on day 6-8, and again on day 13-15.

1.5.2.3.2. Challenge Day 27-29-treated and control group The untreated flank of treated and control animals is cleared of hair (closely-clipped). An occlusive patch or chamber containing the appropriate amount of test substance is applied, at the maximum non-irritant concentration, to the posterior untreated flank of treated and control animals. When relevant, an occlusive patch or chamber with vehicle only is also applied to the anterior untreated flank of both treated and control animals. The patches or chambers are held in contact by a suitable dressing for six hours.

1.5.2.3.3. Observation and grading Blind reading of the test and control animals is encouraged. If it is necessary to clarify the results obtained in the first challenge, a second challenge (i.e. a rechallenge), where appropriate with a new control group, should be considered approximately one week after the first one. A rechallenge may also be performed on the original control group. All skin reactions and any unusual findings, including systemic reactions, resulting from induction and challenge procedures should be observed and recorded according to the Magnusson/Kligman grading scale (See Appendix). Other procedures, e.g. histopathological examination, the measurement of skin fold thickness, may be carried out to clarify doubtful reactions.

approximately 21 hours after removing the patch the challenge area is cleared of hair,

approximately three hours later (approximately 30 hours after application of the challenge patch) the skin reactions are observed and recorded according to the grades shown in the Appendix,

approximately 24 hours after the 30 hour observation (approximately 54 hours after application of the challenge patch) skin reactions are again observed and recorded.

2. DATA (GPMT and Buehler test) Data should be summarised in tabular form, showing for each animal the skin reactions at each observation.

3. REPORTING (GPMT and Buehler test) If a screening assay is performed before the guinea pig test the description or reference of the test (e.g. Mouse Ear Swelling Test (MEST)), including details of the procedure, must be given together with results obtained with the test and reference substances.

3. Test report (GMPT and Buehler test)

The test report shall, if possible, include the following information:

Test animals:

strain of guinea-pig used,

number, age and sex of animals,

source, housing conditions, diet, etc.,

individual weights of animals at the start of the test.

Test conditions:

technique of patch site preparation,

details of patch materials used and patching technique,

result of pilot study with conclusion on induction and challenge concentrations to be used in the test,

details of test substance preparation, application and removal,

justification for choice of vehicle,

vehicle and test substance concentrations used for induction and challenge exposures and the total amount of substance applied for induction and challenge.

Results:

a summary of the results of the latest sensitivity and reliability check (see 1.3) including information on substance, concentration and vehicle used,

on each animal including grading system,

narrative description of the nature and degree effects observed,

any histopathological findings.

Discussion of results.

Conclusions.

4. REFERENCES This method is analogous to OECD TG 406.

B.7. REPEATED DOSE (28 DAYS) TOXICITY (ORAL)

B.7.

1. METHOD

1.

1.1. INTRODUCTION See General introduction Part B.

1.2. DEFINITIONS See General introduction Part B.

1.3. PRINCIPLE OF THE TEST METHOD The test substance is orally administered daily in graduated doses to several groups of experimental animals, one dose level per group for a period of 28 days. During the period of administration the animals are observed closely, each day for signs of toxicity. Animals which die or are killed during the test are necropsied and at the conclusion of the test surviving animals are killed and necropsied. This method places more emphasis on neurological effects as a specific endpoint, and the need for careful clinical observations of the animals, so as to obtain as much information as possible, is stressed. The method should identify chemicals with neurotoxic potential, which may warrant further indepth investigation of this aspect. In addition, the method may give an indication of immunological effects and reproductive organ toxicity.

1.4. DESCRIPTION OF THE TEST METHOD

1.4.

1.4.1. Preparations Healthy young adult animals are randomly assigned to the control and treatment groups. Cages should be arranged in such a way that possible effects due to cage placement are minimised. The animals are identified uniquely and kept in their cages for at least five days prior to the start of the study to allow for acclimatisation to the laboratory conditions. The test substance is administered by gavage or via the diet or drinking water. The method of oral administration is dependent on the purpose of the study, and the physical/chemical properties of the substance. Where necessary, the test substance is dissolved or suspended in a suitable vehicle. It is recommended that, wherever possible, the use of an aqueous solution/suspension be considered first, followed by consideration of a solution/emulsion in oil (e.g. corn oil) and then by possible solution in other vehicles. For vehicles other than water the toxic characteristics of the vehicle must be known. The stability of the test substance in the vehicle should be determined.

1.4.2. Test conditions

1.4.2.

1.4.2.1. Test animals The preferred rodent species is the rat, although other rodent species may be used. Commonly used laboratory strains of young healthy adult animals should be employed. The females should be nulliparous and non-pregnant. Dosing should begin as soon as possible after weaning and, in any case, before the animals are nine weeks old. At the commencement of the study the weight variation of animals used should be minimal and not exceed ± 20 % of the mean weight of each sex. Where a repeated dose oral study is conducted as a preliminary to a long term-study, preferably animals from the same strain and source should be used in both studies.

1.4.2.2. Number and sex At least 10 animals (five female and five male) should be used at each dose level. If interim kills are planned, the number should be increased by the number of animals scheduled to be killed before the completion of the study. In addition, a satellite group of 10 animals (five animals per sex) may be treated with the high dose level for 28 days and observed for reversibility, persistence, or delayed occurrence of toxic effects for 14 days post-treatment. A satellite group of 10 control animals (five animals per sex) is also used.

1.4.2.3. Dose levels Generally, at least three test groups and a control group should be used. Except for treatment with the test substance, animals in the control group should be handled in an identical manner to the test group subjects. If a vehicle is used in administering the test substance, the control group should receive the vehicle in the highest volume used. If from assessment of other data, no effects would be expected at a dose of 1000 mg/kg bw/d, a limit test may be performed. If there are no suitable data available, a range finding study may be performed to aid the determination of the doses to be used. Dose levels should be selected taking into account any existing toxicity and (toxico-) kinetic data available for the test substance or related materials. The highest dose level should be chosen with the aim of inducing toxic effects but not death or severe suffering. Thereafter, a descending sequence of dose levels should be selected with a view to demonstrating any dosage related response and no-observed-adverse effects at the lowest dose level (NOAEL). Two to four fold intervals are frequently optimal for setting the descending dose levels and addition of a fourth test group is often preferable to using very large intervals (e.g. more than a factor of 10) between dosages. For substances administered via the diet or drinking water it is important to ensure that the quantities of the test substance involved do not interfere with normal nutrition or water balance. When the test substance is administered in the diet either a constant dietary concentration (ppm) or a constant dose level in terms of the animals' body weight may be used; the alternative used must be specified. For a substance administered by gavage, the dose should be given at similar times each day, and adjusted as necessary to maintain a constant dose level in terms of animal body weight. Where a repeated dose study is used as a preliminary to a long term-study, a similar diet should be used in both studies.

1.4.2.4. Limit test If a test at one dose level of at least 1000 mg/kg body weight/day or, for dietary or drinking water administration, an equivalent percentage in the diet or drinking water (based upon body weight determinations), using the procedures described for this study, produces no observable toxic effects and if toxicity would not be expected based upon data from structurally related substances, then a full study using three dose levels may not be considered necessary. The limit test applies except when human exposure indicates the need for a higher dose level to be used.

1.4.2.5. Observation period The observation period should be 28 days. Animals in a satellite group scheduled for follow-up observations should be kept for at least a further 14 days without treatment to detect delayed occurrence, or persistence of, or recovery from toxic effects.

1.4.3. Procedure The animals are dosed with the test substance daily seven days each week for a period of 28 days; use of a five-day per week dosing regime needs to be justified. When the test substance is administered by gavage, this should be done in a single dose to the animals using a stomach tube or a suitable intubation cannula. The maximum volume of liquid that can be administered at one time depends on the size of the test animal. The volume should not exceed 1 ml/100 g body weight, except in the case of aqueous solutions where 2 ml/100 g body weight may be used. Except for irritating or corrosive substances, which will normally reveal exacerbated effects with higher concentrations, variability in test volume should be minimised by adjusting the concentration to ensure a constant volume at all dose levels.

1.4.3.

1.4.3.1. General observations General clinical observations should be made at least once a day, preferably at the same time(s) each day and considering the peak period of anticipated effects after dosing. The health condition of the animals should be recorded. At least twice daily, all animals are observed for morbidity and mortality. Moribund animals and animals in severe distress or pain should be removed when noticed, humanely killed and necropsied. Once before the first exposure (to allow for within-subject comparisons), and at least once a week thereafter, detailed clinical observations should be made in all animals. These observations should be made outside the home cage in a standard arena and preferably at the same time, each time. They should be carefully recorded, preferably using scoring systems, explicitly defined by the testing laboratory. Effort should be made to ensure that variations in the test conditions are minimal and that observations are preferably conducted by observers unaware of the treatment. Signs noted should include, but not be limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g. excessive grooming, repetitive circling) or bizarre behaviour (e.g. self-mutilation, walking backwards) should also be recorded. In the fourth exposure week sensory reactivity to stimuli of different types (e.g. auditory, visual and proprioceptive stimuli), assessment of grip strength and motor activity assessment should be conducted. Further details of the procedures that could be followed are given in the literature (see General introduction Part B). Functional observations conducted in the fourth exposure week may be omitted when the study is conducted as a preliminary study to a subsequent subchronic (90-day) study. In that case, the functional observations should be included in this follow-up study. On the other hand, the availability of data on functional observations from the repeated dose study may enhance the ability to select dose levels for a subsequent subchronic study. Exceptionally, functional observations may also be omitted for groups that otherwise reveal signs of toxicity to an extent that would significantly interfere with the functional test performance.

1.4.3.2. Body weight and food/water consumption All animals should be weighed at least once a week. Measurements of food and water consumption should be made at least weekly. If the test substance is administered via the drinking water, water consumption should also be measured at least weekly.

1.4.3.3. Haematology The following haematological examinations should be made at the end of the test period: haematocrit, haemoglobin concentration, erythrocyte count, total and differential leucocyte count, platelet count and a measure of blood clotting time/potential. Blood samples should be taken from a named site just prior to or as part of the procedure for killing the animals, and stored under appropriate conditions.

1.4.3.4. Clinical biochemistry Clinical biochemistry determinations to investigate major toxic effects in tissues and, specifically, effects on kidney and liver, should be performed on blood samples obtained of all animals just prior to or as part of the procedure for killing the animals (apart from those found moribund and/or intercurrently killed). Overnight fasting of the animals prior to blood sampling is recommended. Investigations of plasma or serum shall include sodium, potassium, glucose, total cholesterol, urea, creatinine, total protein and albumin, at least two enzymes indicative of hepatocellular effects (such as alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, gamma glutamyl transpeptidase, and sorbitol dehydrogenase). Measurements of additional enzymes (of hepatic or other origin) and bile acids may provide useful information under certain circumstances. Optionally, the following urine analysis determinations could be performed during the last week of the study using timed urine volume collection; appearance, volume, osmolality or specific gravity, pH, protein, glucose and blood/blood cells. In addition, studies to investigate serum markers of general tissue damage should be considered. Other determinations that should be carried out if the known properties of the test substance may, or are suspected to, affect related metabolic profiles include calcium, phosphate, fasting triglycerides, specific hormones, methaemoglobin and cholinesterase. These need to be identified for substances in certain classes or on a case-by-case basis. Overall, there is a need for a flexible approach, depending on the species and the observed and/or expected effect with a given substance. If historical baseline data are inadequate, consideration should be given to determination of haematological and clinical biochemistry variables before dosing commences.

1.4.3.5. Gross necropsy All animals in the study shall be subjected to a full, detailed gross necropsy, which includes careful examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents. The liver, kidneys, adrenals, testes, epididymides, thymus, spleen, brain and heart of all animals should be trimmed of any adherent tissue, as appropriate, and their wet weight taken as soon as possible after dissection to avoid drying. The following tissues should be preserved in the most appropriate fixation medium for both the type of tissue and the intended subsequent histopathological examination: all gross lesions, brain (representative regions including cerebrum, cerebellum and pons), spinal cord, stomach, small and large intestines (including Peyer's patches), liver, kidneys, adrenals, spleen, heart, thymus, thyroid, trachea and lungs (preserved by inflation with fixative and then immersion), gonads, accessory sex organs (e.g. uterus, prostate), urinary bladder, lymph nodes (preferably one lymph node covering the route of administration and another one distant from the route of administration to cover systemic effects), peripheral nerve (sciatic or tibial) preferably in close proximity to the muscle, and a section of bone marrow (or, alternatively, a fresh mounted bone marrow aspirate). The clinical and other findings may suggest the need to examine additional tissues. Also any organs considered likely to be target organs based on the known properties of the test substance should be preserved.

1.4.3.6. Histopathological examination Full histopathology should be carried out on the preserved organs and tissues of all animals in the control and high dose groups. These examinations should be extended to animals of all other dosage groups, if treatment-related changes are observed in the high dose group. All gross lesions shall be examined. When a satellite group is used, histopathology should be performed on tissues and organs identified as showing effects in the treated groups.

2. DATA Individual data should be provided. Additionally, all data should be summarised in tabular form showing for each test group the number of animals at the start of the test, the number of animals found dead during the test or killed for humane reasons and the time of any death or humane kill, the number showing signs of toxicity, a description of the signs of toxicity observed, including time of onset, duration, and severity of any toxic effects, the number of animals showing lesions, the type of lesions and the percentage of animals displaying each type of lesion. When possible, numerical results should be evaluated by an appropriate and generally acceptable statistical method. The statistical methods should be selected during the design of the study.

3. REPORTING

3. TEST REPORT

The test report shall, if possible, include the following information:

Test animals:

species/strain used,

number, age and sex of animals,

source, housing conditions, diet, etc.,

individual weights of animals at the start of the test in weekly intervals thereafter and at the end of the test.

Test conditions:

justification for choice of vehicle, if other than water,

rationale for dose level selection,

details of test substance formulation/diet preparation, achieved concentration, stability and homogeneity of the preparation,

details of the administration of the test substance,

conversion from diet/drinking water test substance concentration (ppm) to the actual dose (mg/kg body weight/day), if applicable,

details of food and water quality.

Results:

body weight/body weight changes,

food consumption, and water consumption, if applicable,

toxic response data by sex and dose level, including signs of toxicity,

nature, severity and duration of clinic observations (whether reversible or not),

sensory activity, grip strength and motor activity assessments,

haematological tests with relevant base-line values,

clinical biochemistry tests with relevant base-line values,

body weight at killing and organ weight data,

necropsy findings,

a detailed description of all histopathological findings,

absorption data if available,

statistical treatment of results, where appropriate.

Discussion of results.

Conclusions.

4. REFERENCES This method is analogous to OECD TG 407.

B.8. REPEATED DOSE (28 DAYS) TOXICITY (INHALATION)

B.8.

1. METHOD

1.

1.2. DEFINITION See General introduction Part B (B).

1.3. REFERENCE SUBSTANCES None.

1.4. PRINCIPLE OF THE TEST METHOD Several groups of experimental animals are exposed daily for a defined period to the test substance in graduated concentrations, one concentration being used per group, for a period of 28 days. Where a vehicle is used to help generate an appropriate concentration of the test substance in the atmosphere, a vehicle control group should be used. During the period of administration the animals are observed daily to detect signs of toxicity. Animals, which die during the test are necropsied and at the conclusion of the test surviving animals are necropsied.

1.5. QUALITY CRITERIA None.

1.6. DESCRIPTION OF THE TEST METHOD

1.6.

1.6.1. Preparations The animals are kept under the experimental housing and feeding conditions for at least five days prior to the experiment. Before the test, healthy young animals are randomised and assigned to the required number of groups. Where necessary, a suitable vehicle may be added to the test substance to help generate an appropriate concentration of the substance in the atmosphere. If a vehicle or other additive is used to facilitate dosing, it should be known not to produce toxic effects. Historical data can be used if appropriate.

1.6.2. Test conditions

1.6.2.

1.6.2.1. Experimental animals Unless there are contra-indications, the rat is the preferred species. Commonly used laboratory strains of young healthy animals should be employed. At the commencement of the study the range of weight variation in the animals used should not exceed ± 20 % of the appropriate mean value.

1.6.2.2. Number and sex At least 10 animals (five female and five male) should be used for each test group. The females should be nulliparous and non-pregnant. If interim sacrifices are planned, the numbers should be increased by the number of animals scheduled to be sacrificed before the completion of the study. In addition, a satellite group of 10 animals (five animals per sex) may be treated with the high concentration level for 28 days and observed for reversibility, persistence, or delayed occurrence of toxic effects for 14 days post-treatment. A satellite group of 10 control animals (five animals per sex) is also used.

1.6.2.3. Exposure concentration At least three concentrations are required, with a control or a vehicle control (corresponding to the concentration of vehicle at the highest level) if a vehicle is used. Except for treatment with the test substance, animals in the control group should be handled in an identical manner to the test-group animals. The highest concentration should result in toxic effects but no, or few, fatalities. The lowest concentration should not produce any evidence of toxicity. Where there is a usable estimation of human exposure, the lowest concentration should exceed this. Ideally, the intermediate concentration should produce minimal observable toxic effects. If more than one intermediate concentration is used the concentrations should be spaced to produce a gradation of toxic effects. In the low and intermediate groups and in the controls, the incidence of fatalities should be low to permit a meaningful evaluation of the results.

1.6.2.4. Exposure time The duration of daily exposure should be six hours but other periods may be needed to meet specific requirements.

1.6.2.5. Equipment The animals should be tested in inhalation equipment designed to sustain a dynamic airflow of at least 12 air changes per hour to ensure an adequate oxygen content and an evenly distributed exposure atmosphere. Where a chamber is used its design should minimise crowding of the test animals and maximise their exposure by inhalation of the test substance. As a general rule to ensure stability of a chamber atmosphere the total volume of the test animals should not exceed 5 % of the volume of the test chamber. Oro-nasal, head only, or individual whole body chamber exposure may be used; the first two will minimise uptake by other routes.

1.6.2.6. Observation period The experimental animals should be observed daily for signs of toxicity during the entire treatment and recovery period. The time of death and the time at which signs of toxicity appear and disappear should be recorded.

1.6.3. Procedure The animals are exposed to the test substance daily, five to seven days per week, for a period of 28 days. Animals in any satellite groups scheduled for follow-up observations should be kept for a further 14 days without treatment to detect recovery from, or persistence of toxic effects. The temperature at which the test is performed should be maintained at 22 ± 3 oC. Ideally, the relative humidity should be maintained between 30 and 70 %, but in certain instances (e.g. tests of some aerosols) this may not be practicable. Maintenance of a slight negative pressure inside the chamber (≤ 5 mm of water) will prevent leakage of the test substance into the surrounding area. Food and water should be withheld during exposure. A dynamic inhalation system with a suitable analytical concentration control system should be used. To establish suitable exposure concentrations a trial test is recommended. The airflow should be adjusted to ensure that conditions throughout the exposure chamber are homogeneous. The system should ensure that stable exposure conditions are achieved as rapidly as possible. Measurements or monitoring should be made: During and following exposure observations are made and recorded systematically; individual records should be maintained for each animal. All the animals should be observed daily and signs of toxicity recorded including the time of onset, their degree and duration. Observations should include changes in the skin and fur, eyes, mucous membranes, respiratory, circulatory, autonomic and central nervous systems, somatomotor activity and behaviour pattern. Measurements should be made weekly of the animals' weight. It is also recommended that food consumption is measured weekly. Regular observation of the animals is necessary to ensure that animals are not lost from the study due to causes such as cannibalism, autolysis of tissues or misplacement. At the end of the study period, all survivors in the non-satellite treatment groups are necropsied. Moribund animals and animals in severe distress or pain should be removed when noticed, humanely killed and necropsied. The following examinations shall be made at the end of the test on all animals including the controls: Other determinations, which may be necessary for an adequate toxicological evaluation include calcium, phosphorus, chloride, sodium, potassium, fasting glucose analysis of lipids, hormones, acid/base balance, methaemoglobin and cholinesterase activity. Additional clinical biochemistry may be employed, where necessary, to extend the investigation of observed toxic effects.

1.6.3.

(a) of the rate of airflow (continuously),

(b) of the actual concentration of the test substance measured in the breathing zone. During the daily exposure period the concentration should not vary by more than ± 15 % of the mean value. However, in the case of some aerosols, this level of control may not be achievable and a wider range would then be acceptable. During the total duration of the study, the day-to-day concentrations should be held as constant as practicable. For aerosols, at least one particle size analysis should be performed per test group weekly,

(i) haematology, including at least haematocrit, haemoglobin concentration, erythrocyte count, total and differential leucocyte count and a measure of clotting potential;

(ii) clinical blood biochemistry including at least one parameter of liver and kidney function: serum alanine aminotransferase (formerly known as glutamic pyruvic transaminase), serum aspartate aminotransferase (formerly known as glutamic oxaloacetic transaminase), urea nitrogen, albumin, blood creatinine, total bilirubin and total serum protein measurements;

1.6.3.1. Gross necropsy All animals in the study should be subjected to a full gross necropsy. At least the liver, kidneys, adrenals, lungs, and testes should be weighed wet as soon as possible after dissection to avoid drying. Organs and tissues (the respiratory tract, liver, kidneys, spleen, testes, adrenals, heart, and any organs showing gross lesions or changes in size) should be preserved in a suitable medium for possible future histopathological examination. The lungs should be removed intact, weighed and treated with a suitable fixative to ensure that lung structure is maintained.

1.6.3.2. Histopathological examination In the high-concentration group and in the control(s), histological examination should be performed on preserved organs and tissues. Organs and tissues showing defects attributable to the test substance at the highest dosage level should be examined in all lower-dosage groups. Animals in any satellite groups should be examined histologically with particular emphasis on those organs and tissues identified as showing effects in the other treated groups.

2. DATA Data should be summarised in tabular form, showing for each test group the number of animals at the start of the test and the number of animals displaying each type of lesion. All observed results should be evaluated by an appropriate statistical method. Any recognised statistical method may be used.

3. REPORTING

3.

