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)

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 2 000 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 5 000 mg/kg may be considered (see Appendix 3). For reasons of animal welfare concern, testing of animals in GHS Category 5 ranges (2 000 -5 000 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.   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 5 000 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 2 000 mg/kg (or exceptionally 5 000 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.   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

3.1.   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. REFERENCES

• (1)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.

• (2)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.

• (3)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.

• (4)Whitehead, A. and Curnow, R.N (1992) Statistical evaluation of the fixed-dose procedure. Fd. Chem. Toxicol., 30, p. 313-324.

• (5)Stallard, N. and Whitehead, A (1995) Reducing numbers in the fixed-dose procedure. Human Exptl. Toxicol. 14, p. 315-323.

• (6)Stallard, N., Whitehead, A and Ridgeway, P. (2002) Statistical evaluation of the revised fixed dose procedure. Hum. Exp. Toxicol., 21, p. 183-196.

• (7)OECD (2001) Guidance Document on Acute Oral Toxicity Testing. Environmental Health and Safety Monograph Series on Testing and Assessment No 24. Paris

• (8)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.

• (9)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].

• (10)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.

• (11)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.

Appendix 1

FLOW CHART FOR THE SIGHTING STUDY

Image omitted; see official source

Text of image

Starting dose: 5 mg/kg

START

1 animal 5 mg/kg

1 animal 50 mg/kg

1 animal 300 mg/kg

1 animal 2000 mg/kg

Classify GHS

Category 1*

Main study starting Dose (mg/kg):

starting dose: 50 mg/kg

START

1 animal 5 mg/kg

1 animal 50 mg/kg

1 animal 300 mg/kg

1 animal 2000 mg/kg

Classify GHS

Category 1*

Main study starting Dose (mg/kg):

Outcome

death

evident toxicity

no evident toxicity and no death

• for outcome at 5 mg/kg there is an optional supplementary procedure to confirm the GHS classification: see section 1.5.2

Image omitted; see official source

Text of image

Starting dose: 300 mg/kg

START

1 animal 5 mg/kg

1 animal 50 mg/kg

1 animal 300 mg/kg

1 animal 2000 mg/kg

Classify GHS

Category 1*

Main study starting Dose (mg/kg):

starting dose: 2000 mg/kg

START

1 animal 5 mg/kg

1 animal 50 mg/kg

1 animal 300 mg/kg

1 animal 2000 mg/kg

Classify GHS

Category 1*

Main study starting Dose (mg/kg):

Outcome

death

evident toxicity

no evident toxicity and no death

• for outcome at 5 mg/kg there is an optional supplementary procedure to confirm the GHS classification: see section 1.5.2

Appendix 2

FLOW CHART FOR THE MAIN STUDY

Image omitted; see official source

Text of image

Starting dose: 5 mg/kg

START

5 animals 5 mg/kg

5 animals 50 mg/kg*

5 animals 300 mg/kg

5 animals 2000 mg/kg

Classify GHS

Category

5/Unclassified

Starting dose: 50 mg/kg

START

5 animals 5 mg/kg

5 animals 50 mg/kg

5 animals 300 mg/kg*

5 animals 2000 mg/kg

Classify GHS

Category

5/Unclassified

Outcome

≥ 2 deaths

≥ 1 with evident toxicity and / or 1 death

No evident toxicity and no death

Group size

The 5 animals in each main study group will include any animal tested at that dose level in the sighting study

• Animal welfare override

If this dose level caused death in the sighting study, then no further animals will be tested. Go directly to outcome

Image omitted; see official source

Text of image

Starting dose: 300 mg/kg

START

5 animals 5 mg/kg

5 animals 50 mg/kg

5 animals 300 mg/kg

5 animals 2000 mg/kg*

Classify GHS

Category

5/Unclassified

starting dose: 2000 mg/kg

START

5 animals 5 mg/kg

5 animals 50 mg/kg

5 animals 300 mg/kg

5 animals 2000 mg/kg

Classify GHS

Category

5/Unclassified

Outcome

≥ 2 deaths

≥ 1 with evident toxicity and / or 1 death

No evident toxicity and no death

Group size

The 5 animals in each main study group will include any animal tested at that dose level in the sighting study

• Animal welfare override

If this dose level caused death in the sighting study, then no further animals will be tested. Go directly to outcome

Appendix 3

CRITERIA FOR CLASSIFICATION OF TEST SUBSTANCES WITH EXPECTED LD50 VALUES EXCEEDING 2 000 MG/KG WITHOUT THE NEED FOR TESTING.