3.1. TEST REPORT The test report shall, if possible, include the following information:

species, strain, source, environmental conditions, diet, etc.,

test conditions. Description of exposure apparatus including design, type, dimensions, source of air, system for generating aerosols, method of conditioning air, treatment of exhaust air and the method of housing animals in a test chamber when this is used. The equipment for measuring temperature, humidity and, where appropriate, stability of aerosol concentrations or particle size distribution, should be described. Exposure data: These should be tabulated and presented with mean values and a measure of variability (e.g. standard deviation) and shall, if possible, include:

3.1. pt_2

(a) airflow rates through the inhalation equipment;

(b) temperature and humidity of air;

(c) nominal concentrations (total amount of test substance fed into the inhalation equipment divided by the volume of air);

(d) nature of vehicle, if used;

(e) actual concentrations in test breathing zone;

(f) the mass median aerodynamic diameter (MMAD) and the geometric standard deviation (GSD);

toxic response data by sex and concentration,

time of death during the study or whether animals survived to termination,

description of toxic or other effects, no-effect level,

the time of observation of each abnormal sign and its subsequent course,

food and body-weight data,

haematological tests employed and results,

clinical biochemistry tests employed and results,

necropsy findings,

a detailed description of all histopathological findings,

a statistical treatment of results where possible,

discussion of the results,

interpretation of results.

3.2. EVALUATION AND INTERPRETATION See General introduction Part B (D).

4. REFERENCES See General introduction Part B (E).

B.9. REPEATED DOSE (28 DAYS) TOXICITY (DERMAL)

B.9.

1. METHOD

1.

1.1. INTRODUCTION See General introduction Part B (A).

1.2. DEFINITIONS See General introduction Part B (B).

1.3. REFERENCE SUBSTANCES None.

1.4. PRINCIPLE OF THE TEST METHOD The test substance is applied daily to the skin in graduated doses to several groups of experimental animals, one dose per group, for a period of 28 days. During the period of application, the animals are observed daily to detect signs of toxicity. Animals, which die during the test, are necropsied and at the conclusion of the test surviving animals are necropsied.

1.5. QUALITY CRITERIA None.

1.6. DESCRIPTION OF THE TEST METHOD

1.6.

1.6.1. Preparations The animals are kept under the experimental housing and feeding conditions for at least five days prior to the test. Before the test, healthy young animals are randomised and assigned to the treatment and control groups. Shortly before testing, fur is clipped from the dorsal area of the trunk of the test animals. Shaving may be employed but it should be carried out approximately 24 hours before the test. Repeat clipping or shaving is usually needed at approximately weekly intervals. When clipping or shaving the fur, care must be taken to avoid abrading the skin. Not less than 10 % of the body surface area should be clear for the application of the test substance. The weight of the animal should be taken into account when deciding on the area to be cleared and on the dimensions of the covering. When testing solids, which may be pulverised if appropriate, the test substance should be moistened sufficiently with water or, where necessary, a suitable vehicle to ensure good contact with the skin. Liquid test substances are generally used undiluted. Daily application on a five to seven-day per week basis is used.

1.6.2. Test conditions

1.6.2.

1.6.2.1. Experimental animals The adult rat, rabbit or guinea-pig may be used. Other species may be used but their use would require justification. At the commencement of the study, the range of weight variation in the animals used should not exceed ± 20 % of the appropriate mean value.

1.6.2.2. Number and sex At least 10 animals (five female and five male) with healthy skin should be used at each dose level. The females should be nulliparous and non-pregnant. If interim sacrifices are planned, the numbers should be increased by the number of animals scheduled to be sacrificed before the completion of the study. In addition, a satellite group of 10 animals (five animals per sex) may be treated with the high dose level for 28 days and observed for reversibility, persistence, or delayed occurrence of toxic effects for 14 days post-treatment. A satellite group of 10 control animals (five animals per sex) is also used.

1.6.2.3. Dose levels At least three dose levels are required with a control or a vehicle control if a vehicle is used. The exposure period should be at least six hours per day. The application of the test substance should be made at similar times each day, and adjusted at intervals (weekly or bi-weekly) to maintain a constant dose level in terms of animal body-weight. Except for treatment with the test substance, animals in the control group should be handled in an identical manner to the test group subjects. Where a vehicle is used to facilitate dosing, the vehicle control group should be dosed in the same way as the treated groups, and receive the same amount as that received by the highest dose level group. The highest dose level should result in toxic effects but produce no, or few, fatalities. The lowest dose level should not produce any evidence or toxicity. Where there is a usable estimation of human exposure, the lowest level should exceed this. Ideally, the intermediate dose level should produce minimal observable toxic effects. If more than one intermediate dose is used the dose levels should be spaced to produce a gradation of toxic effects. In the low and intermediate groups and in the controls, the incidence of fatalities should be low in order to permit a meaningful evaluation of the results. If application of the test substance produces severe skin irritation, the concentrations should be reduced and this may result in a reduction in, or absence of, other toxic effects at the high dose level. Moreover if the skin has been badly damaged it may be necessary to terminate the study and undertake a new study at lower concentrations.

1.6.2.4. Limit test If a preliminary study at a dose level of 1000 mg/kg, or a higher dose level related to possible human exposure where this is known, produces no toxic effects, further testing may not be considered necessary.

1.6.2.5. Observation period The experimental animals should be observed daily for signs of toxicity. The time of death and the time at which signs of toxicity appear and disappear should be recorded.

1.6.3. Procedure Animals should be caged individually. The animals are treated with the test substance, ideally on seven days per week, for a period of 28 days. Animals in any satellite groups scheduled for follow-up observations should be kept for a further 14 days without treatment to detect recovery from or persistence of toxic effects. Exposure time should be at least six hours per day. The test substance should be applied uniformly over an area, which is approximately 10 % of the total body surface area. With highly toxic substances, the surface area covered may be less but as much of the area as possible should be covered with as thin and uniform a layer as possible. During exposure the test substance is held in contact with the skin with porous gauze dressing and non-irritating tape. The test site should be further covered in a suitable manner to retain the gauze dressing and test substance and ensure that the animals cannot ingest the test substance. Restrainers may be used to prevent the ingestion of the test substance but complete immobilisation is not a recommended method. As an alternative a collar protective device may be used. At the end of the exposure period, residual test substance should be removed, where practicable, using water or some other appropriate method of cleansing the skin. All the animals should be observed daily and signs of toxicity recorded including the time of onset, their degree and duration. Observations should include changes in skin and fur, eyes and mucous membranes as well as respiratory, circulatory, autonomic and central nervous systems, somatomotor activity and behaviour pattern. Measurements should be made weekly of the animals' weight. It is also recommended that food consumption is measured weekly. Regular observation of the animals is necessary to ensure that animals are not lost from the study due to causes such as cannibalism, autolysis of tissues or misplacement. At the end of the study period, all survivors in the non-satellite treatment groups are necropsied. Moribund animals and animals in severe distress or pain should be removed when noticed, humanely killed and necropsied. The following examinations shall be made at the end of the test on all animals including the controls: Other determinations which may be necessary for an adequate toxicological evaluation include calcium, phosphorus, chloride, sodium, potassium, fasting glucose, analysis of lipids, hormones, acid/base balance, methaemoglobin and cholinesterase activity. Additional clinical biochemistry may be employed, where necessary, to extend the investigation of observed effects.

1.6.3.

(1) haematology, including at least haematocrit, haemoglobin concentration, erythrocyte count, total and differential leucocyte count, and a measure of clotting potential;

(2) clinical blood biochemistry including at least one parameter of liver and kidney function: serum alanine aminotransferase (formerly known as glutamic pyruvic transaminase), serum aspartate aminotransferase (formerly known as glutamic oxaloacetic transaminase), urea nitrogen, albumin, blood creatinine, total bilirubin and total serum protein;

1.6.4. Gross necropsy All animals in the study should be subjected to a full gross necropsy. At least the liver, kidneys, adrenals, and testes should be weighed wet as soon as possible after dissection, to avoid drying. Organs and tissues, i.e. normal and treated skin, liver, kidney, spleen, testes, adrenals, heart, and target organs (that is those organs showing gross lesions or changes in size) should be preserved in a suitable medium for possible future histopathological examination.

1.6.5. Histopathological examination In the high dose group and in the control group, histological examination should be performed on the preserved organs and tissues. Organs and tissues showing defects attributable to the test substance at the highest dosage level should be examined in all lower-dosage groups. Animals in the satellite group should be examined histologically with particular emphasis on those organs and tissues identified as showing effects in the other treated groups.

3. REPORTING

3.

3.1. TEST REPORT The test report shall, if possible, include the following information:

animal data (species, strain, source, environmental conditions, diet, etc.),

test conditions (including the type of dressing: occlusive or not-occlusive),

dose levels (including vehicle, if used) and concentrations,

no-effect level, where possible,

toxic response data by sex and dose,

time of death during the study or whether animals survived to termination,

toxic or other effects,

the time of observation of each abnormal sign and its subsequent course,

food and body-weight data,

haematological tests employed and results,

clinical biochemistry tests employed and results,

necropsy findings,

a detailed description of all histopathological findings,

statistical treatment of results where possible,

discussion of the results,

interpretation of the results.

3.2. EVALUATION AND INTERPRETATION See General introduction Part B (D).

4. REFERENCES See General introduction Part B (E).

B.10. MUTAGENICITY — IN VITRO MAMMALIAN CHROMOSOME ABERRATION TEST

B.10.

1. METHOD This method is a replicate of the OECD TG 473, In Vitro Mammalian Chromosome Aberration Test (1997).

1.

1.1. INTRODUCTION The purpose of the in vitro chromosomal aberration test is to identify agents that cause structural chromosome aberrations in cultured mammalian cells (1)(2)(3). Structural aberrations may be of two types, chromosome or chromatid. With the majority of chemical mutagens, induced aberrations are of the chromatid type, but chromosome-type aberrations also occur. An increase in polyploidy may indicate that a chemical has the potential to induce numerical aberrations. However, this method is not designed to measure numerical aberrations and is not routinely used for that purpose. Chromosome mutations and related events are the cause of many human genetic diseases and there is substantial evidence that chromosome mutations and related events causing alterations in oncogenes and tumour-suppressor genes of somatic cells are involved in cancer induction in humans and experimental animals. The in vitro chromosome aberration test may employ cultures of established cell lines, cell strains or primary cell cultures. The cells used are selected on the basis of growth ability in culture, stability of the karyotype, chromosome number, chromosome diversity and spontaneous frequency of chromosome aberrations. Tests conducted in vitro generally require the use of an exogenous source of metabolic activation. This metabolic activation system cannot mimic entirely the mammalian in vivo conditions. Care should be taken to avoid conditions which would lead to positive results which do not reflect intrinsic mutagenicity and may arise from changes in pH, osmolality or high levels of cytotoxicity (4)(5). This test is used to screen for possible mammalian mutagens and carcinogens. Many compounds that are positive in this test are mammalian carcinogens; however, there is not a perfect correlation between this test and carcinogenicity. Correlation is dependent on chemical class and there is increasing evidence that there are carcinogens that are not detected by this test because they appear to act through mechanisms other than direct DNA damage. See also General introduction Part B.

1.2. DEFINITIONS Chromatid-type aberration: structural chromosome damage expressed as breakage of single chromatids or breakage and reunion between chromatids. Chromosome-type aberration: structural chromosome damage expressed as breakage, or breakage and reunion, of both chromatids at an identical site. Endoreduplication: a process in which after an S period of DNA replication, the nucleus does not go into mitosis but starts another S period. The result is chromosomes with four, eight, 16, …chromatids. Gap: an achromatic lesion smaller than the width of one chromatid, and with minimum misalignment of the chromatids). Mitotic index: the ratio of cells in metaphase divided by the total number of cells observed in a population of cells; an indication of the degree of proliferation of that population. Numerical aberration: a change in the number of chromosomes from the normal number characteristic of the cells utilised. Polyploidy: a multiple of the haploid chromosome number (n) other than the diploid number (i.e. 3n, 4n and so on). Structural aberration: a change in chromosome structure detectable by microscopic examination of the metaphase stage of cell division, observed as deletions and fragments, intrachanges or interchanges.

1.3. PRINCIPLE OF THE TEST METHOD Cell cultures are exposed to the test substance both with and without metabolic activation. At predetermined intervals after exposure of cell cultures to the test substance, they are treated with a metaphase-arresting substance (e.g. Colcemid® or colchicine), harvested, stained and metaphase cells are analysed microscopically for the presence of chromosome aberrations.

1.4. DESCRIPTION OF THE TEST METHOD

1.4.

1.4.1. Preparations

1.4.1.

1.4.1.1. Cells A variety of cell lines, strains or primary cell cultures, including human cells, may be used (e.g. Chinese hamster fibroblasts, human or other mammalian peripheral blood lymphocytes).

1.4.1.2. Media and culture conditions Appropriate culture media and incubation conditions (culture vessels, CO2 concentration, temperature and humidity) should be used in maintaining cultures. Established cell lines and strains should be checked routinely for stability in the modal chromosome number and the absence of mycoplasma contamination and should not be used if contaminated. The normal cell cycle time for the cells and culture conditions used should be known.

1.4.1.3. Preparation of cultures Established cell lines and strains: cells are propagated from stock cultures, seeded in culture medium at a density such that the cultures will not reach confluency before the time of harvest, and incubated at 37 oC. Lymphocytes: whole blood treated with an anti-coagulant (e.g. heparin) or separated lymphocytes obtained from healthy subjects are added to the culture medium containing a mitogen (e.g. phytohaemagglutinin) and incubated at 37 oC.

1.4.1.4. Metabolic activation Cells should be exposed to the test substance both in the presence and absence of an appropriate metabolic activation system. The most commonly used system is a cofactor-supplemented post-mitochondrial fraction (S9) prepared from the livers of rodents treated with enzyme-inducing agents such as: Aroclor 1254 (6)(7)(8)(9), or a mixture of phenobarbitone and ß–naphthoflavone (10)(11)(12). The post-mitochondrial fraction is usually used at concentrations in the range from 1-10 % v/v in the final test medium. The condition of a metabolic activation system may depend upon the class of chemical being tested. In some cases it may be appropriate to utilise more than one concentration of post-mitochondrial fraction. A number of developments, including the construction of genetically engineered cell lines expressing specific activating enzymes, may provide the potential for endogenous activation. The choice of the cell lines used should be scientifically justified (e.g. by the relevance of the cytochrome P450 isoenzyme for the metabolism of the test substance).

1.4.1.5. Test substance/Preparation Solid test substances should be dissolved or suspended in appropriate solvents or vehicles and diluted if appropriate prior to treatment of the cells. Liquid test substances may be added directly to the test systems and/or diluted prior to treatment. Fresh preparations of the test substance should be employed unless stability data demonstrate the acceptability of storage.

1.4.2. Test conditions

1.4.2.

1.4.2.1. Solvent/vehicle The solvent/vehicle should not be suspected of chemical reaction with the test substance and should be compatible with the survival of the cells and the S9 activity. If other than well-known solvent/vehicles are used, their inclusion should be supported by data indicating their compatibility. It is recommended that wherever possible, the use of an aqueous solvent/vehicle be considered first. When testing water-unstable substances, the organic solvents used should be free of water. Water can be removed by adding a molecular sieve.

1.4.2.2. Exposure concentrations Among the criteria to be considered when determining the highest concentration are cytotoxicity, solubility in the test system and changes in pH or osmolality. Cytotoxicity should be determined with and without metabolic activation in the main experiment using an appropriate indication of cell integrity and growth, such as degree of confluency, viable cell counts, or mitotic index. It may be useful to determine cytotoxicity and solubility in a preliminary experiment. At least three analysable concentrations should be used. Where cytotoxicity occurs, these concentrations should cover a range from the maximum to little or no toxicity; this will usually mean that the concentrations should be separated by no more than a factor between 2 and √10. At the time of harvesting, the highest concentration should show a significant reduction in degree of confluency, cell count or mitotic index, (all greater than 50 %). The mitotic index is only an indirect measure of cytotoxic/cytostatic effects and depends on the time after treatment. However, the mitotic index is acceptable for suspension cultures in which other toxicity measurements may be cumbersome and impractical. Information on cell cycle kinetics, such as average generation time (AGT), could be used as supplementary information. AGT, however, is an overall average that does not always reveal the existence of delayed subpopulations, and even slight increases in average generation time can be associated with very substantial delay in the time of optimal yield of aberrations. For relatively non-cytotoxic substances, the maximum test concentration should be 5 μl/ml, 5 mg/ml or 0,01 M, whichever is the lowest. For relatively insoluble substances that are not toxic at concentrations lower than the insoluble concentration, the highest dose used should be a concentration above the limit of solubility in the final culture medium at the end of the treatment period. In some cases (e.g. when toxicity occurs only at higher than the lowest insoluble concentration) it is advisable to test at more than one concentration with visible precipitation. It may be useful to assess solubility at the beginning and the end of the treatment, as solubility can change during the course of exposure in the test system due to presence of cells, S9, serum etc. Insolubility can be detected by using the unaided eye. The precipitate should not interfere with the scoring.

1.4.2.3. Negative and positive controls Concurrent positive and negative (solvent or vehicle) controls, both with and without metabolic activation, should be included in each experiment. When metabolic activation is used, the positive control chemical should be the one that requires activation to give a mutagenic response. Positive controls should employ a known clastogen at exposure levels expected to give a reproducible and detectable increase over background, which demonstrates the sensitivity of the test system. Positive control concentrations should be chosen so that the effects are clear but do not immediately reveal the identity of the coded slides to the reader. Examples of positive control substances include: Metabolic Activation condition Substance CAS No EINECS No Absence of exogenous metabolic Activation Methyl methanesulphonate 66-27-3 200-625-0 Ethyl methanesulphonate 62-50-0 200-536-7 Ethyl nitrosourea 759-73-9 212-072-2 Mitomycin C 50-07-7 200-008-6 4-Nitroquinoline-N-oxide 56-57-5 200-281-1 Presence of exogenous metabolic Activation Benzo[a]pyrene 50-32-8 200-028-5 Cyclophosphamide Cyclophosphamide monohydrate 50-18-0 6055-19-2 200-015-4 Other appropriate positive control substances may be used. The use of chemical class-related positive control chemicals should be considered, when available. Negative controls, consisting of solvent or vehicle alone in the treatment medium, and treated in the same way as the treatment cultures, should be included for every harvest time. In addition, untreated controls should also be used unless there are historical control data demonstrating that no deleterious or mutagenic effects are induced by the chosen solvent.

1.4.3. Procedure

1.4.3.

1.4.3.1. Treatment with the test substance Proliferating cells are treated with the test substance in the presence and absence of a metabolic activation system. Treatment of lymphocytes should commence at about 48 hours after mitogenic stimulation.

1.4.3.2. Duplicate cultures should normally be used at each concentration, and are strongly recommended for negative/solvent control cultures. Where minimal variation between duplicate cultures can be demonstrated (13)(14), from historical data, it may be acceptable for single cultures to be used at each concentration. Gaseous or volatile substances should be tested by appropriate methods, such as in sealed culture vessels (15)(16).

1.4.3.3. Culture harvest time In the first experiment, cells should be exposed to the test substance, both with and without metabolic activation, for three to six hours, and sampled at a time equivalent to about 1,5 normal cell cycle length after the beginning of treatment (12). If this protocol gives negative results both with and without activation, an additional experiment without activation should be done, with continuous treatment until sampling at a time equivalent to about 1,5 normal cell cycle lengths. Certain chemicals may be more readily detected by treatment/sampling times longer than 1,5 cycle lengths. Negative results with metabolic activation need to be confirmed on a case-by-case basis. In those cases where confirmation of negative results is not considered necessary, justification should be provided.

1.4.3.4. Chromosome preparation Cell cultures are treated with Colcemid® or colchicine usually for one to three hours prior to harvesting. Each cell culture is harvested and processed separately for the preparation of chromosomes. Chromosome preparation involves hypotonic treatment of the cells, fixation and staining.

1.4.3.5. Analysis All slides, including those of positive and negative controls, should be independently coded before microscopic analysis. Since fixation procedures often result in the breakage of a proportion of metaphase cells with loss of chromosomes, the cells scored should therefore contain a number of centromeres equal to the modal number ± 2 for all cell types. At least 200 well spread metaphases should be scored per concentration and control, equally divided amongst the duplicates, if applicable. This number can be reduced when high number of aberrations is observed. Though the purpose of the test is to detect structural chromosome aberrations, it is important to record polyploidy and endoreduplication when these events are seen.

2. DATA

2.

2.1. TREATMENT OF RESULTS The experimental unit is the cell, and therefore the percentage of cells with structural chromosome aberration(s) should be evaluated. Different types of structural chromosome aberrations should be listed with their numbers and frequencies for experimental and control cultures. Gaps are recorded separately and reported but generally not included in the total aberration frequency. Concurrent measures of cytotoxicity for all treated and negative control cultures in the main aberration experiments should also be recorded. Individual culture data should be provided. Additionally, all data should be summarised in tabular form. There is no requirement for verification of a clear positive response. Equivocal results should be clarified by further testing preferably using modification of experimental conditions. The need to confirm negative results has been discussed in 1.4.3.3. Modification of study parameters to extend the range of conditions assessed should be considered in follow-up experiments. Study parameters that might be modified include the concentration spacing and the metabolic activation conditions.

2.2. EVALUATION AND INTERPRETATION OF RESULTS There are several criteria for determining a positive result, such as a concentration-related increase or a reproducible increase in the number of cells with chromosome aberrations. Biological relevance of the results should be considered first. Statistical methods may be used as an aid in evaluating the test results (3)(13). Statistical significance should not be the only determining factor for a positive response. An increase in the number of polyploid cells may indicate that the test substance has the potential to inhibit mitotic processes and to induce numerical chromosome aberrations. An increase in the number of cells with endoreduplicated chromosomes may indicate that the test substance has the potential to inhibit cell cycle progression (17)(18). A test substance for which the results do not meet the above criteria is considered non-mutagenic in this system. Although most experiments will give clearly positive or negative results, in rare cases the data set will preclude making a definite judgement about the activity of the test substance. Results may remain equivocal or questionable regardless of the number of times the experiment is repeated. Positive results from the in vitro chromosome aberration test indicate that the test substance induces structural chromosome aberrations in cultured mammalian somatic cells. Negative results indicate that, under the test conditions, the test substance does not induce chromosome aberrations in cultured mammalian somatic cells.