Criteria for hazard Category 5 are intended to enable the identification of test substances which are of relatively low acute toxicity hazard but which, under certain circumstances may present a danger to vulnerable populations. These substances are anticipated to have an oral or dermal LD50 in the range of 2 000 -5 000 mg/kg or equivalent doses for other routes. Test substances could be classified in the hazard category defined by: 2 000 mg/kg < LD50 < 5 000 mg/kg (Category 5 in the GHS) in the following cases:

• (a) if directed to this category by any of the testing schemes of Appendix 2, based on mortality incidences

• (b) if reliable evidence is already available that indicates the LD50 to be in the range of Category 5 values; or other animal studies or toxic effects in humans indicate a concern for human health of an acute nature;

• (c) through extrapolation, estimation or measurement of data if assignment to a more hazardous class is not warranted; and
• — reliable information is available indicating significant toxic effects in humans, or
• — any mortality is observed when tested up to Category 4 values by the oral route, or
• — where expert judgement confirms significant clinical signs of toxicity, when tested up to Category 4 values, except for diarrhoea, piloerection or an ungroomed appearance, or
• — where expert judgement confirms reliable information indicating the potential for significant acute effects from the other animal studies.

TESTING AT DOSES ABOVE 2 000 MG/KG

Exceptionally, and only when justified by specific regulatory needs, the use of an additional upper fixed dose level of 5 000 mg/kg may be considered. Recognising the need to protect animal welfare, testing at 5 000 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 animal or human health (9).

Sighting study

The decision rules governing the sequential procedure presented in Appendix 1 are extended to include a 5 000 mg/kg dose level. Thus, when a sighting study starting dose of 5 000 mg/kg is used outcome A (death) will require a second animal to be tested at 2 000 mg/kg; outcomes B and C (evident toxicity or no toxicity) will allow the selection of 5 000 mg/kg as the main study starting dose. Similarly, if a starting dose other than 5 000 mg/kg is used then testing will progress to 5 000 mg/kg in the event of outcomes B or C at 2 000 mg/kg; a subsequent 5 000 mg/kg outcome A will dictate a main study starting dose of 2 000 mg/kg and outcomes B and C will dictate a main study starting dose of 5 000 mg/kg.

Main study

The decision rules governing the sequential procedure presented in Appendix 2 are extended to include a 5 000 mg/kg dose level. Thus, when a main study starting dose of 5 000 mg/kg is used, outcome A (≥ 2 deaths) will require the testing of a second group at 2 000 mg/kg; outcome B (evident toxicity and/or ≤ 1 death) or C (no toxicity) will result in the substance being unclassified according to GHS. Similarly, if a starting dose other than 5 000 mg/kg is used then testing will progress to 5 000 mg/kg in the event of outcome C at 2 000 mg/kg; a subsequent 5 000 mg/kg outcome A will result in the substance being assigned to GHS Category 5 and outcomes B or C will lead to the substance being unclassified.

Appendix 4

TEST METHOD B.1 bis

Guidance on classification according to the EU scheme to cover the transition period until full implementation of the Globally Harmonised Classification System (GHS) (taken from reference (8))

Image omitted; see official source

Text of image

Starting dose: 5 mg/kg

START

5 animals 5 mg/kg

5 animals 50 mg/kg

5 animals 300 mg/kg

5 animals 2000 mg/kg

Classify EU

starting dose: 50 mg/kg

START

5 animals 5 mg/kg

5 animals 50 mg/kg

5 animals 300 mg/kg *

5 animals 2000 mg/kg

Classify EU

Outcome

≥ 2 deaths

≥ 1 with evident toxicity and/or 1 death

No evident toxicity and no death

T+ = very toxic

T = toxic

H = harmful

U = unclassified

• Animal welfare override If this dose level caused death in the sighting study, then no futher animals will be tested. Go directely to outcome

Group size The 5 animals in each main study group will include any animal tested at that dose level in the study

Image omitted; see official source

Text of image

Starting dose: 300 mg/kg

START

5 animals 5 mg/kg

5 animals 50 mg/kg

5 animals 300 mg/kg

5 animals 2000 mg/kg*

Classify EU

Starting dose: 2000 mg/kg

START

5 animals 5 mg/kg

5 animals 50 mg/kg

5 animals 300 mg/kg

5 animals 2000 mg/kg

Classify EU

Outcome

≥ 2 deaths

≥ 1 with evident toxicity and / or 1 death

No evident toxicity and no death

T+ = very toxic

T = toxic

H = harmful

U + unclassified

Group size

The 5 animals in each main study group will include any animal tested at that dose level in the sighting study

• Animal welfare override

If this dose level caused death in the sighting study, then no further animals will be tested. Go directly to outcome

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

1. METHOD

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

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).