3. REPORTING

3. TEST REPORT

The test report must include the following information:

Solvent/Vehicle:

justification for choice of vehicle,

solubility and stability of the test substance in solvent/vehicle, if known.

Cells:

type and source of cells,

karyotype features and suitability of the cell type used,

absence of mycoplasma, if applicable,

information on cell cycle length,

sex of blood donors, whole blood or separated lymphocytes, mitogen used,

number of passages, if applicable,

methods for maintenance of cell culture, if applicable,

modal number of chromosomes.

Test conditions:

identity of metaphase arresting substance, its concentration and duration of cell exposure,

rationale for selection of concentrations and number of cultures including, e.g. cytotoxicity data and solubility limitations, if available,

composition of media, CO2 concentration if applicable,

concentration of test substance,

volume of vehicle and test substance added,

incubation temperature,

incubation time,

duration of treatment,

cell density at seeding, if appropriate,

type and composition of metabolic activation system, including acceptability criteria,

positive and negative controls,

methods of slide preparation,

criteria for scoring aberrations,

number of metaphases analysed,

methods for the measurements of toxicity,

criteria for considering studies as positive, negative or equivocal.

Results:

signs of toxicity, e.g. degree of confluency, cell cycle data, cell counts, mitotic index,

signs of precipitation,

data on pH and osmolality of the treatment medium, if determined,

definition for aberrations, including gaps,

number of cells with chromosome aberrations and type of chromosome aberrations given separately for each treated and control culture,

changes in ploidy if seen,

dose-response relationship, where possible,

statistical analyses, if any,

concurrent negative (solvent/vehicle) and positive control data,

historical negative (solvent/vehicle) and positive control data, with ranges, means and standard deviations.

Discussion of results.

Conclusions.

4.(1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)

REFERENCES Evans, H. J., (1976) Cytological Methods for Detecting Chemical Mutagens. In: Chemical mutagens, Principles and Methods for their Detection, Vol. 4, Hollaender, A. (ed) Plenum Press, New York and London, p. 1-29. Ishidate, M. Jr. and Sofuni, T., (1985). The In Vitro Chromosomal Aberration Test Using Chinese Hamster Lung (CHL) Fibroblast Cells in Culture. In: Progress in Mutation Research, Vol. 5, Ashby, J. et al., (Eds) Elsevier Science Publishers, Amsterdam-New York-Oxford, p. 427-432. Galloway, S.M., Armstrong, M.J., Reuben, C., Colman, S., Brown, B., Cannon, C., Bloom, A.D., Nakamura, F., Ahmed, M., Duk, S., Rimpo, J., Margolin, G.H., Resnick, MA, Anderson, G. and Zeiger, E., (1978). Chromosome aberration and sister chromatic exchanges in Chinese hamster ovary cells: Evaluation of 108 chemicals. Environs. Molec. Mutagen 10 (suppl. 10), p. 1-175. Scott, D., Galloway, S.M., Marshall, R.R., Ishidate, M.Jr., Brusick, D., Ashby, J. and Myhr, B.C., (1991) Genotoxicity under Extreme Culture Conditions. A report from ICPEMC Task Group 9. Mutation Res, 257, p. 147-204. Morita, T., Nagaki, T., Fukuda, I. and Okumura, K., (1992). Clastogenicity of low pH to Various Cultured Mammalian Cells. Mutation Res., 268, p. 297-305. Ames, B.N., McCann, J. and Yamasaki, E., (1975). Methods for Detecting Carcinogens and Mutagens with the Salmonella/Mammalian Microsome Mutagenicity Test. Mutation Res., 31, p. 347-364. Maron, D.M. and Ames, B.N., (1983) Revised Methods for the Salmonella Mutagenicity Test. Mutation Res., 113, p. 173-215. Natarajan, A.T., Tates, A.D., van Buul, P.P.W., Meijers, M. and de Vogel, N., (1976) Cytogenetic Effects of Mutagen/Carcinogens after Activation in a Microsomal System in Vitro, I. Induction of Chromosome Aberrations and Sister Chromatid Exchange by Diethylnitrosamine (DEN) and Dimethylnitrosamine (DMN) in CHO Cells in the Presence of Rat-Liver Microsomes. Mutation Res., 37, p. 83-90. Matsuoka, A., Hayashi, M. and Ishidate, M. Jr., (1979) Chromosomal Aberration Tests on 29 Chemicals Combined with S9 Mix In Vitro. Mutation Res., 66, p. 277-290. Elliot, B.M., Combes, R.D., Elcombe, C.R., Gatehouse, D.G., Gibson, G.G., Mackay, J.M. and Wolf, R.C., (1992). Report of UK Environmental Mutagen Society Working Party. Alternatives to Aroclor 1254-induced S9 in In Vitro Genotoxicity Assays. Mutagenesis, 7, p. 175-177. Matsushima, T., Sawamura, M., Hara, K. and Sugimura, T., (1976) A Safe Substitute for Polychlorinated Biphenyls as an Inducer of Metabolic Activation Systems. In: de Serres, F.J., Fouts, J.R., Bend, J.R. and Philpot, R.M. (eds) In Vitro Metabolic Activation in Mutagenesis Testing, Elsevier, North-Holland, p. 85-88. Galloway, S.M., Aardema, M.J., Ishidate, M.Jr., Ivett, J.L., Kirkland, D.J., Morita, T., Mosesso, P., Sofuni, T., (1994) Report from Working Group on in In Vitro Tests for Chromosomal Aberrations. Mutation Res., 312, p. 241-261. Richardson, C., Williams, D.A., Allen, J.A., Amphlett, G., Chanter, D.O. and Phillips, B., (1989) Analysis of Data from In Vitro Cytogenetic Assays. In: Statistical Evaluation of Mutagenicity Test Data. Kirkland, D.J., (ed) Cambridge University Press, Cambridge, p. 141-154. Soper, K.A. and Galloway, S.M., (1994) Replicate Flasks are not Necessary for In Vitro Chromosome Aberration Assays in CHO Cells. Mutation Res., 312, p. 139-149. Krahn, D.F., Barsky, F.C. and McCooey, K.T., (1982) CHO/HGPRT Mutation Assay: Evaluation of Gases and Volatile Liquids. In: Tice, R.R., Costa, D.L., Schaich, K.M. (eds). Genotoxic Effects of Airborne Agents. New York, Plenum, p. 91-103. Zamora, P.O., Benson, J.M., Li, A.P. and Brooks, A.L., (1983) Evaluation of an Exposure System Using Cells Grown on Collagen Gels for Detecting Highly Volatile Mutagens in the CHO/HGPRT Mutation Assay. Environmental Mutagenesis, 5, p. 795-801. Locke-Huhle, C., (1983) Endoreduplication in Chinese hamster cells during alpha-radiation induced G2 arrest. Mutation Res., 119, p. 403-413. Huang, Y., Change, C. and Trosko, J.E., (1983) Aphidicolin-induced endoreduplication in Chinese hamster cells. Cancer Res., 43, p. 1362-1364.

B.11. MUTAGENICITY — IN VIVO MAMMALIAN BONE MARROW CHROMOSOME ABERRATION TEST

B.11.

1. METHOD This method is a replicate of the OECD TG 475, Mammalian Bone Marrow Chromosome Aberration Test (1997).

1.

1.1. INTRODUCTION The mammalian in vivo chromosome aberration test is used for the detection of structural chromosome aberrations induced by the test substance to the bone marrow cells of animals, usually rodents (1)(2)(3)(4). Structural chromosome aberrations may be of two types, chromosome or chromatid. An increase in polyploidy may indicate that a chemical has the potential to induce numerical aberrations. With the majority of chemical mutagens, induced aberrations are of the chromatid-type, but chromosome-type aberrations also occur. Chromosome mutations and related events are the cause of many human genetic diseases and there is substantial evidence that chromosome mutations and related events causing alterations in oncogenes and tumour-suppressor genes are involved in cancer in humans and experimental systems. Rodents are routinely used in this test. Bone marrow is the target tissue in this test, since it is a highly vascularised tissue, and it contains a population of rapidly cycling cells that can be readily isolated and processed. Other species and target tissues are not the subject of this method. This chromosome aberration test is especially relevant to assessing mutagenic hazard in that it allows consideration of factors of in vivo metabolism, pharmacokinetics and DNA-repair processes although these may vary among species and among tissues. An in vivo test is also useful for further investigation of a mutagenic effect detected by in vitro test. If there is evidence that the test substance, or a reactive metabolite, will not reach the target tissue, it is not appropriate to use this test. See also General introduction Part B.

1.2. DEFINITIONS Chromatid-type aberration: structural chromosome damage expressed as breakage of single chromatids or breakage and reunion between chromatids. Chromosome-type aberration: structural chromosome damage expressed as breakage, or breakage and reunion, of both chromatids at an identical site. Endoreduplication: a process in which after an S period of DNA replication, the nucleus does not go into mitosis but starts another S period. The result is chromosomes with four, eight, 16, ...chromatids. Gap: an achromatic lesion smaller than the width of one chromatid, and with minimum misalignment of the chromatid(s). Numerical aberration: a change in the number of chromosomes from the normal number characteristic of the cells utilised. Polyploidy: a multiple of the haploid chromosome number (n) other than the diploid number (i.e. 3n, 4n and so on). Structural aberration: a change in chromosome structure detectable by microscopic examination of the metaphase stage of cell division, observed as deletions and fragments, intrachanges or interchanges.

1.3. PRINCIPLE OF THE TEST METHOD Animals are exposed to the test substance by an appropriate route of exposure and are sacrificed at appropriate times after treatment. Prior to sacrifice, animals are treated with a metaphase arresting agent (e.g. Colcemid® or colchicine). Chromosome preparations are then made from the bone marrow cells and stained, and metaphase cells are analysed for chromosome aberrations.

1.4. DESCRIPTION OF THE TEST METHOD

1.4.

1.4.1. Preparations

1.4.1.

1.4.1.1. Selection of animal species Rats, mice and Chinese hamsters are commonly used, although any appropriate mammalian species may be used. Commonly used laboratory strains of young healthy adult animals should be employed. At the commencement of the study, the weight variation of animals should be minimal and not exceed ± 20 % of the mean weight of each sex.

1.4.1.2. Housing and feeding conditions General conditions referred in the General introduction to Part B are applied although the aim for humidity should be 50-60 %.

1.4.1.3. Preparation of the animals Healthy young adult animals are randomly assigned to the control and treatment groups. Cages should be arranged in such a way that possible effects due to cage placement are minimised. The animals are identified uniquely. The animals are acclimated to the laboratory conditions for at least five days.

1.4.1.4. Preparation of doses Solid test substances should be dissolved or suspended in appropriate solvents or vehicles and diluted, if appropriate, prior to dosing of the animals. Liquid test substances may be dosed directly or diluted prior to dosing. Fresh preparations of the test substance should be employed unless stability data demonstrate the acceptability of storage.

1.4.2. Test conditions

1.4.2.

1.4.2.1. Solvent/Vehicle The solvent/vehicle should not produce toxic effects at the dose levels used, and should not be suspected of chemical reaction with the test substance. If other than well-known solvents/vehicles are used, their inclusion should be supported with data indicating their compatibility. It is recommended that wherever possible, the use of an aqueous solvent/vehicle should be considered first.

1.4.2.2. Controls Concurrent positive and negative (solvent/vehicle) controls should be included for each sex in each test. Except for treatment with the test substance, animals in the control groups should be handled in an identical manner to the animals in the treated groups. Positive controls should produce structural aberrations in vivo at exposure levels expected to give a detectable increase over background. Positive control doses should be chosen so that the effects are clear but do not immediately reveal the identity of the coded slides to the reader. It is acceptable that the positive control be administered by a route different from the test substance and sampled at only a single time. The use of chemical class related positive control chemicals may be considered, when available. Examples of positive control substances include: Substance CAS No EINECS No Ethyl methanesulphonate 62-50-0 200-536-7 Ethyl nitrosourea 759-73-9 212-072-2 Mitomycin C 50-07-7 200-008-6 Cyclophosphamide Cyclophosphamide monohydrate 50-18-0 6055-19-2 200-015-4 Triethylenemelamine 51-18-3 200-083-5 Negative controls, treated with solvent or vehicle alone, and otherwise treated in the same way as the treatment groups, should be included for every sampling time, unless acceptable inter-animal variability and frequencies of cells with chromosome aberrations are available from historical control data. If single sampling is applied for negative controls, the most appropriate time is the first sampling time. In addition, untreated controls should also be used unless there are historical or published control data demonstrating that no deleterious or mutagenic effects are induced by the chosen solvent/vehicle.

1.5. PROCEDURE

1.5.

1.5.1. Number and sex of animals Each treated and control group must include at least five analysable animals per sex. If at the time of the study there are data available from studies in the same species and using the same route of exposure that demonstrate that there are no substantial differences in toxicity between sexes, then testing in a single sex will be sufficient. Where human exposure to chemicals may be sex-specific, as for example with some pharmaceutical agents, the test should be performed with animals of the appropriate sex.

1.5.2. Treatment schedule Test substances are preferably administered as a single treatment. Test substances may also be administered as a split dose, i.e. two treatments on the same day separated by no more than a few hours, to facilitate administering a large volume of material. Other dose regimens should be scientifically justified. Samples should be taken at two separate times following treatment on one day. For rodents, the first sampling interval is 1,5 normal cell cycle length (the latter being normally 12-18 hr) following treatment. Since the time required for uptake and metabolism of the test substance as well as its effect on cell cycle kinetics can affect the optimum time for chromosome aberration detection, a later sample collection 24 hr after the first sample time is recommended. If dose regimens of more than one day are used, one sampling time at 1,5 normal cell cycle lengths after the final treatment should be used. Prior to sacrifice, animals are injected intraperitoneally with an appropriate dose of a metaphase arresting agent (e.g. Colcemid® or colchicine). Animals are sampled at an appropriate interval thereafter. For mice this interval is approximately three to five hours; for Chinese hamsters this interval is approximately four to five hours. Cells are harvested from the bone marrow and analysed for chromosome aberrations.

1.5.3. Dose levels If a range finding study is performed because there are no suitable data available, it should be performed in the same laboratory, using the same species, strain, sex and treatment regimen to be used in the main study (5). If there is toxicity, three dose levels are used for the first sampling time. These dose levels should cover a range from the maximum to little or no toxicity. At the later sampling time only the highest dose needs to be used. The highest dose is defined as the dose producing signs of toxicity such that higher dose levels, based on the same dosing regimen, would be expected to produce lethality. Substances with specific biological activities at low non-toxic doses (such as hormones and mitogens) may be exceptions to the dose-setting criteria and should be evaluated on a case-by-case basis. The highest dose may also be defined as a dose that produces some indication of toxicity in the bone marrow (e.g. greater than 50 % reduction in mitotic index).

1.5.4. Limit test If a test at one dose level of at least 2000 mg/kg body weight using a single treatment, or as two treatments on the same day, produces no observable toxic effects, and if genotoxicity would not be expected based on data from structurally related substances, then a full study using three dose levels may not be considered necessary. For studies of a longer duration, the limit dose is 2000 mg/kg/body weight/day for treatment up to 14 days, and 1000 mg/kg/body weight/day for treatment longer than 14 days. Expected human exposure may indicate the need for a higher dose level to be used in the limit test.

1.5.5. Administration of doses The test substance is usually administered by gavage using a stomach tube or a suitable intubation cannula, or by intraperitoneal injection. Other routes of exposure may be acceptable where they can be justified. The maximum volume of liquid that can be administered by gavage or injection at one time depends on the size of the test animal. The volume should not exceed 2 ml/100 g body weight. The use of volumes higher than these must be justified. Except for irritating or corrosive substances which will normally reveal exacerbated effects with higher concentrations, variability in test volume should be minimised by adjusting the concentration to ensure a constant volume at all dose levels.

1.5.6. Chromosome preparation Immediately after sacrifice, bone marrow is obtained, exposed to hypotonic solution and fixed. The cells are then spread on slides and stained.

1.5.7. Analysis The mitotic index should be determined as a measure of cytotoxicity in at least 1000 cells per animal for all treated animals (including positive controls) and untreated negative control animals. At least 100 cells should be analysed for each animal. This number could be reduced when high numbers of aberrations are observed. All slides, including those of positive and negative controls, should be independently coded before microscopic analysis. Since slide preparation procedures often result in the breakage of a proportion of metaphases with loss of chromosomes, the cells scored should therefore contain a number of centromeres equal to the number 2n ± 2.

2. DATA

2.

2.1. TREATMENT OF RESULTS Individual animal data should be presented in tabular form. The experimental unit is the animal. For each animal the number of cells scored, the number of aberrations per cell and the percentage of cells with structural chromosome aberration(s) should be evaluated. Different types of structural chromosome aberrations should be listed with their numbers and frequencies for treated and control groups. Gaps are recorded separately and reported but generally not included in the total aberration frequency. If there is no evidence for a difference in response between the sexes, the data from both sexes may be combined for statistical analysis.

2.2. EVALUATION AND INTERPRETATION OF RESULTS There are several criteria for determining a positive result, such as a dose-related increase in the relative number of cells with chromosome aberrations or a clear increase in the number of cells with aberrations in a single dose group at a single sampling time. Biological relevance of the results should be considered first. Statistical methods may be used as an aid in evaluating the test results (6). Statistical significance should not be the only determining factor for a positive response. Equivocal results should be clarified by further testing preferably using a modification of experimental conditions. An increase in polyploidy may indicate that the test substance has the potential to induce numerical chromosome aberrations. An increase in endoreduplication may indicate that the test substance has the potential to inhibit cell cycle progression (7)(8). A test substance for which the results do not meet the above criteria is considered non-mutagenic in this test. Although most experiments will give clearly positive or negative results, in rare cases the data set will preclude making a definite judgement about the activity of the test substance. Results may remain equivocal or questionable regardless of the number of experiments performed. Positive results from the in vivo chromosome aberration test indicate that a substance induces chromosome aberrations in the bone marrow of the species tested. Negative results indicate that, under the test conditions, the test substance does not induce chromosome aberrations in the bone marrow of the species tested. The likelihood that the test substance or its metabolites reach the general circulation or specifically the target tissue (e.g. systemic toxicity) should be discussed.

3. REPORTING

3.

3.1. TEST REPORT The test report must include the following information:

Solvent/Vehicle:

justification for choice of vehicle,

solubility and stability of the test substance in solvent/vehicle, if known,

Test animals:

species/strain used,

number, age and sex of animals,

source, housing conditions, diet, etc.,

individual weight of the animals at the start of the test, including body weight range, mean and standard deviation for each group,

Test conditions:

positive and negative (vehicle/solvent) controls,

data from range-finding study, if conducted,

rationale for dose level selection,

details of test substance preparation,

details of the administration of the test substance,

rationale for route of administration,

methods for verifying that the test substance reached the general circulation or target tissue, if applicable,

conversion from diet/drinking water test substance concentration (ppm) to the actual dose (mg/kg body weight/day), if applicable,

details of food and water quality,

detailed description of treatment and sampling schedules,

methods for measurements of toxicity,

identity of metaphase arresting substance, its concentration and duration of treatment,

methods of slide preparation,

criteria for scoring aberrations,

number of cells analysed per animal,

criteria for considering studies as positive, negative or equivocal.

Results:

signs of toxicity,

mitotic index,

type and number of aberrations, given separately for each animal,

total number of aberrations per group with means and standard deviations,

number of cells with aberrations per group with means and standard deviations,

changes in ploidy, if seen,

dose-response relationship, where possible,

statistical analyses, if any,

concurrent negative control data,

historical negative control data with ranges, means and standard deviations,

concurrent positive control data.

Discussion of the results.

Conclusions.

4.(1)(2)(3)(4)(5)(6)(7)(8)

REFERENCES Adler, I.D., (1984) Cytogenetic Tests in Mammals. In: Mutagenicity Testing: a Practical Approach. S. Venitt and J.M. Parry (Eds). IRL Press, Oxford, Washington D.C., p. 275-306. Preston, R.J., Dean, B.J., Galloway, S., Holden, H., McFee, A.F. and Shelby, M. (1987). Mammalian In Vivo Cytogenetic Assays: Analysis of Chromosome Aberrations in Bone Marrow Cells. Mutation Res., 189, p. 157-165. Richold, M., Chandley, A., Ashby, J., Gatehouse, D.G., Bootman, J. and Henderson, L., (1990) In Vivo Cytogenetic Assays. In: D.J. Kirkland (Ed.) Basic Mutagenicity Tests, UKEMS Recommended Procedures. UKEMS Sub-Committee on Guidelines for Mutagenicity Testing. Report. Part I revised. Cambridge University Press, Cambridge, New York, Port Chester, Melbourne, Sydney, p. 115-141. Tice, R.R., Hayashi, M., MacGregor, J.T., Anderson, D., Blakey, D.H., Holden, H.E., Kirsch-Volders, M., Oleson Jr., F.B., Pacchierotti, F., Preston, R.J., Romagna, F., Shimada, H., Sutou, S. and Vannier, B., (1994) Report from the Working Group on the In Vivo Mammalian Bone Marrow Chromosomal Aberration Test. Mutation Res., 312, p. 305-312. Fielder, R.J., Allen, J.A., Boobis, A.R., Botham, P.A., Doe, J., Esdaile, D.J., Gatehouse, D.G., Hodson-Walker, G., Morton, D.B., Kirkland, D.J. and Richold, M. (1992). Report of British Toxicology Society/UK Environmental Mutagen Society Working Group: Dose setting in In vivo Mutagenicity Assays. Mutagenesis, 7, p. 313-319. Lovell, D.P., Anderson, D., Albanese, R., Amphlett, G.E., Clare, G., Ferguson, R., Richold, M., Papworth, D.G. and Savage, J.R.K. (1989). Statistical Analysis of In Vivo Cytogenetic Assays. In: UKEMS Sub-Committee on Guidelines for Mutagenicity Testing. Report Part III. Statistical Evaluation of Mutagenicity Test Data. D.J. Kirkland, (Ed.) Cambridge University Press, Cambridge. p. 184-232. Locke-Huhle, C., (1983) Endoreduplication in Chinese hamster cells during alpha-radiation induced G2 arrest. Mutation Res. 119, p. 403-413. Huang, Y., Change, C. and Trosko, J.E., (1983) Aphidicolin-induced endoreduplication in Chinese hamster cells. Cancer Res., 43, p. 1362-1364.

B.12. MUTAGENICITY — IN VIVO MAMMALIAN ERYTHROCYTE MICRONUCLEUS TEST

B.12.

1. METHOD This method is a replicate of the OECD TG 474, Mammalian Erythrocyte Micronucleus Test (1997).

1.

1.1. INTRODUCTION The mammalian in vivo micronucleus test is used for the detection of damage induced by the test substance to the chromosomes or the mitotic apparatus of erythroblasts by analysis of erythrocytes as sampled in bone marrow and/or peripheral blood cells of animals, usually rodents. The purpose of the micronucleus test is to identify substances that cause cytogenetic damage, which results in the formation of micronuclei containing lagging chromosome fragments or whole chromosomes. When a bone marrow erythroblast develops into a polychromatic erythrocyte, the main nucleus is extruded; any micronucleus that has been formed may remain behind in the otherwise anucleated cytoplasm. Visualisation of micronuclei is facilitated in these cells because they lack a main nucleus. An increase in the frequency of micronucleated polychromatic erythrocytes in treated animals is an indication of induced chromosome damage. The bone marrow of rodents is routinely used in this test since polychromatic erythrocytes are produced in that tissue. The measurement of micronucleated immature (polychromatic) erythrocytes in peripheral blood is equally acceptable in any species in which the inability of the spleen to remove micronucleated erythrocytes has been demonstrated, or which has shown an adequate sensitivity to detect agents that cause structural or numerical chromosome aberrations. Micronuclei can be distinguished by a number of criteria. These include identification of the presence or absence of a kinetochore or centromeric DNA in the micronuclei. The frequency of micronucleated immature (polychromatic) erythrocytes is the principal endpoint. The number of mature (normochromatic) erythrocytes in the peripheral blood that contain micronuclei among a given number of mature erythrocytes can also be used as the endpoint of the assay when animals are treated continuously for four weeks or more. This mammalian in vivo micronucleus test is especially relevant to assessing mutagenic hazard in that it allows consideration of factors of in vivo metabolism, pharmacokinetics and DNA-repair processes although these may vary among species, among tissues and among genetic endpoints. An in vivo assay is also useful for further investigation of a mutagenic effect detected by an in vitro system. If there is evidence that the test substance, or a reactive metabolite, will not reach the target tissue, it is not appropriate to use this test. See also General introduction Part B.

1.2. DEFINITIONS Centromere (Kinetochore): region(s) of a chromosome with which spindle fibers are associated during cell division, allowing orderly movement of daughter chromosomes to the poles of the daughter cells. Micronuclei: small nuclei, separate from and additional to the main nuclei of cells, produced during telophase of mitosis (meiosis) by lagging chromosome fragments or whole chromosomes. Normochromatic erythrocyte: mature erythrocyte that lacks ribosomes and can be distinguished from immature, polychromatic erythrocytes by stains selective for ribosomes. Polychromatic erythrocyte: immature erythrocyte, in an intermediate stage of development, that still contains ribosomes and therefore can be distinguished from mature, normochromatic erythrocytes by stains selective for ribosomes.

1.3. PRINCIPLE OF THE TEST METHOD Animals are exposed to the test substance by an appropriate route. If bone marrow is used, the animals are sacrificed at appropriate times after treatment, the bone marrow extracted, and preparations made and stained (1)(2)(3)(4)(5)(6)(7). When peripheral blood is used, the blood is collected at appropriate times after treatment and smear preparations are made and stained (4)(8)(9)(10). For studies with peripheral blood, as little time as possible should elapse between the last exposure and cell harvest. Preparations are analysed for the presence of micronuclei.

1.4. DESCRIPTION OF THE TEST METHOD

1.4.

1.4.1. Preparations

1.4.1.

1.4.1.1. Selection of animal species Mice or rats are recommended if bone marrow is used, although any appropriate mammalian species may be used. When peripheral blood is used, mice are recommended. However, any appropriate mammalian species may be used provided it is a species in which the spleen does not remove micronucleated erythrocytes or a species, which has shown an adequate sensitivity to detect agents that cause structural or numerical chromosome aberrations. Commonly used laboratory strains of young healthy animals should be employed. At the commencement of the study, the weight variation of animals should be minimal and not exceed ±20 % of the mean weight of each sex.

1.4.1.2. Housing and feeding conditions General conditions referred in the General introduction to Part B are applied although the aim for humidity should be 50-60 %.

1.4.1.3. Preparation of the animals Healthy young adult animals are randomly assigned to the control and treatment groups. The animals are identified uniquely. The animals are acclimated to the laboratory conditions for at least five days. Cages should be arranged in such a way that possible effects due to cage placement are minimised.

1.4.2. Test conditions

1.4.2.

1.4.2.1. Solvent/Vehicle The solvent/vehicle should not produce toxic effects at the dose levels used, and should not be suspected of chemical reaction with the test substance. If other than well-known solvents/vehicles are used, their inclusion should be supported with reference data indicating their compatibility. It is recommended that wherever possible, the use of an aqueous solvent/vehicle should be considered first.

1.4.2.2. Controls Concurrent positive and negative (solvent/vehicle) controls should be included for each sex in each test. Except for treatment with the test substance, animals in the control groups should be handled in an identical manner to animals of the treatment groups. Positive controls should produce micronuclei in vivo at exposure levels expected to give a detectable increase over background. Positive control doses should be chosen so that the effects are clear but do not immediately reveal the identity of the coded slides to the reader. It is acceptable that the positive control be administered by a route different from the test substance and sampled at only a single time. In addition, the use of chemical class-related positive control chemicals may be considered, when available. Examples of positive control substances include: Substance CAS No EINECS No Ethyl methanesulphonate 62-50-0 200-536-7 N-ethyl-N-nitrosourea 759-73-9 212-072-2 Mitomycin C 50-07-7 200-008-6 Cyclophosphamide Cyclophosphamide monohydrate 50-18-0 6055-19-2 200-015-4 Triethylenemelamine 51-18-3 200-083-5 Negative controls, treated with solvent or vehicle alone, and otherwise treated in the same way as the treatment groups should be included for every sampling time, unless acceptable inter-animal variability and frequencies of cells with micronuclei are demonstrated by historical control data. If single sampling is applied for negative controls, the most appropriate time is the first sampling time. In addition, untreated controls should also be used unless there are historical or published control data demonstrating that no deleterious or mutagenic effects are induced by the chosen solvent/vehicle. If peripheral blood is used, a pre-treatment sample may also be acceptable as a concurrent negative control, but only in the short peripheral blood studies (e.g. 1-3 treatment(s)) when the resulting data are in the expected range for the historical control.

1.5. PROCEDURE

1.5.

1.5.1. Number and sex of animals Each treated and control group must include at least five analysable animals per sex (11). If at the time of the study there are data available from studies in the same species and using the same route of exposure that demonstrate that there are no substantial differences between sexes in toxicity, then testing in a single sex will be sufficient. Where human exposure to chemicals may be sex-specific, as for example with some pharmaceutical agents, the test should be performed with animals of the appropriate sex.

1.5.2. Treatment schedule No standard treatment schedule (i.e. one, two or more treatments at 24 h intervals) can be recommended. The samples from extended dose regimens are acceptable as long as a positive effect has been demonstrated for this study or, for a negative study, as long as toxicity has been demonstrated or the limit dose has been used, and dosing continued until the time of sampling. Test substances may also be administered as a split dose, i.e., two treatments on the same day separated by no more than a few hours, to facilitate administering a large volume of material. The test may be performed in two ways: Other sampling times may be used in addition, when relevant.

1.5.2.

(a) animals are treated with the test substance once. Samples of bone marrow are taken at least twice, starting not earlier than 24 hours after treatment, but not extending beyond 48 hours after treatment with appropriate intervals between samples. The use of sampling times earlier than 24 hours after treatment should be justified. Samples of peripheral blood are taken at least twice, starting not earlier than 36 hours after treatment, with appropriate intervals following the first sample, but not extending beyond 72 hours. When a positive response is recognised at one sampling time, additional sampling is not required.

(b) if two or more daily treatments are used (e.g. two or more treatments at 24 hour intervals), samples should be collected once between 18 and 24 hours following the final treatment for the bone marrow and once between 36 and 48 hours following the final treatment for the peripheral blood (12);

1.5.3. Dose levels If a range finding study is performed because there are no suitable data available, it should be performed in the same laboratory, using the same species, strain, sex, and treatment regimen to be used in the main study (13). If there is toxicity, three dose levels are used for the first sampling time. These dose levels should cover a range from the maximum to little or no toxicity. At the later sampling time only the highest dose needs to be used. The highest dose is defined as the dose producing signs of toxicity such that higher dose levels, based on the same dosing regimen, would be expected to produce lethality. Substances with specific biological activities at low non-toxic doses (such as hormones and mitogens) may be exceptions to the dose-setting criteria and should be evaluated on a case-by-case basis. The highest dose may also be defined as a dose that produces some indication of toxicity in the bone marrow (e.g. a reduction in the proportion of immature erythrocytes among total erythrocytes in the bone marrow or peripheral blood).

1.5.4. Limit test If a test at one dose level of at least 2000 mg/kg body weight using a single treatment, or as two treatments on the same day, produces no observable toxic effects, and if genotoxicity would not be expected based upon data from structurally related substances, then a full study using three dose levels may not be considered necessary. For studies of a longer duration, the limit dose is 2000 mg/kg/body weight/day for treatment up to 14 days, and 1000 mg/kg/body weight/day for treatment longer than 14 days. Expected human exposure may indicate the need for a higher dose level to be used in the limit test.

1.5.5. Administration of doses The test substance is usually administered by gavage using a stomach tube or a suitable intubation cannula, or by intraperitoneal injection. Other routes of exposure may be acceptable where they can be justified. The maximum volume of liquid that can be administered by gavage or injection at one time depends on the size of the test animal. The volume should not exceed 2 ml/100 g body weight. The use of volumes higher than these must be justified. Except for irritating or corrosive substances, which will normally reveal exacerbated effects with higher concentrations, variability in test volume should be minimised by adjusting the concentration to ensure a constant volume at all dose levels.

1.5.6. Bone marrow/blood preparation Bone marrow cells are usually obtained from the femurs or tibias immediately following sacrifice. Commonly, cells are removed from femurs or tibias, prepared and stained using established methods. Peripheral blood is obtained from the tail vein or other appropriate blood vessel. Blood cells are immediately stained supravitally (8)(9)(10) or smear preparations are made and then stained. The use of a DNA specific stain (e.g. acridine orange (14) or Hoechst 33258 plus pyronin-Y (15)) can eliminate some of the artifacts associated with using a non DNA specific stain. This advantage does not preclude the use of conventional stains (e.g. Giemsa). Additional systems (e.g. cellulose columns to remove nucleated cells (16)) can also be used provided that these systems have been shown to adequately work for micronucleus preparation in the laboratory.

1.5.7. Analysis The proportion of immature among total (immature + mature) erythrocytes is determined for each animal by counting a total of at least 200 erythrocytes for bone marrow and 1000 erythrocytes for peripheral blood (17). All slides, including those of positive and negative controls, should be independently coded before microscopic analysis. At least 2000 immature erythrocytes per animal are scored for the incidence of micronucleated immature erythrocytes. Additional information may be obtained by scoring mature erythrocytes for micronuclei. When analysing slides, the proportion of immature erythrocytes among total erythrocytes should not be less than 20 % of the control value. When animals are treated continuously for four weeks or more, at least 2000 mature erythrocytes per animal can also be scored for the incidence of micronuclei. Systems for automated analysis (image analysis and cell suspensions flow cytometry) are acceptable alternatives to manual evaluation if appropriately justified and validated.

2. DATA

2.

2.1. TREATMENT OF RESULTS Individual animal data should be presented in tabular form. The experimental unit is the animal. The number of immature erythrocytes scored, the number of micronucleated immature erythrocytes, and the number of immature among total erythrocytes should be listed separately for each animal analysed. When animals are treated continuously for four weeks or more, the data on mature erythrocytes should also be given if it is collected. The proportion of immature among total erythrocytes and, if considered applicable, the percentage of micronucleated erythrocytes is given for each animal. If there is no evidence for a difference in response between the sexes, the data from both sexes may be combined for statistical analysis.

2.2. EVALUATION AND INTERPRETATION OF RESULTS There are several criteria for determining a positive result, such as a dose-related increase in the number of micronucleated cells or a clear increase in the number of micronucleated cells in a single dose group at a single sampling time. Biological relevance of the results should be considered first. Statistical methods may be used as an aid in evaluating the test results (18)(19). Statistical significance should not be the only determining factor for a positive response. Equivocal results should be clarified by further testing preferably using a modification of experimental conditions. A test substance for which the results do not meet the above criteria is considered non-mutagenic in this test. Although most experiments will give clearly positive or negative results, in rare cases the data set will preclude making a definite judgement about the activity of the test substance. Results may remain equivocal or questionable regardless of the number of times the experiment is repeated. Positive results in the micronucleus test indicate that the substance induces micronuclei which are the result of chromosomal damage or damage to the mitotic apparatus in the erythroblasts of the test species. Negative results indicate that, under the test conditions, the test substance does not produce micronuclei in the immature erythrocytes of the test species. The likelihood that the test substance or its metabolites reach the general circulation or specifically the target tissue (e.g. systemic toxicity) should be discussed.

3. REPORTING

3. TEST REPORT

The test report should include the following information:

Solvent/Vehicle:

justification for choice of vehicle,

solubility and stability of the test substance in solvent/vehicle, if known.

Test animals:

species/strain used,

number, age and sex of animals,

source, housing conditions, diet, etc.,

individual weight of the animals at the start of the test, including body weight range, mean and standard deviation for each group.

Test conditions:

positive and negative (vehicle/solvent) control data,

data from range-finding study, if conducted,

rationale for dose level selection,

details of test substance preparation,

details of the administration of the test substance,

rationale for route of administration,

methods for verifying that the test substance reached the general circulation or target tissue, if applicable,

conversion from diet/drinking water test substance concentration (ppm) to the actual dose (mg/kg body weight/day), if applicable,

details of food and water quality,

detailed description of treatment and sampling schedules,

methods of slide preparation,

methods for measurements of toxicity,

criteria for scoring micronucleated immature erythrocytes,

number of cells analysed per animal,

criteria for considering studies as positive, negative or equivocal.

Results:

signs of toxicity,

proportion of immature erythrocytes among total erythrocytes,

number of micronucleated immature erythrocytes, given separately for each animal,

mean ± standard deviation of micronucleated immature erythrocytes per group,

dose-response relationship, where possible,

statistical analyses and methods applied,

concurrent and historical negative control data,

concurrent positive control data.

— Discussion of the results.

— Conclusions.

4.(1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)

REFERENCES Heddle, J.A., (1973) A Rapid In Vivo Test for Chromosomal Damage, Mutation Res., 18, p. 187-190. Schmid, W., (1975) The Micronucleus Test, Mutation Res., 31, p. 9-15. Heddle, J.A., Salamone, M.F., Hite, M., Kirkhart, B., Mavournin, K., MacGregor, J.G. and Newell, G.W. (1983). The Induction of Micronuclei as a Measure of Genotoxicity. Mutation Res., 123, p. 61-118. Mavournin, K.H., Blakey, D.H., Cimino, M.C., Salamone, M.F. and Heddle, J.A., (1990) The In Vivo Micronucleus Assay in Mammalian Bone Marrow and Peripheral Blood. A report of the U.S. Environmental Protection Agency Gene-Tox Program, Mutation Res., 239, p. 29-80. MacGregor, J.T., Schlegel, R. Choy, W.N., and Wehr, C.M., (1983) Micronuclei in Circulating Erythrocytes: A Rapid Screen for Chromosomal Damage During Routine Toxicity Testing in Mice. Iin: Developments in Science and Practice of Toxicology, Ed. A.W. Hayes, R.C. Schnell and T.S. Miya, Elsevier, Amsterdam, p. 555-558. MacGregor, J.T., Heddle, J.A. Hite, M., Margolin, G.H., Ramel, C., Salamone, M.F., Tice, R.R., and Wild, D., (1987) Guidelines for the Conduct of Micronucleus Assays in Mammalian Bone Marrow Erytrocytes. Mutation Res., 189, p. 103-112. MacGregor, J.T., Wehr, C.M., Henika, P.R., and Shelby, M.E., (1990) The in vivo Erythrocyte Micronucleus Test: Measurement at Steady State Increases Assay Efficiency and Permits Integration with Toxicity Studies. Fund. Appl. Toxicol., 14, p. 513-522. Hayashi, M., Morita, T., Kodama, Y., Sofuni, T. and Ishidate, M. Jr., (1990) The Micronucleus Assay with Mouse Peripheral Blood Reticulocytes Using Acridine Orange-Coated Slides. Mutation Res., 245, p. 245-249. The Collaborative Study Group for the Micronucleus Test, (1992) Micronucleus Test with Mouse Peripheral Blood Erythrocytes by Acridine Orange Supravital Staining: The Summary Report of the 5th Collaborative Study by CSGMT/JEMMS. MMS. Mutation Res., p. 278, 83-98. The Collaborative Study Group for the Micronucleus Test (CSGMT/JEMMS. MMS: The Mammalian Mutagenesis Study Group of the Environmental Mutagen Society of Japan), (1995) Protocol recommended for the short-term mouse peripheral blood micronucleus test. Mutagenesis, 10, p. 153-159. Hayashi, M., Tice, R.R., MacGregor, J.T., Anderson, D., Blackey, D.H., Kirsch-Volders, M., Oleson, Jr. F. B., Pacchierotti, F., Romagna, F., Shimada, H., Sutou, S. and Vannier, B., (1994) In Vivo Rodent Erythrocyte Micronucleus Assay. Mutation Res., 312, p. 293-304. Higashikuni, N. and Sutou, S., (1995) An optimal, generalised sampling time of 30 ± 6 h after double dosing in the mouse peripheral blood micronucleus test. Mutagenesis, 10, p. 313-319. Fielder, R.J., Allen, J.A., Boobis, A.R., Botham, P.A., Doe, J., Esdaile, D.J., Gatehouse, D.G., Hodson-Walker, G., Morton, D.B., Kirkland, D.J. and Rochold, M., (1992) Report of British Toxicology Society/UK Environmental Mutagen Society Working Group: Dose Setting in In Vivo Mutagenicity Assays. Mutagenesis, 7, p. 313-319. Hayashi, M., Sofuni, T. and Ishidate, M. Jr., (1983) An Application of Acridine Orange Fluorescent Staining to the Micronucleus Test. Mutation Res., 120, p. 241-247. MacGregor, J.T., Wehr, C.M. and Langlois, R.G., (1983) A Simple Fluorescent Staining Procedure for Micronuclei and RNA in Erythrocytes Using Hoechst 33258 and Pyronin Y. Mutation Res., 120, p. 269-275. Romagna, F. and Staniforth, C.D., (1989) The automated bone marrow micronucleus test. Mutation Res., 213, p. 91-104. Gollapudi, B. and McFadden, L.G., (1995) Sample size for the estimation of polychromatic to normochromatic erythrocyte ratio in the bone marrow micronucleus test. Mutation Res., 347, p. 97-99. Richold, M., Ashby, J., Bootman, J., Chandley, A., Gatehouse, D.G. and Henderson, L., (1990) In Vivo Cytogenetics Assay. In: D.J. Kirkland (Ed.) Basic Mutagenicity tests. UKEMS Recommended Procedures. UKEMS Sub-Committee on Guidelines for Mutagenicity Testing. Report, Part I, revised. Cambridge University Press, Cambridge, New York, Port Chester, Melbourne, Sydney, p. 115-141. Lovell, D.P., Anderson, D., Albanese, R., Amphlett, G.E., Clare, G., Ferguson, R., Richold, M., Papworth, D.G. and Savage, J.R.K., (1989) Statistical Analysis of In Vivo Cytogenetic Assays. In: D.J. Kirkland (Ed.) Statistical Evaluation of Mutagenicity Test Data. UKEMS Sub-Committee on Guidelines for Mutagenicity Testing. Report, Part III. Cambridge University Press, Cambridge, New York, Port Chester, Melbourne, Sydney, p. 184-232.

B.13/14. MUTAGENICITY: REVERSE MUTATION TEST USING BACTERIA

B.13/14.

1. METHOD This method is a replicate of the OECD TG 471, Bacterial Reverse Mutation Test (1997).

1.

1.1. INTRODUCTION The bacterial reverse mutation test uses amino-acid requiring strains of Salmonella typhimurium and Escherichia coli to detect point mutations, which involve substitution, addition or deletion of one or a few DNA base pairs (1)(2)(3). The principle of this bacterial reverse mutation test is that it detects mutations, which revert mutations present in the test strains and restore the functional capability of the bacteria to synthesise an essential amino acid. The revertant bacteria are detected by their ability to grow in the absence of the amino-acid required by the parent test strain. Point mutations are the cause of many human genetic diseases and there is substantial evidence that point mutations in oncogenes and tumour-suppressor genes of somatic cells are involved in tumour formation in humans and experimental animals. The bacterial reverse mutation test is rapid, inexpensive and relatively easy to perform. Many of the test strains have several features that make them more sensitive for the detection of mutations including responsive DNA sequences at the reversion sites, increased cell permeability to large molecules and elimination of DNA repair systems or enhancement of error-prone DNA repair processes. The specificity of the test strains can provide some useful information on the types of mutations that are induced by genotoxic agents. A very large data base of results for a wide variety of structures is available for bacterial reverse mutation tests and well-established methodologies have been developed for testing chemicals with different physico-chemical properties, including volatile compounds. See also General introduction Part B.

1.2. DEFINITIONS A reverse mutation test in either Salmonella typhimurium or Escherichia coli detects mutation in an amino-acid requiring strain (histidine or tryptophan, respectively) to produce a strain independent of an outside supply of amino-acid. Base pair substitution mutagens are agents that cause a base change in DNA. In a reversion test this change may occur at the site of the original mutation, or at a second site in the bacterial genome. Frameshift mutagens are agents that cause the addition or deletion of one or more base pairs in the DNA, thus changing the reading frame in the RNA.

1.3. INITIAL CONSIDERATIONS The bacterial reverse mutation test utilises prokaryotic cells, which differ from mammalian cells in such factors as uptake, metabolism, chromosome structure and DNA repair processes. Tests conducted in vitro generally require the use of an exogenous source of metabolic activation. In vitro metabolic activation systems cannot mimic entirely the mammalian in vivo conditions. The test therefore does not provide direct information on the mutagenic and carcinogenic potency of a substance in mammals. The bacterial reverse mutation test is commonly employed as an initial screen for genotoxic activity and, in particular, for point mutation-inducing activity. An extensive database has demonstrated that many chemicals that are positive in this test also exhibit mutagenic activity in other tests. There are examples of mutagenic agents, which are not detected by this test; reasons for these shortcomings can be ascribed to the specific nature of the endpoint detected, differences in metabolic activation, or differences in bioavailability. On the other hand, factors, which enhance the sensitivity of the bacterial reverse mutation test can lead to an overestimation of mutagenic activity. The bacterial reverse mutation test may not be appropriate for the evaluation of certain classes of chemicals, for example highly bactericidal compounds (e.g. certain antibiotics) and those which are thought (or known) to interfere specifically with the mammalian cell replication system (e.g. some topoisomerase inhibitors and some nucleoside analogues). In such cases, mammalian mutation tests may be more appropriate. Although many compounds that are positive in this test are mammalian carcinogens, the correlation is not absolute. It is dependent on chemical class and there are carcinogens that are not detected by this test because they act through other, non-genotoxic, mechanisms or mechanisms absent in bacterial cells.

1.4. PRINCIPLE OF THE TEST METHOD Suspensions of bacterial cells are exposed to the test substance in the presence and in the absence of an exogenous metabolic activation system. In the plate incorporation method, these suspensions are mixed with an overlay agar and plated immediately onto minimal medium. In the preincubation method, the treatment mixture is incubated and then mixed with an overlay agar before plating onto minimal medium. For both techniques, after two or three days of incubation, revertant colonies are counted and compared to the number of spontaneous revertant colonies on solvent control plates. Several procedures for performing the bacterial reverse mutation test have been described. Among those commonly used are the plate incorporation method (1)(2)(3)(4), the preincubation method (2)(3)(5)(6)(7)(8), the fluctuation method (9)(10), and the suspension method (11). Modifications for the testing of gases or vapours have been described (12). The procedures described in the method pertain primarily to the plate incorporation and preincubation methods. Either of them is acceptable for conducting experiments both with and without metabolic activation. Some substances may be detected more efficiently using the preincubation method. These substances belong to chemical classes that include short chain aliphatic nitrosamines, divalent metals, aldehydes, azo-dyes and diazo compounds, pyrollizidine alkaloids, allyl compounds and nitro compounds (3). It is also recognised that certain classes of mutagens are not always detected using standard procedures such as the plate incorporation method or preincubation method. These should be regarded as special cases and it is strongly recommended that alternative procedures should be used for their detection. The following special cases could be identified (together with examples of procedures that could be used for their detection): azo-dyes and diazo compounds (3)(5)(6)(13), gases and volatile chemicals (12)(14)(15)(16) and glycosides (17)(18). A deviation from the standard procedure needs to be scientifically justified.

1.5. DESCRIPTION OF THE TEST METHOD

1.5.

1.5.1. Preparations

1.5.1.

1.5.1.1. Bacteria Fresh cultures of bacteria should be grown up to the late exponential or early stationary phase of growth (approximately 109 cells per ml). Cultures in late stationary phase should not be used. It is essential that the cultures used in the experiment contain a high titre of viable bacteria. The titre may be demonstrated either from historical control data on growth curves, or in each assay through the determination of viable cell numbers by a plating experiment. The recommended incubation temperature is 37 oC. At least five strains of bacteria should be used. These should include four strains of S. typhimurium (TA 1535; TA 1537 or TA97a or TA97; TA98; and TA100) that have been shown to be reliable and reproducibly responsive between laboratories. These four S. typhimurium strains have GC base pairs at the primary reversion site and it is known that may not detect certain oxidising mutagens, cross-linking agents and hydrazines. Such substances may be detected by E. coli WP2 strains or S. typhimurium TA102 (19), which have an AT base pair at the primary reversion site. Therefore the recommended combination of strains is: In order to detect cross-linking mutagens it may be preferable to include TA102 or to add a DNA repair-proficient strain of E. coli [e.g. E. coli WP2 or E. coli WP2 (pKM101)] Established procedures for stock culture preparation, marker verification and storage should be used. The amino-acid requirement for growth should be demonstrated for each frozen stock culture preparation (histidine for S. typhimurium strains, and tryptophan for E. coli strains). Other phenotypic characteristics should be similarly checked, namely: the presence or absence of R-factor plasmids where appropriate [i.e. ampicillin resistance in strains TA98, TA100 and TA97a or TA97, WP2 uvrA and WP2 uvrA (pKM101), and ampicillin + tetracycline resistance in strain TA102]; the presence of characteristic mutations (i.e. rfa mutation in S. typhimurium through sensitivity to crystal violet, and uvrA mutation in E. coli or uvrB mutation in S. typhimurium, through sensitivity to ultraviolet light) (2)(3). The strains should also yield spontaneous revertant colony plate counts within the frequency ranges expected from the laboratory's historical control data and preferably within the range reported in the literature.

S. typhimurium TA1535, and

S. typhimurium TA1537 or TA97 or TA97a, and

S. typhimurium TA98, and

S. typhimurium TA100, and

E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102.

1.5.1.2. Medium An appropriate minimal agar (e.g. containing Vogel-Bonner minimal medium E and glucose), and an overlay agar containing histidine and biotin or tryptophan to allow for a few cell divisions, is used (1)(2)(9).

1.5.1.3. Metabolic activation Bacteria should be exposed to the test substance both in the presence and absence of an appropriate metabolic activation system. The most commonly used system is a cofactor-supplemented post-mitochondrial fraction (S9) prepared from the livers of rodents treated with enzyme-inducing agents such as Aroclor 1254 (1)(2) or a combination of Phenobarbitone and ß-naphthoflavone (18)(20)(21). The post-mitochondrial fraction is usually used at concentrations in the range from 5 to 30 % v/v in the S9-mix. The choice and condition of a metabolic activation system may depend upon the class of chemical being tested. In some cases, it may be appropriate to utilise more than one concentration of post-mitochondrial fraction. For azo-dyes and diazo-compounds, using a reductive metabolic activation system may be more appropriate (6)(13).

1.5.1.4. Test substance/Preparation Solid test substances should be dissolved or suspended in appropriate solvents or vehicles and diluted if appropriate prior to treatment of the bacteria. Liquid test substances may be added directly to the test systems and/or diluted prior to treatment. Fresh preparations should be employed unless stability data demonstrate the acceptability of storage. The solvent/vehicle should not be suspected of chemical reaction with the test substance and should be compatible with the survival of the bacteria and the S9 activity (22). If other than well-known solvent/vehicles are used, their inclusion should be supported by data indicating their compatibility. It is recommended that wherever possible, the use of an aqueous solvent/vehicle be considered first. When testing water-unstable substances, the organic solvents used should be free of water.

1.5.2. Test conditions

1.5.2.

1.5.2.1. Test strains (see 1.5.1.1)

1.5.2.2. Exposure concentration Amongst the criteria to be taken into consideration when determining the highest amount of the test substance to be used are the cytotoxicity and the solubility in the final treatment mixture. It may be useful to determine toxicity and insolubility in a preliminary experiment. Cytotoxicity may be detected by a reduction in the number of revertant colonies, a clearing or diminution of the background lawn, or the degree of survival of treated cultures. The cytotoxicity of a substance may be altered in the presence of metabolic activation systems. Insolubility should be assessed as precipitation in the final mixture under the actual test conditions and evident to the unaided eye. The recommended maximum test concentration for soluble non-cytotoxic substances is 5 mg/plate or 5 μl/plate. For non-cytotoxic substances that are not soluble at 5 mg/plate or 5 μl/plate, one or more concentrations tested should be insoluble in the final treatment mixture. Test substances that are cytotoxic already below 5 mg/plate or 5 μl/plate should be tested up to a cytotoxic concentration. The precipitate should not interfere with the scoring. At least five different analysable concentrations of the test substance should be used with approximately half log (i.e. √10) intervals between test points for an initial experiment. Smaller intervals may be appropriate when a concentration-response is being investigated. Testing above the concentration of 5 mg/plate or 5 μl/plate may be considered when evaluating substances containing substantial amounts of potentially mutagenic impurities.

1.5.2.3. Negative and positive controls Concurrent strain-specific positive and negative (solvent or vehicle) controls, both with and without metabolic activation, should be included in each assay. Positive control concentrations that demonstrate the effective performance of each assay should be selected. For assays employing a metabolic activation system, the positive control reference substance(s) should be selected on the basis of the type of bacteria strains used. The following substances are examples of suitable positive controls for assays with metabolic activation: CA numbers EINECS numbers Names 781-43-1 212-308-4 9,10-dimethylanthracene 57-97-6 200-359-5 7,12-dimethylbenz[a]anthracene 50-32-8 200-028-5 benzo[a]pyrene 613-13-8 210-330-9 2-aminoanthracene 50-18-0 cyclophosphamide 6055-19-2 200-015-4 cyclophosphamide monohydrate The following substance is a suitable positive control for the reductive metabolic activation method: CA numbers EINECS numbers Names 573-58-0 209-358-4 Congo Red 2-Aminoanthracene should not be used as the sole indicator of the efficacy of the S9-mix. If 2-aminoanthracene is used, each batch of S9 should also be characterised with a mutagen that requires metabolic activation by microsomal enzymes, e.g. benzo[a]pyrene, dimethylbenzanthracene. The following substances are examples of strain-specific positive controls for assays performed without exogenous metabolic activation system: CAS numbers EINECS numbers Names Strain 26628-22-8 247-852-1 Sodium azide TA 1535 and TA 100 607-57-8 210-138-5 2-nitrofluorene TA 98 90-45-9 201-995-6 9-aminoacridine TA 1537, TA 97 and TA 97a 17070-45-0 241-129-4 ICR 191 TA 1537, TA 97 and TA 97a 80-15-9 201-254-7 Cumene hydroperoxide TA 102 50-07-7 200-008-6 Mitomycin C WP2 uvrA and TA102 70-25-7 200-730-1 N-ethyl-N-nitro-N-nitrosoguanidine WP2, WP2uvrA and WP2uvrA(pKM101) 56-57-5 200-281-1 4-nitroquinoline-1-oxide WP2, WP2uvrA and WP2uvrA(pKM101) 3688-53-7 Furylfuramide (AF2) plasmid-containing strains Other appropriate positive control reference substances may be used. The use of chemical class-related positive control chemicals should be considered, when available. Negative controls, consisting of solvent or vehicle alone, without test substance, and otherwise treated in the same way as the treatment groups, should be included. In addition, untreated controls should also be used unless there are historical control data demonstrating that no deleterious or mutagenic effects are induced by the chosen solvent.

1.5.3. Procedure For the plate incorporation method (1)(2)(3)(4), without metabolic activation, usually 0,05 ml or 0,1 ml of the test solutions, 0,1 ml of fresh bacterial culture (containing approximately 108 viable cells) and 0,5 ml of sterile buffer are mixed with 2,0 ml of overlay agar. For the assay with metabolic activation, usually 0,5 ml of metabolic activation mixture containing an adequate amount of post-mitochondrial fraction (in the range from 5 to 30 % v/v in the metabolic activation mixture) are mixed with the overlay agar (2,0 ml), together with the bacteria and test substance/test solution. The contents of each tube are mixed and poured over the surface of a minimal agar plate. The overlay agar is allowed to solidify before incubation. For the preincubation method (2)(3)(5)(6), the test substance/test solution is preincubated with the test strain (containing approximately 108 viable cells) and sterile buffer or the metabolic activation system (0,5 ml) usually for 20 min. or more at 30-37 oC prior to mixing with the overlay agar and pouring onto the surface of a minimal agar plate. Usually, 0,05 or 0,1 ml of test substance/test solution, 0,1 ml of bacteria, and 0,5 ml of S9-mix or sterile buffer are mixed with 2,0 ml of overlay agar. Tubes should be aerated during pre-incubation by using a shaker. For an adequate estimate of variation, triplicate plating should be used at each dose level. The use of duplicate plating is acceptable when scientifically justified. The occasional loss of a plate does not necessarily invalidate the assay. Gaseous or volatile substances should be tested by appropriate methods, such as in sealed vessels (12)(14)(15)(16).

1.5.4. Incubation All plates in a given assay should be incubated at 37 oC for 48-72 hours. After the incubation period, the number of revertant colonies per plate is counted.

2. DATA

2.

2.1. TREATMENT OF RESULTS Data should be presented as the number of revertant colonies per plate. The number of revertant colonies on both negative (solvent control, and untreated control if used) and positive control plates should also be given. Individual plate counts, the mean number of revertant colonies per plate and the standard deviation should be presented for the test substance and positive and negative (untreated and/or solvent) controls. There is no requirement for verification of a clear positive response. Equivocal results should be clarified by further testing preferably using a modification of experimental conditions. Negative results need to be confirmed on a case-by-case basis. In those cases where confirmation of negative results is not considered necessary, justification should be provided. Modification of study parameters to extend the range of conditions assessed should be considered in follow-up experiments. Study parameters that might be modified include the concentration spacing, the method of treatment (plate-incorporation or liquid pre-incubation), and metabolic activation conditions.

2.2. EVALUATION AND INTERPRETATION OF RESULTS There are several criteria for determining a positive result, such as a concentration-related increase over the range tested and/or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation system (23). Biological relevance of the results should be considered first. Statistical methods may be used as an aid in evaluating the test results (24). However, statistical significance should not be the only determining factor for a positive response. A test substance for which the results do not meet the above criteria is considered non-mutagenic in this test. Although most experiments will give clearly positive or negative results, in rare cases the data set will preclude making a definite judgement about the activity of the test substance. Results may remain equivocal or questionable regardless of the number of times the experiment is repeated. Positive results from the bacterial reverse mutation test indicate that the substance induces point mutations by base substitutions or frameshifts in the genome of either Salmonella typhimurium and/or Escherichia coli. Negative results indicate that under the test conditions, the test substance is not mutagenic in the tested species.

3. REPORTING

3. TEST REPORT

The test report must include the following information:

Solvent/Vehicle:

justification for choice of solvent/vehicle,

solubility and stability of the test substance in solvent/vehicle, if known.

Strains:

strains used,

number of cells per culture,

strain characteristics.

Test conditions:

amount of test substance per plate (mg/plate or μl/plate) with rationale for selection of dose and number of plates per concentration,

media used,

type and composition of metabolic activation system, including acceptability criteria,

treatment procedures.

Results:

signs of toxicity,

signs of precipitation,

individual plate counts,

the mean number of revertant colonies per plate and standard deviation,

dose-response relationship, where possible,

statistical analyses, if any,

concurrent negative (solvent/vehicle) and positive control data, with ranges, means and standard deviations,

historical negative (solvent/vehicle) and positive control data with ranges, means and standard deviations.

Discussion of results.

Conclusions.

4.(1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)

REFERENCES Ames, B.N., McCann, J. and Yamasaki E., (1975) Methods for Detecting Carcinogens and Mutagens with the Salmonella/Mammalian-Microsome Mutagenicity Test. Mutation Res., 31, p. 347-364. Maron, D.M. and Ames, B.N., (1983) Revised Methods for the Salmonella Mutagenicity Test. Mutation Res., 113, p. 173-215. Gatehouse, D., Haworth, S., Cebula, T., Gocke, E., Kier, L., Matsushima, T., Melcion, C., Nohmi, T., Venitt, S. and Zeiger, E., (1994) Recommendations for the Performance of Bacterial Mutation Assays. Mutation Res., 312, p. 217-233. Kier, L.D., Brusick D.J., Auletta, A.E., Von Halle, E.S., Brown, M.M., Simmon, V.F., Dunkel, V., McCann, J., Mortelmans, K., Prival, M., Rao, T.K. and Ray V., (1986) The Salmonella typhimurium/Mammalian Microsomal Assay: A Report of the U.S. Environmental Protection Agency Gene-Tox Program. Mutation Res., 168, p. 69-240. Yahagi, T., Degawa, M., Seino, Y.Y., Matsushima, T., Nagao, M., Sugimura, T. and Hashimoto, Y., (1975) Mutagenicity of Carcinogen Azo Dyes and their Derivatives, Cancer Letters, 1, p. 91-96. Matsushima, M., Sugimura, T., Nagao, M., Yahagi, T., Shirai, A. and Sawamura, M., (1980) Factors Modulating Mutagenicity Microbial Tests. In: Short-term Test Systems for Detecting Carcinogens. Ed. Norpoth K.H. and Garner, R.C., Springer, Berlin-Heidelberg-New York. p. 273-285. Gatehouse, D.G., Rowland, I.R., Wilcox, P., Callender, R.D. and Foster, R., (1980) Bacterial Mutation Assays. In: Basic Mutagenicity Tests: UKEMS Part 1 Revised. Ed. D.J. Kirkland, Cambridge University Press, p. 13-61. Aeschacher, H.U., Wolleb, U. and Porchet, L., (1987) Liquid Preincubation Mutagenicity Test for Foods. J. Food Safety, 8, p. 167-177. Green, M.H.L., Muriel, W.J. and Bridges, B.A., (1976) Use of a simplified fluctuation test to detect low levels of mutagens. Mutation Res., 38, p. 33-42. Hubbard, S.A., Green, M.H.L., Gatehouse, D. and J.W. Bridges (1984) The Fluctuation Test in Bacteria. In: Handbook of Mutagenicity Test Procedures. 2nd Edition. Ed. Kilbey, B.J., Legator, M., Nichols, W. and Ramel, C., Elsevier, Amsterdam-New York-Oxford, p. 141-161. Thompson, E.D. and Melampy, P.J., (1981) An Examination of the Quantitative Suspension Assay for Mutagenesis with Strains of Salmonella typhimurium. Environmental Mutagenesis, 3, p. 453-465. Araki, A., Noguchi, T., Kato, F. and T. Matsushima (1994) Improved Method for Mutagenicity Testing of Gaseous Compounds by Using a Gas Sampling Bag. Mutation Res., 307, p. 335-344. Prival, M.J., Bell, S.J., Mitchell, V.D., Reipert, M.D. and Vaughn, V.L., (1984) Mutagenicity of Benzidine and Benzidine-Congener Dyes and Selected Monoazo Dyes in a Modified Salmonella Assay. Mutation Res., 136, p. 33-47. Zeiger, E., Anderson, B.E., Haworth, S., Lawlor, T. and Mortelmans, K., (1992) Salmonella Mutagenicity Tests. V. Results from the Testing of 311 Chemicals. Environ. Mol. Mutagen., 19, p. 2-141. Simmon, V., Kauhanen, K. and Tardiff, R.G., (1977) Mutagenic Activity of Chemicals Identified in Drinking Water. In Progress in Genetic Toxicology, D. Scott, B. Bridges and F. Sobels (Eds.) Elsevier, Amsterdam, p. 249-258. Hughes, T.J., Simmons, D.M., Monteith, I.G. and Claxton, L.D., (1987) Vaporisation Technique to Measure Mutagenic Activity of Volatile Organic Chemicals in the Ames/Salmonella Assay. Environmental Mutagenesis, 9, p. 421-441. Matsushima, T., Matsumoto, A., Shirai, M., Sawamura, M., and Sugimura, T., (1979) Mutagenicity of the Naturally Occurring Carcinogen Cycasin and Synthetic Methylazoxy Methane Conjugates in Salmonella typhimurium. Cancer Res., 39, p. 3780-3782. Tamura, G., Gold, C., Ferro-Luzzi, A. and Ames, B.N., (1980) Fecalase: A Model for Activation of Dietary Glycosides to Mutagens by Intestinal Flora. Proc. Natl. Acad. Sci. USA, 77, p. 4961-4965. Wilcox, P., Naidoo, A., Wedd, D.J. and Gatehouse, D.G., (1990) Comparison of Salmonella typhimurium TA 102 with Escherichia coli WP2 Tester strains. Mutagenesis, 5, p. 285-291. Matsushima, T., Sawamura, M., Hara, K. and Sugimura, T., (1976) A Safe Substitute for Polychlorinated Biphenyls as an Inducer or Metabolic Activation Systems. In: In vitro metabolic Activation in Mutagenesis Testing Eds. F.J. de Serres et al. Elsevier, North Holland, p. 85-88. Elliot, B.M., Combes, R.D., Elcombe, C.R., Gatehouse, D.G., Gibson, G.G., Mackay, J.M. and Wolf, R.C., (1992) Alternatives to Aroclor 1254-induced S9 in in vitro Genotoxicity Assays. Mutagenesis, 7, p. 175-177. Maron, D., Katzenellenbogen, J. and Ames, B.N., (1981) Compatibility of Organic Solvents with the Salmonella/Microsome Test. Mutation Res., p. 88343-350. Claxton, L.D., Allen, J., Auletta, A., Mortelmans, K., Nestmann, E. and Zeiger, E., (1987) Guide for the Salmonella typhimurium/Mammalian Microsome Tests for Bacterial Mutagenicity. Mutation Res. 189, p. 83-91. Mahon, G.A.T., Green, M.H.L., Middleton, B., Mitchell, I., Robinson, W.D. and Tweats, D.J., (1989) Analysis of Data from Microbial Colony Assays. In: UKEMS Sub-Committee on Guidelines for Mutagenicity Testing. Part II. Statistical Evaluation of Mutagenicity Test Data. Ed. Kirkland, D.J., Cambridge University Press, p. 28-65.

B.15. MUTAGENICITY TESTING AND SCREENING FOR CARCINOGENICITY GENE MUTATION — SACCHAROMYCES CEREVISIAE

B.15.

1. METHOD

1.

1.1. INTRODUCTION See General introduction Part B.

1.2. DEFINITION See General introduction Part B.

1.3. REFERENCE SUBSTANCES None.

1.4. PRINCIPLE OF THE TEST METHOD A variety of haploid and diploid strains of the yeast Saccharomyces cerevisiae can be used to measure the production of gene mutations induced by chemical agents with and without metabolic activation. Forward mutation systems in haploid strains, such as the measurement of mutation from red, adenine-requiring mutants (ade-1, ade-2) to double adenine-requiring white mutants and selective systems such as the induction of resistance to canavnaine and cycloheximide, have been utilised. The most extensively validated reverse mutation system involves the use of the haploid strain XV 185-14C which carries the ochre nonsense mutations ade 2-1, arg 4-17, lys 1-1 and trp 5-48, which are reversible by base substitution mutagens that induce site specific mutations or ochre suppressor mutations. XV 185-14C also carries the his 1-7 marker, a missense mutation reverted mainly by second site mutations, and the marker hom 3-10 which is reverted by frameshift mutagens. In diploid strains of S. cerevisiae the only extensively used strain is D7 which is homozygous for ilv 1-92.

1.5. QUALITY CRITERIA None.

1.6. DESCRIPTION OF THE TEST METHOD

1.6. Preparations

Solutions of test chemicals and control should be prepared just prior to testing, using an appropriate vehicle. In the case of organic compounds, which are not water soluble, not more than a 2 % solution v/v of organic solvents such as ethanol, acetone or dimethylsulphoxide (DMSO) should be used. The final concentration of the vehicle should not significantly affect cell viability and growth characertistics.

Metabolic activation

Cells should be exposed to test chemicals both in the presence and absence of an appropriate exogenous metabolic activation system. The most commonly used system is a co-factor supplemented post-mitochondrial fraction from the livers of rodents pre-treated with enzyme inducing agents. The use of other species, tissues, post-mitochondrial fractions, or procedures may also be appropriate for metabolic activation.

Test conditions

Tester strains

The haploid strain XV 185-14C and the diploid strain D7 are the most used in gene mutation studies. Other strains may also be appropriate.

Media

Appropriate culture media are used for the determination of survival and mutant numbers.

Use of negative and positive controls

Positive, untreated and solvent controls should be performed concurrently. Appropriate positive control chemicals should be used for each specific mutational endpoint.

Exposure concentration

At least five adequately spaced concentrations of the test substance should be used. For toxic substances, the highest concentration tested should not reduce survival below 5 to 10 %. Relatively water-insoluble substances should be tested up to their limit of solubility, using appropriate procedures. For freely water-soluble non-toxic substances, the upper concentration should be determined on a case by case basis.

Incubation conditions

The plates are incubated four to seven days at 28 to 30 oC in the dark.

Spontaneous mutation frequencies

Sub-cultures should be used with spontaneous mutation frequencies within the accepted normal range.

Number of replicates

At least three replicate plates should be used per concentration for the assay of prototrophs produced by gene mutation and for cell viability. In the case of experiments using markers such as hom 3-10 with a low mutation rate, the number of plates used must be increased to provide statistically relevant data.

Procedure

Treatment of S. cerevisiae strains is usually performed in a liquid test procedure involving either stationary or growing cells. Initial experiments should be carried out on growing cells: 1-5 × 107 cells/ml are exposed to the test chemical for up to 18 hours at 28 to 37 oC with shaking; an adequate amount of metabolic activation system is added during treatment when appropriate. At the end of the treatment, cells are centrifuged, washed and seeded upon an appropriate culture medium. After incubation, plates are scored for survival and the induction of gene mutation. If the first experiment is negative, then a second experiment should be carried out using stationary phase cells. If the first experiment is positive it is confirmed in an appropriate independent experiment.

2. DATA Data should be presented in tabular form indicating the number of colonies counted, number of mutants, survival and mutant frequency. All results should be confirmed in an independent experiment. The dara should be evaluated using appropriate statistical methods.

3. REPORTING

3.

3.1. TEST REPORT The test report shall, if possible, contain the following information:

strain used,

test conditions: stationary phase or growing cells, compositions of media, incubation temperature and duration, metabolic activation system,

treatment conditions: exposure levels, procedure and duration of treatment, treatment temperature, positive and negative controls,

number of colonies counted, number of mutants, survival and mutant frequency, dose/response relationship if applicable, statistical evaluation of data,

discussion of results,

interpretation of results.

3.2. EVALUATION AND INTERPRETATION See General introduction Part B.

4. REFERENCES See General introduction Part B.

B.16. MITOTIC RECOMBINATION — SACCHAROMYCES CEREVISIAE

B.16.

1. METHOD

1.

1.1. INTRODUCTION See General introduction Part B.

1.2. DEFINITION See General introduction Part B.

1.3. REFERENCE SUBSTANCES None.

1.4. PRINCIPLE OF THE TEST METHOD Mitotic recombination in Saccharomyces cerevisiae can be detected between genes (or more generally between a gene and its centromere) and within genes. The former event is called mitotic crossing-over and generates reciprocal products whereas the latter event is most frequently non-reciprocal and is called gene conversion. Crossing-over is generally assayed by the production of recessive homozygous colonies or sectors produced in a heterozygous strain, whereas gene conversion is assayed by the production of prototrophic revertants produced in an auxotrophic heteroallelic strain carrying two different defective alleles of the same gene. The most commonly used strains for the detection of mitotic gene conversion are D4 (heteroallelic at ade 2 and trp 5) D7 (heteroallelic at trp 5) BZ34 (heteroallelic at arg 4) and JDl (heteroallelic at his 4 and trp 5). Mitotic crossing-over producing red and pink homozygous sectors can be assayed in D5 or in D7 (which also measures mitotic gene conversion and reverse mutation at ilv 1-92) both strains being heteroallelic for complementing alleles of ade 2.

1.5. QUALITY CRITERIA None.

1.6. DESCRIPTION OF THE TEST METHOD

1.6. Preparations

Solutions of test chemicals and control or reference compounds should be prepared just prior to testing, using an appropriate vehicle. With organic compounds that are water insoluble not more than a 2 % solution v/v of organic solvents such as ethanol, acetone or dimethylsulphoxide (DMSO) should be used. The final concentration of the vehicle should not significantly affect cell viability and growth characteristics.

Metabolic activation

Cells should be exposed to test chemicals both in the presence and absence of an appropriate exogenous metabolic activation system. The system most commonly used is a co-factor supplemented post-mitochondrial fraction from the livers of rodents pre-treated with enzyme inducing agents. The use of other species, tissues, post-mitochondrial fractions, or procedures may also be appropriate for metabolic activation.

Test conditions

Tester strains

The most frequently used strains are the diploids D4, D5, D7 and JD1. The use of other strains may be appropriate.

Media

Appropriate culture media are used for the determination of survival and the frequency of mitotic recombination.

Use of negative and positive controls

Positive, untreated and solvent controls should be performed concurrently. Appropriate positive control chemicals should be used for each specific recombination endpoint.

Exposure concentrations

At least five adequately spaced concentrations of the test substance should be used. Among the factors to be taken into consideration are cytotoxicity and solubility. The lowest concentration must have no effect on celf viability. For toxic chemicals, the highest concentration tested should not reduce survival below 5 to 10 %. Relatively water-insoluble chemicals should be tested up to the limit of solubility using appropriate procedures. For freely water-soluble non-toxic substances the upper concentration should be determined on a case by case basis. Cells may be exposed to test chemicals in either the stationary phase or during growth for periods of up to 18 hours. However, for long treatment times cultures should be microscopically inspected for spore formation, the presence of which invalidates the test.

Incubation conditions

The plates are incubated in the dark for four to seven days at 28 to 30 oC. Plates used for the assay of red and pink homozygous sectors produced by mitotic crossing-over should be kept in a refrigerator (about 4 oC) for a further one to two days before scoring to allow for the development of the appropriate pigmented colonies.

Spontaneous mitotic recombination frequencies

Sub-cultures should be used with spontaneous mitotic recombination mutation frequencies within the accepted normal range.

Number of replicates

A minimum of three replicate plates should be used per concentration for the assay of prototrophs produced by mitotic gene conversion and for viability. In the case of the assay of recessive homozygosis produced by mitotic crossing-over, the plate number should be increased to provide an adequate number of colonies.

Procedures

Treatment of S. cerevisiae strains is usually performed in a liquid test procedure involving either stationary or growing cells. Initial experiments should be done on growing cells. 1-5 × 107 celles/ml are exposed to the test chemical for up to 18 hours at 28 to 37 oC with shaking; an adequate amount of metabolic activation system is added during treatment when appropriate. At the end of the treatment, cells are centrifuged, washed and seeded upon appropriate culutre medium. After incubation plates are scored for survival and the induction of mitotic recombination. If the first experiment is negative, then a second experiment should be carried out using stationary phase cells. If the first experiment is positive it is confirmed in an independent experiment.

2. DATA Data should be presented in tabular form indicating the number of colonies counted, the number of recombinants, survival and the frequency of recombinants. Results should be confirmed in an independent experiment. The data should be evaluated using appropriate statistical methods.

3. REPORTING

3.

3.1. TEST REPORT The test report shall, if possible, contain the following information:

strain used,

test conditions: stationary phase or growing cells, composition of media, incubation temperature and duration, metabolic activation system,

treatment conditions: exposure concentration, procedure and duration of treatment, treatment temperature, positive and negative controls,

number of colonies counted, number of recombinants; survival and recombination frequency, dose/response relationship if applicable, statistical evaluation of data,

discussion of the results,

interpretation of the results.

3.2. EVALUATION AND INTERPRETATION See General introduction Part B.

4. REFERENCES See General introduction Part B.

B.17. MUTAGENICITY — IN VITRO MAMMALIAN CELL GENE MUTATION TEST

B.17.

1. METHOD This method is a replicate of the OECD TG 476, In Vitro Mammalian Cell Gene Mutation Test (1997).

1.

1.1. INTRODUCTION The in vitro mammalian cell gene mutation test can be used to detect gene mutations induced by chemical substances. Suitable cell lines include L5178Y mouse lymphoma cells, the CHO, CHO-AS52 and V79 lines of Chinese hamster cells, and TK6 human lymphoblastoid cells (1). In these cell lines the most commonly-used genetic endpoints measure mutation at thymidine kinase (TK) and hypoxanthine-guanine phosphoribosyl transferase (HPRT), and a transgene of xanthine-guanine phosphoribosyl transferase (XPRT). The TK, HPRT and XPRT mutation tests detect different spectra of genetic events. The autosomal location of TK and XPRT may allow the detection of genetic events (e.g. large deletions) not detected at the HPRT locus on X-chromosomes (2)(3)(4)(5)(6). In the in vitro mammalian cell gene mutation test, cultures of established cell lines or cell strains can be used. The cells used are selected on the basis of growth ability in culture and stability of the spontaneous mutation frequency. Tests conducted in vitro generally require the use of an exogenous source of metabolic activation. This metabolic activation system cannot mimic entirely the mammalian in vivo conditions. Care should be taken to avoid conditions, which would lead to results not reflecting intrinsic mutagenicity. Positive results, which do not reflect intrinsic mutagenicity may arise from changes in pH, osmolality or high levels of cytotoxicity (7). This test is used to screen for possible mammalian mutagens and carcinogens. Many compounds that are positive in this test are mammalian carcinogens; however, there is not a perfect correlation between this test and carcinogenicity. Correlation is dependent on chemical class and there is increasing evidence that there are carcinogens that are not detected by this test because they appear to act through other, non genotoxic mechanisms or mechanisms absent in bacterial cells (6). See also General introduction Part B.

1.2. DEFINITIONS Forward mutation: a gene mutation from the parental type to the mutant form which gives rise to an alteration or a loss of the enzymatic activity of the function of the encoded protein. Base pair substitution mutagens: substances, which cause substitution of one or several base pairs in the DNA. Frameshift mutagens: Substances, which cause the addition or deletion of single or multiple base pairs in the DNA molecule. Phenotypic expression time: a period during which unaltered gene products are depleted from newly mutated cells. Mutant frequency: the number of mutant cells observed divided by the number of viable cells. Relative total growth: increase in cell number over time compared to a control population of cells; calculated as the product of suspension growth relative to the negative control times cloning efficiency relative to negative control. Relative suspension growth: increase in cell number over the expression period relative to the negative control. Viability: the cloning efficiency of the treated cells at the time of plating in selective conditions after the expression period. Survival: the cloning efficiency of the treated cells when plated at the end of the treatment period; survival is usually expressed in relation to the survival of the control cell population.

1.3. PRINCIPLE OF THE TEST METHOD Cells deficient in thymidine kinase (TK) due to the mutation TK+/- -> TK-/- are resistant to the cytotoxic effects of the pyrimidine analogue trifluorothymidine (TFT). Thymidine kinase proficient cells are sensitive to TFT, which causes the inhibition of cellular metabolism and halts further cell division. Thus mutant cells are able to proliferate in the presence of TFT, whereas normal cells, which contain thymidine kinase, are not. Similarly, cells deficient in HPRT or XPRT are selected by resistance to 6-thioguanine (TG) or 8-azaguanine (AG). The properties of the test substance should be considered carefully if a base analogue or a compound related to the selective agent is tested in any of the mammalian cell gene mutation tests. For example, any suspected selective toxicity by the test substance for mutant and non-mutant cells should be investigated. Thus, performance of the selection system/agent must be confirmed when testing chemicals structurally related to the selective agent (8). Cells in suspension or monolayer culture are exposed to the test substance, both with and without metabolic activation, for a suitable period of time and subcultured to determine cytotoxicity and to allow phenotypic expression prior to mutant selection (9)(10)(11)(12)(13). Cytotoxicity is usually determined by measuring the relative cloning efficiency (survival) or relative total growth of the cultures after the treatment period. The treated cultures are maintained in growth medium for a sufficient period of time, characteristic of each selected locus and cell type, to allow near-optimal phenotypic expression of induced mutations. Mutant frequency is determined by seeding known numbers of cells in medium containing the selective agent to detect mutant cells and in medium without selective agent to determine the cloning efficiency (viability). After a suitable incubation time, colonies are counted. The mutant frequency is derived from the number of mutant colonies in selective medium and the number of colonies in non-selective medium.

1.4. DESCRIPTION OF THE TEST METHOD

1.4.

1.4.1. Preparations

1.4.1.

1.4.1.1. Cells A variety of cell types are available for use in this test including subclones of L5178Y, CHO, CHO-AS52, V79 or TK6 cells. Cell types used in this test should have a demonstrated sensitivity to chemical mutagens, a high cloning efficiency and a stable spontaneous mutant frequency. Cells should be checked for mycoplasma contamination and should not be used if contaminated. The test should be designed to have a predetermined sensitivity and power. The number of cells, cultures and concentrations of test substance used should reflect these defined parameters (14). The minimal number of viable cells surviving treatment and used at each stage in the test should be based on the spontaneous mutation frequency. A general guide is to use a cell number, which is at least 10 times the inverse of the spontaneous mutation frequency. However, it is recommended to utilise at least 106 cells. Adequate historical data on the cell system used should be available to indicate consistent performance of the test.

1.4.1.2. Media and culture conditions Appropriate culture media, and incubation conditions (culture vessels, temperature, CO2 concentration, and humidity) should be used. Media should be chosen according to the selective systems and cell type used in the test. It is particularly important that culture conditions should be chosen that ensure optimal growth of cells during the expression period and colony forming ability of both mutant and non-mutant cells.

1.4.1.3. Preparation of cultures Cell are propagated from stock cultures, seeded in culture medium and incubated at 37 oC. Prior to use in this test, cultures may need to be cleansed of pre-existing mutant cells.

1.4.1.4. Metabolic activation Cells should be exposed to the test substance both in the presence and absence of an appropriate metabolic activation system. The most commonly used system is a cofactor-supplemented post-mitochondrial fraction (S9) prepared from the livers of rodents treated with enzyme-inducing agents such as Aroclor 1254 (15)(16)(17)(18) or a combination of phenobarbitone and ß–naphthoflavone (19)(20). The post-mitochondrial fraction is usually used at concentrations in the range from 1-10 % v/v in the final test medium. The choice and condition of a metabolic activation system may depend upon the class of chemical being tested. In some cases it may be appropriate to utilise more than one concentration of post-mitochondrial fraction. A number of developments, including the construction of genetically engineered cell lines expressing specific activating enzymes, may provide the potential for endogenous activation. The choice of the cell lines used should be scientifically justified (e.g. by the relevance of the cytochrome P450 isoenzyme for the metabolism of the test substance).

1.4.2. Test conditions

1.4.2.

1.4.2.1. Solvent/Vehicle The solvent/vehicle should not be suspected of chemical reaction with the test substance and should be compatible with the survival of the cells and the S9 activity. If other than well-known solvent/vehicles are used, their inclusion should be supported by data indicating their compatibility. It is recommended that wherever possible, the use of an aqueous solvent/vehicle be considered first. When testing water-unstable substances, the organic solvents used should be free of water. Water can be removed by adding a molecular sieve.

1.4.2.3. Controls Concurrent positive and negative (solvent or vehicle) controls, both with and without metabolic activation should be included in each experiment. When metabolic activation is used, the positive control chemical should be the one that requires activation to give a mutagenic response. Examples of positive control substances include: Metabolic activation condition Locus Substance CAS No EINECS No Absence of exogenous metabolic activation HPRT Ethyl methanesulphonate 62-50-0 200-536-7 Ethyl nitrosourea 759-73-9 212-072-2 TK (small and large colonies) Methyl methanesulphonate 66-27-3 200-625-0 XPRT Ethyl methanesulphonate 62-50-0 200-536-7 Ethyl nitrosourea 759-73-9 212-072-2 Presence of exogenous metabolic activation HPRT 3-Methylcholanthrene 56-49-5 200-276-4 N-Nitrosodimethylamine 62-75-9 200-549-8 7,12-Dimethylbenzanthracene 57-97-6 200-359-5 TK (small and large colonies) Cyclophosphamide 50-18-0 200-015-4 Cyclophosphamide monohydrate 6055-19-2 Benzo[a]pyrene 50-32-8 200-028-5 3-Methylcholanthrene 56-49-5 200-276-5 XPRT N-Nitrosodimethylamine (for high levels of S-9) 62-75-9 200-549-8 Benzo[a]pyrene 50-32-8 200-028-5 Other appropriate positive control reference substances may be used, e.g. if a laboratory has a historical data base on 5-Bromo 2'-deoxyuridine (CAS n. 59-14-3, Einecs n. 200-415-9), this reference substance could be used as well. The use of chemical class-related positive control chemicals should be considered, when available. Negative controls, consisting of solvent or vehicle alone in the treatment medium, and treated in the same way as the treatment groups, should be included. In addition, untreated controls should also be used unless there are historical control data demonstrating that no deleterious or mutagenic effects are induced by the chosen solvent.

1.4.3. Procedure

1.4.3.

1.4.3.1. Treatment with the test substance Proliferating cells should be exposed to the test substance both with and without metabolic activation. Exposure should be for a suitable period of time (usually three to six hours is effective). Exposure time may be extended over one or more cell cycles. Either duplicate or single treated cultures may be used at each concentration tested. When single cultures are used, the number of concentrations should be increased to ensure an adequate number of cultures for analysis (e.g. at least eight analysable concentrations). Duplicate negative (solvent) control cultures should be used. Gaseous or volatile substances should be tested by appropriate methods, such as in sealed culture vessels (21)(22).

1.4.3.2. Measurement of survival, viability and mutant frequency At the end of the exposure period, cells are washed and cultured to determine survival and to allow for expression of the mutant phenotype. Measurement of cytotoxicity by determining the relative cloning efficiency (survival) or relative total growth of the cultures is usually initiated after the treatment period. Each locus has a defined minimum time requirement to allow near optimal phenotypic expression of newly induced mutants (HPRT and XPRT require at least six to eight days, and TK at least two days). Cells are grown in medium with and without selective agent(s) for determination of numbers of mutants and cloning efficiency, respectively. The measurement of viability (used to calculate mutant frequency) is initiated at the end of the expression time by plating in non-selective medium. If the test substance is positive in the L5178Y TK+/- test, colony sizing should be performed on at least one of the test cultures (the highest positive concentration) and on the negative and positive controls. If the test substance is negative in the L5178Y TK+/- test, colony sizing should be performed on the negative and positive controls. In studies using TK6TK+/-, colony sizing may also be performed.

2. DATA

2.

2.1. TREATMENT OF RESULTS Data should include cytotoxicity and viability determination, colony counts and mutant frequencies for the treated and control cultures. In the case of a positive response in the L5178Y TK+/- test, colonies are scored using the criteria of small and large colonies on at least one concentration of the test substance (highest positive concentration) and on the negative and positive control. The molecular and cytogenetic nature of both large and small colony mutants has been explored in detail (23)(24). In the TK+/- test, colonies are scored using the criteria of normal growth (large) and slow growth (small) colonies (25). Mutant cells that have suffered the most extensive genetic damage have prolonged doubling times and thus form small colonies. This damage typically ranges in scale from the losses of the entire gene to karyotypically visible chromosome aberrations. The induction of small colony mutants has been associated with chemicals that induce gross chromosome aberrations (26). Less seriously affected mutant cells grow at rates similar to the parental cells and form large colonies. Survival (relative cloning efficiencies) or relative total growth should be given. Mutant frequency should be expressed as number of mutant cells per number of surviving cells. Individual culture data should be provided. Additionally, all data should be summarised in tabular form. There is no requirement for verification of a clear positive response. Equivocal results should be clarified by further testing preferably using modification of experimental conditions. Negative results need to be confirmed on a case-by-case basis. In those cases where confirmation of negative results is not considered necessary, justification should be provided. Modification of study parameters to extend the range of conditions assessed should be considered in follow-up experiments for either equivocal or negative results. Study parameters that might be modified include the concentration spacing and the metabolic activation conditions.

2.2. EVALUATION AND INTERPRETATION OF RESULTS There are several criteria for determining a positive result, such as a concentration-related increase or a reproducible increase in mutant frequency. Biological relevance of the results should be considered first. Statistical methods may be used as an aid in evaluating the test results. Statistical significance should not be the only determining factor for a positive response. A test substance for which the results do not meet the above criteria is considered non-mutagenic in this system. Although most studies will give clearly positive or negative results, in rare cases the data set will preclude making a definite judgement about the activity of the test substance. Results may remain equivocal or questionable regardless of the number of times the experiment is repeated. Positive results from the in vitro mammalian cell gene mutation test indicate that the test substance induces gene mutations in the cultured mammalian cells used. A positive concentration response that is reproducible is most meaningful. Negative results indicate that, under the test conditions, the test substance does not induce gene mutations in the cultured mammalian cells used.

3. REPORTING

3. TEST REPORT

The test report must include the following information:

Solvent/Vehicle:

justification for choice of vehicle/solvent,

solubility and stability of the test substance in solvent/vehicle, if known,

Cells:

type and source of cells,

number of cell cultures,

number of cell passages, if applicable,

methods for maintenance of cell culture, if applicable,

absence of mycoplasma.

Test conditions:

rationale for selection of concentrations and number of cultures including, e.g. cytotoxicity data and solubility limitations, if available,

composition of media, CO2 concentration,

concentration of test substance,

volume of vehicle and test substance added,

incubation temperature,

incubation time,

duration of treatment,

cell density during treatment,

type and composition of metabolic activation system, including acceptability criteria,

positive and negative controls,

length of expression period (including number of cells seeded, and subcultures and feeding schedules, if appropriate),

selective agents,

criteria for considering tests as positive, negative or equivocal,

methods used to enumerate numbers of viable and mutant cells.

definition of colonies of which size and type are considered (including criteria for small and large colonies, as appropriate).

Results:

signs of toxicity,

signs of precipitation,

data on pH and osmolality during the exposure to the test substance, if determined,

colony size if scored for at least negative and positive controls,

laboratory's adequacy to detect small colony mutants with the L5178Y TK+/- system, where appropriate,

dose-response relationship, where possible,

statistical analyses, if any,

concurrent negative (solvent/vehicle) and positive control data,

historical negative (solvent/vehicle) and positive control data with ranges, means and standard deviations,

mutant frequency.

Discussion of results.

Conclusions.

4.(1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)

REFERENCES Moore, M.M., DeMarini, D.M., DeSerres, F.J. and Tindall, K.R. (Eds.), (1987) Banbury Report 28: Mammalian Cell Mutagenesis, Cold Spring Harbor Laboratory, New York. Chu, E.H.Y. and Malling, H.V., (1968) Mammalian Cell Genetics. II. Chemical Induction of Specific Locus Mutations in Chinese Hamster Cells In Vitro, Proc. Natl. Acad. Sci., USA, 61, p. 1306-1312. Liber, H.L. and Thilly, W.G., (1982) Mutation Assay at the Thymidine Kinase Locus in Diploid Human Lymphoblasts. Mutation Res., 94, p. 467-485. Moore, M.M., Harington-Brock, K., Doerr, C.L. and Dearfield, K.L., (1989) Differential Mutant Quantitation at the Mouse Lymphoma TK and CHO HGPRT Loci. Mutagenesis, 4, p. 394-403. Aaron, C.S. and Stankowski, Jr.L.F., (1989) Comparison of the AS52/XPRT and the CHO/HPRT Assays: Evaluation of Six Drug Candidates. Mutation Res., 223, p. 121-128. Aaron, C.S., Bolcsfoldi, G., Glatt, H.R., Moore, M., Nishi, Y., Stankowski, Jr.L.F., Theiss, J. and Thompson, E., (1994) Mammalian Cell Gene Mutation Assays Working Group Report. Report of the International Workshop on Standardisation of Genotoxicity Test Procedures. Mutation Res., 312, p. 235-239. Scott, D., Galloway, S.M., Marshall, R.R., Ishidate, M., Brusick, D., Ashby, J. and Myhr, B.C., (1991) Genotoxicity Under Extreme Culture Conditions. A report from ICPEMC Task Group 9. Mutation Res., 257, p. 147-204. Clive, D., McCuen, R., Spector, J.F.S., Piper, C. and Mavournin, K.H., (1983) Specific Gene Mutations in L5178Y Cells in Culture. A Report of the U.S. Environmental Protection Agency Gene-Tox Program. Mutation Res., 115, p. 225-251. Li, A.P., Gupta, R.S., Heflich, R.H. and Wasson, J.S., (1988) A Review and Analysis of the Chinese Hamster Ovary/Hypoxanthine Guanine Phosphoribosyl Transferase System to Determine the Mutagenicity of Chemical Agents: A Report of Phase III of the U.S. Environmental Protection Agency Gene-Tox Program. Mutation Res., 196, p. 17-36. Li, A.P., Carver, J.H., Choy, W.N., Hsie, A.W., Gupta, R.S., Loveday, K.S., O'Neill, J.P., Riddle, J.C., Stankowski, L.F. Jr. and Yang, L.L., (1987) A Guide for the Performance of the Chinese Hamster Ovary Cell/Hypoxanthine-Guanine Phosphoribosyl Transferase Gene Mutation Assay. Mutation Res., 189, p. 135-141. Liber, H.L., Yandell, D.W and Little, J.B., (1989) A Comparison of Mutation Induction at the TK and HPRT Loci in Human Lymphoblastoid Cells: Quantitative Differences are Due to an Additional Class of Mutations at the Autosomal TK Locus. Mutation Res., 216, p. 9-17. Stankowski, L.F. Jr., Tindall, K.R. and Hsie, A.W., (1986) Quantitative and Molecular Analyses of Ethyl Methanosulphonate -and ICR 191- Induced Molecular Analyses of Ethyl Methanosulphonate -and ICR 191-Induced Mutation in AS52 Cells. Mutation Res., 160, p. 133-147. Turner, N.T., Batson, A.G. and Clive, D., (1984) Procedures for the L5178Y/TK+/- — TK+/- Mouse Lymphoma Cell Mutagenicity Assay. In: Kilbey, B.J. et al (eds.) Handbook of Mutagenicity Test Procedures, Elsevier Science Publishers, New York, p. 239-268. Arlett, C.F., Smith, D.M., Clarke, G.M., Green, M.H.L., Cole, J., McGregor, D.B. and Asquith, J.C., (1989) Mammalian Cell Gene Mutation Assays Based upon Colony Formation. In: Statistical Evaluation of Mutagenicity Test Data, Kirkland, D.J., Ed., Cambridge University Press, p. 66-101. Abbondandolo, A., Bonatti, S., Corti, G., Fiorio, R., Loprieno, N. and Mazzaccaro, A., (1977) Induction of 6-Thioguanine-Resistant Mutants in V79 Chinese Hamster Cells by Mouse-Liver Microsome-Activated Dimethylnitrosamine. Mutation Res., 46, p. 365-373. Ames, B.N., McCann, J. and Yamasaki, E., (1975) Methods for Detecting Carcinogens and Mutagens with the Salmonella/Mammalian-Microsome Mutagenicity Test. Mutation Res., 31, p. 347-364. Clive, D., Johnson, K.O., Spector, J.F.S., Batson, A.G. and Brown M.M.M., (1979) Validation and Characterisation of the L5178Y/TK+/- Mouse Lymphoma Mutagen Assay System. Mutat. Res., 59, p. 61-108. Maron, D.M. and Ames, B.N., (1983) Revised Methods for the Salmonella Mutagenicity Test. Mutation Res., 113, p. 173-215. Elliott, B.M., Combes, R.D., Elcombe, C.R., Gatehouse, D.G., Gibson, G.G., Mackay, J.M. and Wolf, R.C., (1992) Alternatives to Aroclor 1254-Induced S9 in In Vitro Genotoxicity Assays. Mutagenesis, 7, p. 175-177. Matsushima, T., Sawamura, M., Hara, K. and Sugimura, T., (1976) A Safe Substitute for Polychlorinated Biphenyls as an Inducer of Metabolic Activation Systems. In: In vitro Metabolic Activation in Mutagenesis Testing, de Serres, F.J., Fouts, J.R., Bend, J.R. and Philpot, R.M. (eds), Elsevier, North-Holland, p. 85-88. Krahn, D.F., Barsky, F.C. and McCooey, K.T., (1982) CHO/HGPRT Mutation Assay: Evaluation of Gases and Volatile Liquids. In: Tice, R.R., Costa, D.L., Schaich, K.M. (eds). Genotoxic Effects of Airborne Agents. New York, Plenum, p. 91-103. Zamora, P.O., Benson, J.M., Li, A.P. and Brooks, A.L., (1983) Evaluation of an Exposure System Using Cells Grown on Collagen Gels for Detecting Highly Volatile Mutagens in the CHO/HGPRT Mutation Assay. Environmental Mutagenesis, 5, p. 795-801. Applegate, M.L., Moore, M.M., Broder, C.B., Burrell, A. and Hozier, J.C., (1990) Molecular Dissection of Mutations at the Heterozygous Thymidine Kinase Locus in Mouse Lymphoma Cells. Proc. Natl. Acad. Sci. USA, 87, p. 51-55. Moore, M.M., Clive, D., Hozier, J.C., Howard, B.E., Batson, A.G., Turner, N.T. and Sawyer, J., (1985) Analysis of Trifluorothymidine-Resistant (TFTr) Mutants of L5178Y/TK+/- Mouse Lymphoma Cells. Mutation Res., 151, p. 161-174. Yandell, D.W., Dryja, T.P. and Little, J.B., (1990) Molecular Genetic Analysis of Recessive Mutations at a Heterozygous Autosomal Locus in Human Cells. Mutation Res., 229, p. 89-102. Moore, M.M. and Doerr, C.L., (1990) Comparison of Chromosome Aberration Frequency and Small-Colony TK-Deficient Mutant Frequency in L5178Y/TK+/- - 3.7.2C Mouse Lymphoma Cells. Mutagenesis, 5, p. 609-614.

B.18. DNA DAMAGE AND REPAIR — UNSCHEDULED DNA SYNTHESIS — MAMMALIAN CELLS IN VITRO

B.18.

1. METHOD

1.

1.1. INTRODUCTION See General introduction Part B.

1.2. DEFINITION See General introduction Part B.

1.3. REFERENCE SUBSTANCES None.

1.4. PRINCIPLE OF THE TEST METHOD The Unscheduled DNA Synthesis (UDS) test measures the DNA repair synthesis after excision and removal of a stretch of DNA containing the region of damage induced by chemical and physical agents. The test is based on the incorporation of tritium labelled thymidine (3H-TdR) into the DNA of mammalian cells, which are not in the S phase of the cell cycle. The uptake of 3H-TdR may be determined by autoradiography or by liquid scintillation counting (LSC) of DNA from the treated cells. Mammalian cells in culture, unless primary rat hepatocytes are used, are treated with the test agent with and without an exogenous metabolic activation system. UDS may also be measured in in vivo systems.

1.5. QUALITY CRITERIA None.

1.6. DESCRIPTION OF THE TEST METHOD

1.6. Preparations

Test chemicals and control or reference substances should be prepared in growth medium or dissolved or suspended in appropriate vehicles and then further diluted in growth medium for use in the assay. The final concentration of the vehicle should not affect cell viability. Primary cultures of rat hepatocytes, human lymphocytes or established cell lines (e.g. human diploid fibroblasts) may be used in the assay. Cells should be exposed to the test chemical both in the presence and absence of an appropriate metabolic activation system.

Test conditions

Number of cultures

At least two cell cultures for autoradiography and six cultures (or less if scientifically justified) for LSC UDS determinations are necessary for each experimental point.

Use of negative and positive controls

Concurrent positive and negative (untreated and/or vehicle) controls with and without metabolic activation should be included in each experiment. Examples of positive controls for the rat hepatocyte assay include 7,12- dimethylbenzathracene (7,12- DMBA) or 2-acetylaminofluorene (2-AAF). In the case of established cell lines 4-nitroquinoline-N-oxide (4-NQO) is an example of a positive control for both the autoradiographic and LSC assays performed without metabolic activation; N-dimethylnitrosamine is an example of a positive control compound when metabolic activation systems are used.

Exposure concentrations

Multiple concentrations of the test substance over a range adequate to define the response should be used. The highest concentration should elicit some cytotoxic effects. Relatively water-insoluble compounds should be tested up to the limit of solubility. For freely water-soluble non-toxic chemicals, the upper test chemical concentration should be determined on a case-by-case basis.

Cells

Appropriate growth media, CO2 concentration, temperature and humidity should be used in maintaining cultures. Established cell lines should be periodically checked for Mycoplasma contamination.

Metabolic activation

A metabolic activation system is not used with primary hepatocyte cultures. Established cell lines and lymphocytes are exposed to test substance both in the presence and absence of an appropriate metabolic activation system.

Procedure

Preparation of cultures

Established cell lines are generated from stock cultures (e.g. by trypsinisation or by shaking off), seeded in culture vessels at appropriate density, and incubated at 37 oC. Short-term cultures of rat hepatocytes are established by allowing freshly dissociated hepatocytes in an appropriate medium to attach themselves to the growing surface. Human lymphocyte cultures are set up using appropriate techniques.

Treatment of the cultures with the test substance Primary rat hepatocytes

Freshly isolated rat hepatocytes are treated with the test substance in a medium containing 3H-TdR for an appropriate length of time. At the end of the treatment period, medium should be drained off the cells, which are then rinsed fixed and dried. Slides should be dipped in autoradiographic emulsion (alternative stripping film may be used), exposed, developed, stained and counted.

Established cell lines and lymphocytes

Autoradiographic techniques: cell cultures are exposed to the test substance for appropriate durations followed by treatment with 3H-TdR. The times will be governed by the nature of the substance, the activity of metabolising systems and the type of cells. To detect the peak of UDS, 3H-TdR should be added either simultaneously with the test subsrance or within a few minutes after exposure to the test substance. The choice between these two procedures will be influenced by possible interactions between test substance and 3H-TdR. In order to discriminate between UDS and semi-conservative DNA replication, the latter can be inhibited, for example, by the use of an arginine-deficient medium, low serum content or by hydroxyurea in the culture medium. LSC measurements of UDS: prior to treatment with test substance, entry of cells into S-phase should be blocked as described above; cells should then be exposed to test chemical as described for autoradiography. At the end of the incubation period, DNA should be extracted from the cells and the total DNA content, and the extent of 3H-TdR, incorporation determined. It should be noted that, where human lymphocytes are used in the above techniques, the suppression of semi-conservative DNA replication is unnecessary in unstimulated cultures.

Analysis

Autoradiographic determinations

In determining UDS in cells in culture, S-phase nuclei are not counted. At least 50 cells per concentration should be counted. Slides should be coded before counting. Several widely separated random fields should be counted on each slide. The amount of 3H-TdR incorporation in the cytoplasm should be determined by counting three nucleus-sized areas in the cyptoplasm of each cell counted.

LSC determinations

An adequate number of cultures should be used at each concentration and in the controls in LSC UDS determinations. All results should be confirmed in an independent experiment.

2. DATA Data should be presented in tabular form.

2.

2.1. AUTORADIOGRAPHIC DETERMINATIONS The extent of 3H-TdR incorporation in the cytoplasm and the number of grains found over the cell nucleus should be recorded separately. Mean, median and mode may be used to describe the distribution of the extent of 3H-TdR incorporation in the cytoplasm and the number of grains per nucleus.

2.2. LSC DETERMINATIONS For LSC determinations, 3H-TdR incorporation should be reported as dpm/μg DNA. The mean dpm/μg DNA with standard deviation may be used to describe the distribution of incorporation. Data should be evaluated using appropriate statistical methods.

3. REPORTING

3.

3.1. TEST REPORT The test report shall, if possible, contain the following information:

cells used, density and passage number at time of treatment, number of cell cultures,

methods used for maintenance of cell cultures including medium, temperature and CO2 concentration,

test substance, vehicle, concentrations and rationale for selection of concentrations used in the assay,

details of metabolic activation systems,

treatment schedule,

positive and negative controls,

autoradiographic technique used,

procedures used to block entry of cells into S-phase,

procedures used for DNA extraction and determination of total DNA content in LSC determination,

dose/response relationship, where possible,

statistical evaluation,

discussion of results,

interpretation of results.

3.2. VALUATION AND INTERPRETATION See General introduction Part B.

4. REFERENCES See General introduction Part B.

B.19. SISTER CHROMATID EXCHANGE ASSAY IN VITRO

B.19.

1. METHOD

1.

1.1. INTRODUCTION See General introduction Part B.

1.2. DEFINITION See General introduction Part B.

1.3. REFERENCE SUBSTANCES None.

1.4. PRINCIPLE OF THE TEST METHOD The Sister Chromatid Exchange (SCE) assay is a short-term test for the detection of reciprocal exchanges of DNA between two sister chromatids of a duplicating chromosome. SCEs represent the interchange of DNA replication products at apparently homologous loci. The exchange process presumably involves DNA breakage and reunion, although little is known about its molecular basis. Detection of SCEs requires some means of differentially labelling sister chromatids and this can be achieved by incorporation of bromodeoxyuridine (BrdU) into chromosomal DNA for two cell cycles. Mammalian cells in vitro are exposed to the test chemical with and without a mammalian exogenous metabolic activation system, if appropriate, and cultured for two rounds of replication in BrdU-containing medium. After treatment with a spindle inhibitor (e.g. colchicine) to accumulate cells in a metaphase-like stage of mitosis (c-metaphase), cells are harvested and chromosome preparations are made.

1.5. QUALITY CRITERIA None.

1.6. DESCRIPTION OF THE TEST METHOD

1.6.

1.6.1. Preparations

Primary cultures, (human lymphocytes) or established cell lines (e.g. Chinese hamster ovary cells) may be used in the assay. Cell lines should be checked for Mycoplasma contamination,

appropriate culture media and incubation conditions (e.g. temperature, culture vessels, CO2 concentration and humidity) should be used,

test substances may be prepared in culture media or dissolved or suspended in appropriate vehicles prior to treatment of the cells. The final concentration of a vehicle in the culture system should not significantly affect, cell viability or growth rate and effects on SCE frequency should be monitored by a solvent control,

cells should be exposed to the test substance both in the presence and absence of an exogenous mammalian metabolic activation system. Alternatively, where cell types with intrinsic metabolic activity are used, the rate and nature of the activity should be appropriate to the chemical class being tested.

1.6.2. Test conditions

1.6.2. Number of cultures

At least duplicate cultures should be used for each experimental point.

Use of negative and positive controls

Positive controls, using both a direct acting compound and a compound requiring metabolic activation should be included in each experiment; a vehicle control should also be used. The following are examples of substances which might be used as positive controls: When appropriate, an additional positive control of the same chemical class as the chemical under test may be included.

direct acting compound:

ethylmethanesulphonate,

indirect acting compound:

cyclophosphamide.

Exposure concentrations

At least three adequately spaced concentrations of the test substance should be used. The highest concentration should give rise to a significant toxic effect but must still allow adequate cell replication to occur. Relatively water-insoluble substances should be tested up to the limit of solubility using appropriate procedures. For freely water-soluble non-toxic substances the upper test substance concentration should be determined on a case-by-case basis.

1.6.3. Procedure

1.6.3. Preparation of cultures

Established cell lines are generated from stock cultures (e.g. by trypsinisation or by shaking off), seeded in culture vessels at appropriate density and incubated at 37 oC. For monolayer cultures, the number of cells per culture vessel should be adjusted so that the cultures are not much more than 50 % confluent at the time of harvest. Alternatively, cells may be used in suspension culture. Human lymphocyte cultures are set up from heparinized blood, using appropriate techniques, and incubated at 37 oC.

Treatment

Cells in an exponential stage of growth are exposed to the test substance for a suitable period of time; in most cases one to two hours may be effective, but the treatment time may be extended up to two complete cell cycles in certain cases. Cells without sufficient instrinsic metabolic activity should be exposed to the test chemical in the presence and absence of an appropriate metabolic activation system. At the end of the exposure period, cells are washed free of test substance and cultured for two rounds of replication in the presence of BrdU. As an alternative procedure cells may be exposed simultaneously to the test chemical and BrdU for the complete culture time of two cell cycles. Human lymphocyte cultures are treated while they are in a semisynchronous condition. Cells are analysed in their second post-treatment division, ensuring that the most sensitive cell cycle stages have been exposed to the chemical. All cultures to which BrdU is added should be handled in darkness or in dim light from incandescent lamps up to the harvesting of cells in order to minimise photolysis of BrdU-containing DNA.

Harvesting of cells

Cell cultures are treated with a spindle inhibitor (e.g. colchicine) one to four hours prior to harvesting. Each culture is harvested and processed separately for the preparation of chromosomes.

Chromosome preparation and staining

Chromosome preparations are made by standard cytogenetic techniques. Staining of slides to show SCEs can be performed by several techniques, (e.g. the fluorescence plus Giemsa method).

Analysis

The number of cells analysed should be based on the spontaneous control frequency of SCE. Usually, at least 25 well-spread metaphases per culture are analysed for SCEs. Slides are coded before analysis. In human lymphocytes only metaphases containing 46 centromeres are analysed. In established cell lines only metaphases containing ± 2 centromeres of the modal number are analysed. It should be stated whether or not centromeric switch of label is, scored as an SCE. The results should be confirmed in an independent experiment.

2. DATA Data should be presented in tabular form. The number of SCEs for each metaphase and the number of SCEs per chromosome for each metaphase should be listed separately for all treated and control cultures. The data should be evaluated using appropriate statistical methods.

3. REPORTING

3.

3.1. TEST REPORT The test report shall, if possible, contain the following information:

cells used, methods of maintenance of cell culture,

test conditions: composition of media, CO2 concentration, concentration of test substance, vehicle used, incubation temperature, treatment time, spindle inhibitor used, its concentration and the duration of treatment with it, type of mammalian activation system used, positive and negative controls,

number of cell cultures per experimental point,

details of the technique used for slide preparation,

number of metaphases analysed (data given separately for each culture),

mean number of SCE per cell and per chromosome (data given separately for each culture),

criteria for scoring SCE,

rationale for dose selection,

dose-response relationship, if applicable,

statistical evaluation,

discussion of results,

interpretation of results.

3.2. EVALUATION AND INTERPRETATION See General introduction Part B.

4. REFERENCES See General introduction Part B.

B.20. SEX-LINKED RECESSIVE LETHAL TEST IN DROSOPHILA MELANOGASTER

B.20.

1. METHOD

1.

1.1. INTRODUCTION See General introduction Part B.

1.2. DEFINITION See General introduction Part B.

1.3. REFERENCE SUBSTANCES None.

1.4. PRINCIPLES OF THE TEST METHOD The sex-linked recessive lethal (SLRL) test using Drosophila melanogaster detects the occurrence of mutations, both point mutations and small deletions, in the germ line of the insect. This test is a forward mutation assay capable of screening for mutations at about 800 loci on the X-chromosome; this represents about 80 % of all X-chromosal loci. The X-chromosome represents approximately one-fifth of the entire haploid genome. Mutations in the X-chromosome of Drosophila melanogaster are phenotypically expressed in males carrying the mutant gene. When the mutation is lethal in the hemizygous condition, its presence is inferred from the absence of one class of male offspring out of the two that are normally produced by a heterozygous female. The SLRL test takes advantage of these facts by means of specially marked and arranged chromosomes.

1.5. QUALITY CRITERIA None.

1.6. DESCRIPTION OF THE TEST METHOD

1.6. Preparations

Stocks

Males of a well-defined wild-type stock and females of the Muller-5 stock may be used. Other appropriately marked female stocks with multiple inverted X-chromosomes may also be used.

Test substance

Test substances should be dissolved in water. Substances, which are insoluble in water may be dissolved or suspended in appropriate vehicles (e.g. a mixture of ethanol and Tween-60 or 80), then diluted in water or saline prior to administration. Dimethylsulphoxide (Dmso) should be avoided as a vehicle.

Number of animals

The test should be designed with a predetermined sensitivity and power. The spontaneous mutant frequency observed in the appropriate control will influence strongly the number of treated chromosomes that must be analysed.

Route of administration

Exposure may be oral, by injection or by exposure to gases or vapours. Feeding of the test substance may be done in sugar solution. When necessary, substances may be dissolved in a 0,7 % NaCl solution and injected into the thorax or abdomen.

Use of negative and positive controls

Negative (vehicle) and positive controls should be included. However, if appropriate laboratory historical control data are available, no concurrent controls are needed.

Exposure levels

Three exposure levels should be used. For a preliminary assessment one exposure level of the test substance may be used, that exposure level being either the maximum tolerated concentration or that producing some indication of toxicity. For non-toxic substances exposure to the maximum practicable concentration should be used.

Procedure

Wild-type males (three to five days old) are treated with the test substance and mated individually to an excess of virgin females from the Muller-5 stock or from another appropriately marked (with multiple inverted X-chromosomes) stock. The females are replaced with fresh virgins every two to three days to cover the entire germ cell cycle. The offspring of these females are scored for lethal effects corresponding to the effects on mature sperm, mid or late-stage spermatids, early spermatids, spermatocytes and spermatogonia at the time of treatment. Heterozygous F1 females from the above crosses are allowed to mate individually (i.e. one female per vial) with their brothers. In the F2 generation, each culture is scored for the absence of wild-type males. If a culture appears to have arisen from an F1 female carrying a lethal in the parental X-chromosome (i.e. no males with the treated chromosome are observed) daughters of that female with the same genotype should be tested to ascertain whether the lethality is repeated in the next generation.

2. DATA Data should be tabulated to show the number of X-chromosomes tested, the number of non-fertile males and the number of lethal chromosomes at each exposure concentration and for each mating period for each male treated. Numbers of clusters of different sizes per male should be reported. These results should be confirmed in a separate experiment. Appropriate statistical methods should be used in evaluation sex-linked recessive lethal tests. Clustering of recessive lethals originating from one male should be considered and evaluated in an appropriate statistical manner.

3. REPORTING

3.

3.1. TEST REPORT The test report shall, if possible, contain the following information:

stock: Drosophila stocks or strains used, age of insects, number of males treated, number of sterile males, number of F2 cultures established, number of F2 cultures without progeny, number of chromosomes carrying a lethal detected at each germ cell stage,

criteria for establishing the size of treated groups,

test conditions. detailed description of treatment and sampling schedule, exposure levels, toxicity data, negative (solvent) and positive controls, if appropriate,

criteria for scoring lethal mutations,

exposure/effect relationship where possible,

evaluation of data,

discussion of results,

interpretation of results.

3.2. EVALUATION AND INTERPRETATION See General introduction Part B.

4. REFERENCES See General introduction Part B.

B.21. IN VITRO MAMMALIAN CELL TRANSFORMATION TESTS

B.21.

1. METHOD

1.

1.1. INTRODUCTION See General introduction Part B.

1.2. DEFINITION See General introduction Part B.

1.3. REFERENCE SUBSTANCES None.

1.4. PRINCIPLE OF THE TEST METHOD Mammalian cell culture systems may be used to detect phenotypic changes in vitro induced by chemical substances associated with malignant transformation in vivo. Widely used cells include C3H10T1/2, 3T3, SHE, Fischer rat and the tests rely on changes in cell morphology, focus formation or changes in anchorage dependence in semi-solid agar. Less widely used systems exist which detect other physiological or morphological changes in cells following exposure to carcinogenic chemicals. None of the in vitro test endpoints has an established mechanistic link with cancer. Some of the test systems are capable of detecting tumour promotors. Cytotoxicity may be determined by measuring the effect of the test material on colony-forming abilities (cloning efficiency) or growth rates of the cultures. The measurement of cytotoxicity is to establish that exposure to the test chemical has been toxicologically relevant but cannot be used to calculate transformation: frequency in all assays since some may involve prolonged incubation and/or replating.

1.5. QUALITY CRITERIA None.

1.6. DESCRIPTION OF THE TEST METHOD

1.6. Preparations

Cells

A variety of cell lines or primary cells are available depending on the transformation test being used. The investigator must ensure that the cells in the test being performed exhibit the appropriate phenotypic change after exposure to known carcinogens and that the test, in the investigator's laboratory, is of proven and documented validity and reliability.

Medium

Media and experimental conditions should be used that are appropriate to the transformation assay in use.

Test substance

Test substances may be prepared in culture media or dissolved or suspended in appropriate vehicles prior to treatment of the cells. The final concentration of the vehicle in the culture system should not affect cell viability, growth rate or transformation incidence.

Metabolic activation

Cells should be exposed to the test substance both in the presence and absence of an appropriate metabolic activation system. Alternatively, when cell types are used that possess intrinsic metabolic activity, the nature of the activity should be known to be appropriate to the chemical class being tested.

Test conditions

Use of negative and positive controls

Positive controls, using both a direct-acting compound and a compound requiring metabolic activation should be included in each experiment; a negative (vehicle) control should also be used. The following are examples of substances, which might be used as positive controls: When appropriate, an additional positive control of the same chemical class as the compound under test should be included.

Direct-acting chemicals:

Ethylmethanesulphonate,

β-propiolactone,

Compounds requiring metabolic activation:

2-acetylaminofluorene,

4-dimethylaminoazobenzene,

7,12-dimethylbenzanthracene.

Exposure concentrations

Several concentrations of the test substance should be used. These concentrations should yield a concentration-related toxic effect, the highest concentration producing a low level of survival and the survival in the lowest concentration being approximately the same as that in the negative control. Relatively water-insoluble substances should be tested up to the limit of solubility using appropriate procedures. For freely water-soluble non-toxic substances the upper test substance concentration should be determined on a case-by-case basis.

Procedure

Cells should be exposed for a suitable period of time depending on the test system in use, and this may involve re-dosing accompanied by a change of medium (and if necessary, fresh metabolic activation mixture) if exposure is prolonged. Cells without sufficient intrinsic metabolic activity should be exposed to the test substance in the presence and absence of an appropriate metabolic activation system. At the end of the exposure period, cells are washed free of test substance and cultured under conditions appropriate for the appearance of the transformed phenotype being monitored and the incidence of transformation determined. All results are confirmed in an independent experiment.

2. DATA Data should be presented in tabular form and may take a variety of forms according to the assay being used e.g. plate counts, positive plates or numbers of transformed cells. Where appropriate, survival should be expressed as a percentage of control levels and transformation frequency expressed as the number of transformants per number of survivors. Data should be evaluated using appropriate statistical methods.

3. REPORTING

3.

3.1. TEST REPORT The test report shall, if possible, contain the following information:

cell type used, number of cell cultures, methods for maintenance of cell cultures,

test conditions: concentration of test substance, vehicle used, incubation time, duration and frequency of treatment, cell density during treatment, type of exogenous metabolic activation system used, positive and negative controls, specification of phenotype being monitored, selective system used (if appropriate), rational for dose selection,

method used to enumerate viable and transformed cells,

statistical evaluation,

discussion of results,

interpretation of results.

3.2. EVALUATION AND INTERPRETATION See General introduction Part B.

4. REFERENCES See General introduction Part B.

B.22.

1. METHOD

1.

1.1. INTRODUCTION See General introduction Part B.

1.2. DEFINITION See General introduction Part B.

1.3. REFERENCE SUBSTANCES None.

1.4. PRINCIPLE OF THE TEST METHOD Dominant lethal effects cause embryonic or foetal death. Induction of dominant lethals by exposure to a chemical substance indicates that the substance has affected germinal tissue of the test species. It is generally accepted that dominant lethals are due to chromosomal damage (structural and numerical anomalies). Embryonic death if females are treated may also be the result of toxic effects. Generally, male animals are exposed to the test compound and mated to untreated virgin females. The various germ cell stages can be tested separately by the use of sequential mating intervals. The increase of dead implants per female in the treated group over the dead implants per female in the control group reflects the post-implantational loss. Pre-implantational loss can be estimated based on corpora lutea counts or by comparing the total implants per female in treated and control groups. The total dominant lethal effect is the sum of pre- and post-implantational loss. The calculation of the total dominant lethal effect is based on comparison of the live implants per female in the test group to the live implants per female in the control group. A reduction in the number of implants at certain intervals may be the result of cell killing (i.e. of spermatocytes and/or spermatogonia).

1.5. QUALITY CRITERIA None.

1.6. DESCRIPTION OF THE TEST METHOD

1.6. Preparations

When possible, test substances should be dissolved or suspended in isotonic saline. Chemicals insoluble in water may be dissolved or suspended in appropriate vehicles. The vehicle used should neither interfere with the test chemical nor produce toxic effects. Fresh preparations of the test chemical should be employed.

Test conditions

Route of administration

The test compound should generally be administered only once. Based on toxicological information a repeated treatment schedule can be employed. The usual routes of administration are oral intubation or intraperitoneal injection. Other routes of administration may be appropriate.

Experimental animals

Rats or mice are recommended as the test species. Healthy fully sexually mature animals are randomised and assigned to treatment and control groups.

Number and sex

An adequate number of treated males should be used, taking into account the spontaneous variation of the biological character being evaluated. The number chosen should be based on the pre-determined sensitivity of detection and power of significance. For example in a typical test, the number of males in each dose group should be sufficient to provide between 30 and 50 pregnant females per mating interval.

Use of negative and positive controls

Generally concurrent positive and negative (vehicle) controls should be included in each experiment. When acceptable positive control results are available from experiments conducted recently in the same laboratory these results can be used instead of a concurrent positive control. Positive control substances should be used at an appropriate low dose (e.g. MMS, intraperitoneally, at 10 mg/kilogram) to demonstrate the test sensitivity.

Dose levels

Normally, three dose levels should be used. The high dose should produce signs of toxicity or reduced fertility in the treated animals. In certain cases a single high dose level may be sufficient.

Limit test

Non-toxic substances should be tested at 5 g/kilogram on a single administration or at 1 g/kilogram/day on repeated administration.

Procedure

Several treatment schedules are available. Single administration of the test substance is the most widely used. Other treatment schedules may be used. Individual males are mated sequentially to one or two untreated virgin females at appropriate intervals after treatment. Females should be left with the males for at least the duration of one oestrous cycle or until mating has occurred as determined by the presence of sperm in the vagina or by the presence of a vaginal plug. The number of matings following treatment is governed by the treatment schedule and should ensure that all germ cell stages are sampled after treatment. Females are sacrificed in the second half of pregnancy and uterine contents are examined to determine the number of dead and live implants. The ovaries may be examined to determine the number of corpora lutea.

2. DATA Data should be tabulated to show the number of males, the number of pregnant females, and the number of non-pregnant females. Results of each mating, including the identity of each male and female, should be reported individually. For each female, week of mating, dose level received by the males, the frequencies of live implants and of dead implants should be recorded. The calculation of the total dominant lethal effect is based on comparison of the live implants per female in the test group to the live implants per female in the control group. The ratio of dead to live implants from the treated group compared to the same ratio from the control group is analysed to indicate the post-implantation loss. If the data are recorded as early and late deaths, the tables should make that clear. If pre-implantation loss is estimated, it should be reported. Pre-implantation loss can be calculated as a discrepancy between the number of corpora lutea and the number of implants or as a reduction in the average number of implants per uterus in comparison with control matings. Data are evaluated using appropriate statistical methods.

species, strain, age and weights of animals used, number of animals of each sex in experimental and control groups,

test substance, vehicle, dose levels tested and rationale for dose selection, negarive and positive controls, toxicity data,

route and treatment schedule,

mating schedule,

method used to determine that mating has occurred,

time of sacrifice,

3238 more provisions are not shown here; each has its own address.

Versions

2008-05-30

Cited by

Regulation (EU) No 528/2012 of the European Parliament and of the Council of 22 May 2012 concerning the making available on the market and use of biocidal products (Text with EEA relevance) Regulation · Grade B
Information required to support the approval of an active substance is listed in the table below. Conditions for not requiring a specific test that are set out in the appropriate test methods in the Regulation (EC) No 440/2008 and are not repeated in column 3, also apply. Column 1 Information required Column 2 All data is CDS unless indicated as ADS Column 3 Specific rules for adaptation from standard information concerning some of the information requirements that may require recourse to testing of vertebrates APPLICANT Name and address Contact person Active substance manufacturer (name, addr
Regulation (EU) No 528/2012 of the European Parliament and of the Council of 22 May 2012 concerning the making available on the market and use of biocidal products (Text with EEA relevance) Regulation · Grade B
Information required to support the approval of an active substance is listed in the table below. Conditions for not requiring a specific test that are set out in the appropriate test methods in Regulation (EC) No 440/2008 that are not repeated in column 3, also apply. Column 1 Information required Column 2 All data is CDS unless indicated as ADS Column 3 Specific rules for adaptation from standard information concerning some of the information requirements that may require recourse to testing of vertebrates APPLICANT Name and address Contact person Manufacturer (name, address and location of
Regulation (EU) No 528/2012 of the European Parliament and of the Council of 22 May 2012 concerning the making available on the market and use of biocidal products (Text with EEA relevance) Regulation · Grade B
This Annex sets out the information requirements for the preparation of the dossier referred to in point (a) of Article 6(1). The data elements set down in this Annex comprise a Core Data Set (CDS) and an Additional Data Set (ADS). The data elements belonging to the CDS are considered as the basic data which should, in principle, be provided for all active substances. However, in some cases the physical or chemical properties of the substance may mean that it is impossible or unnecessary to provide specific data elements belonging to the CDS. With regard to the ADS, the data elements to be pro
Regulation (EU) No 528/2012 of the European Parliament and of the Council of 22 May 2012 concerning the making available on the market and use of biocidal products (Text with EEA relevance) Regulation · Grade B
Information required to support the authorisation of a biocidal product is listed in the table below. For each information requirement set down in this Annex the indications given in columns 1 and 3 of Annex II for the same information requirement shall also apply. Column 1 Information required: Column 2 All data is CDS unless indicated as ADS Column 3 Specific rules for adaptation from standard information concerning some of the information requirements that may require recourse to testing of vertebrates APPLICANT Name and address, etc. Contact person Manufacturer and formulator of the biocid
Regulation (EU) No 528/2012 of the European Parliament and of the Council of 22 May 2012 concerning the making available on the market and use of biocidal products (Text with EEA relevance) Regulation · Grade B
This Annex sets out the information requirements that shall be included in the dossier for the biocidal product accompanying an application for the approval of an active substance in accordance with point (b) of Article 6(1) and the dossier accompanying an application for the authorisation of a biocidal product in accordance with point (a) of Article 20(1). The data elements set down in this Annex comprise a Core Data Set (CDS) and an Additional Data Set (ADS). The data elements belonging to the CDS are considered as the basic data which should, in principle, be provided for all biocidal produ
Commission Regulation (EC) No 761/2009 of 23 July 2009 amending, for the purpose of its adaptation to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) (Text with EEA relevance) Regulation · Grade B
Commission Regulation (EC) No 440/2008 contains the test methods for the purposes of the determination of the physico-chemical properties, toxicity and eco-toxicity of substances to be applied for the purposes of Regulation (EC) No 1907/2006.
Commission Regulation (EC) No 761/2009 of 23 July 2009 amending, for the purpose of its adaptation to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) (Text with EEA relevance) Regulation · Grade B
It is necessary to update Regulation (EC) No 440/2008 to include changes to certain test methods and to include several new test methods adopted by the OECD. Stakeholders have been consulted on this proposal. Those amendments adapt the methods in question to scientific and technical progress.
Commission Regulation (EC) No 761/2009 of 23 July 2009 amending, for the purpose of its adaptation to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) (Text with EEA relevance) Regulation · Grade B
It is appropriate to update Regulation (EC) No 440/2008 to include with priority a new in vitro test method for skin irritation, in order to obtain a reduction of the number of animals to be used for experimental purposes, in accordance with Council Directive 86/609/EEC of 24 November 1986 on the approximation of laws, regulations and administrative provisions of the Member States regarding the protection of animals used for experimental and other scientific purposes. Although a draft in vitro test method for skin irritation is still under discussion within the OECD, it is appropriate in this
Commission Regulation (EC) No 761/2009 of 23 July 2009 amending, for the purpose of its adaptation to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) (Text with EEA relevance) Regulation · Grade B
Regulation (EC) No 440/2008 should therefore be amended accordingly.
Commission Regulation (EC) No 761/2009 of 23 July 2009 amending, for the purpose of its adaptation to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) (Text with EEA relevance) Regulation · Grade B
The Annex to Regulation (EC) No 440/2008 is amended as follows:
Commission Regulation (EU) 2016/266 of 7 December 2015 amending, for the purpose of its adaptation to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) (Text with EEA relevance) Regulation · Grade B
Commission Regulation (EC) No 440/2008 contains the test methods for the purposes of the determination of the physicochemical properties, toxicity and ecotoxicity of chemicals to be applied for the purposes of Regulation (EC) No 1907/2006.
Commission Regulation (EU) 2016/266 of 7 December 2015 amending, for the purpose of its adaptation to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) (Text with EEA relevance) Regulation · Grade B
It is necessary to update Regulation (EC) No 440/2008 to include new and updated test methods recently adopted by the OECD in order to take into account technical progress, and to ensure the reduction in the number of animals to be used for experimental purposes, in accordance with Directive 2010/63/EU of the European Parliament and of the Council. Stakeholders have been consulted on this draft.
Commission Regulation (EU) 2016/266 of 7 December 2015 amending, for the purpose of its adaptation to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) (Text with EEA relevance) Regulation · Grade B
Regulation (EC) No 440/2008 should therefore be amended accordingly.
Commission Regulation (EU) 2016/266 of 7 December 2015 amending, for the purpose of its adaptation to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) (Text with EEA relevance) Regulation · Grade B
The Annex to Regulation (EC) No 440/2008 is amended in accordance with the Annex to this Regulation.
Commission Regulation (EU) 2016/266 of 7 December 2015 amending, for the purpose of its adaptation to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) (Text with EEA relevance) Regulation · Grade B
The Annex to Regulation (EC) No 440/2008 is amended as follows:
Commission Decision of 24 June 2011 on establishing the ecological criteria for the award of the EU Ecolabel to lubricants (notified under document C(2011) 4447) (Text with EEA relevance) (2011/381/EU) Decision · Grade B
Requirements for the lubricant and its main components Acute aquatic toxicity data of the main components and the mixture shall be provided. Acute aquatic toxicity data for each main component shall be stated on each of the following two trophic levels: algae and daphnia. The critical concentration for the acute aquatic toxicity for each main component shall be at least 100 mg/L. Acute aquatic toxicity data for the applied lubricant shall be stated on each of the following three trophic levels: algae, daphnia and fish. The critical concentration for the acute aquatic toxicity for a lubricant i
Commission Decision of 24 June 2011 on establishing the ecological criteria for the award of the EU Ecolabel to lubricants (notified under document C(2011) 4447) (Text with EEA relevance) (2011/381/EU) Decision · Grade B
Requirements for each stated substance present above 0,10 % (w/w) Chronic toxicity test results in the form of No Observed Effect Concentration (NOEC) data shall be stated on each of the following two aquatic trophic levels: daphnia and fish. In case chronic toxicity test results are missing, acute aquatic toxicity tests results shall be provided for each of the following two trophic levels; algae and daphnia. One or more substances exhibiting a certain degree of aquatic toxicity are allowed in each of the five lubricant categories for a cumulative mass concentration as indicated in Table 1. A
Commission Decision of 24 June 2011 on establishing the ecological criteria for the award of the EU Ecolabel to lubricants (notified under document C(2011) 4447) (Text with EEA relevance) (2011/381/EU) Decision · Grade B
In a 28-day biodegradation study according Part C.4 of the Annex to Regulation (EC) No 440/2008, OECD 306, OECD 310 the following levels of biodegradation are achieved: In these ultimately biodegradable tests the 10-day window principle will not necessarily apply. If the substance reaches the biodegradation pass level within 28 days but not within the 10-day time-window, a slower degradation rate is assumed.
Commission Decision of 24 June 2011 on establishing the ecological criteria for the award of the EU Ecolabel to lubricants (notified under document C(2011) 4447) (Text with EEA relevance) (2011/381/EU) Decision · Grade B
The BOD5/ThOD or BOD5/COD ratio ≥ 0,5. The BOD5/(ThOD or COD) ratio can only be used if no data based on Part C.4 of the Annex to Regulation (EC) No 440/2008, OECD 306 or OECD 310 or any other equivalent test methods are available. The BOD5 shall be assessed according to Part C.5 of the Annex to Regulation (EC) No 440/2008 or equivalent methods while the COD shall be assessed according to Part C.6 of the Annex to Regulation (EC) No 440/2008 or equivalent methods.
Commission Decision of 24 June 2011 on establishing the ecological criteria for the award of the EU Ecolabel to lubricants (notified under document C(2011) 4447) (Text with EEA relevance) (2011/381/EU) Decision · Grade B
a biodegradation > 70 % in the Part C.9 of the Annex to Regulation (EC) No 440/2008 or OECD 302 C test for inherent biodegradation or equivalent methods, or