LD 50 (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 (2 000 or 5 000 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.;

• — 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.

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.

1.4.   DESCRIPTION OF THE METHOD

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.   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 2 000 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 (2 000 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 5 000 mg/kg body weight may be considered (see Appendix 2). For reasons of animal welfare concern, testing of animals in GHS Category 5 ranges (2 000 -5 000 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 2 000 mg/kg body weight may be carried out with six animals (three animals per step). Exceptionally a limit test at one dose level of 5 000 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.   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.

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

3.1.   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. REFERENCES

• (1)Roll R., Höfer-Bosse Th. And Kayser D (1986) New Perspectives in Acute Toxicity Testing of Chemicals. Toxicol. Lett., Suppl. 31, p. 86.

• (2)Roll R., Riebschläger M., Mischke U. and Kayser D (1989) Neue Wege zur Bestimmung der akuten Toxizität von Chemikalien. Bundesgesundheitsblatt 32, p. 336-341.

• (3)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.

• (4)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.

• (5)Diener W., and Schlede E., (1999) Acute Toxicity Class Methods: Alterations to LD/LC50 Tests. ALTEX 16, p. 129-134.

• (6)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.

• (7)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.

• (8)OECD, (2001) Guidance Document on Acute Oral Toxicity Testing. Environmental Health and Safety Monograph Series on Testing and Assessment No 24. Paris.

• (9)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.

• (10)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].

• (11)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.

• (12)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.

Appendix 1

PROCEDURE TO BE FOLLOWED FOR EACH OF THE STARTING DOSES

GENERAL REMARKS

For each starting dose, the respective testing schemes as included in this Appendix outline the procedure to be followed.

• — Appendix 1 a: starting dose is 5 mg/kg bw,

• — Appendix 1 b: starting dose is 50 mg/kg bw,

• — Appendix 1 c: starting dose is: 300 mg/kg bw,

• — Apendix 1 d: starting dose is: 2 000 mg/kg bw.

Depending on the number of humanely killed or dead animals, the test procedure follows the indicated arrows.

Appendix 1A

TEST PROCEDURE WITH A STARTING DOSE OF 5 MG/KG BODY WEIGHT

Image omitted; see official source

Text of image

Start

5 mg/kg 3 animals

50 mg/kg 3 animals

300 mg/kg 3 animals

2000 mg/kg 3 animals

5 mg/kg 3 animals

50 mg/kg 3 animals

300 mg/kg 3 animals

2000 mg/kg 3 animals

GHS

Category 1

0 - 5

Category 2

5 - 50

Category 3

50 - 300

Category 4

300 - 2000

Category 5

2000 - 5000

∞

LD50 cut-off mg/kg b.w.

• per step 3 animals of a single sex (normally females) are used

• 0, 1, 2, 3: Number of moribund or dead animals at each step

• GHS: Globally Harmonised Classification System (mg/kg b.w.)

• ∞: unclassified

• Testing at 5000 mg/kg b.w.: see Appendix 2

Appendix 1B

TEST PROCEDURE WITH A STARTING DOSE OF 50 MG/KG BODY WEIGHT

Image omitted; see official source

Text of image

Start

5 mg/kg 3 animals

50 mg/kg 3 animals

300 mg/kg 3 animals

2000 mg/kg 3 animals

5 mg/kg 3 animals

50 mg/kg 3 animals

300 mg/kg 3 animals

2000 mg/kg 3 animals

GHS

Category 1

0 - 5

Category 2

5 - 50

Category 3

50 - 300

Category 4

300 - 2000

Category 5

2000 - 5000

∞

3 (at 50) at the 1^st step

other

LD50 cut-off mg/kg b.w.

• per step 3 animals of a single sex (normally females) are used

• 0, 1, 2, 3: Number of moribund or dead animals at each step

• GHS: Globally Harmonised Classification System (mg/kg b.w.)

• ∞: unclassified

• Testing at 5000 mg/kg b.w.: see Appendix 2

Appendix1C

TEST PROCEDURE WITH A STARTING DOSE OF 300 MG/KG BODY WEIGHT

Image omitted; see official source

Text of image

Start

5 mg/kg 3 animals

50 mg/kg 3 animals

300 mg/kg 3 animals

2000 mg/kg 3 animals

5 mg/kg 3 animals

50 mg/kg 3 animals

300 mg/kg 3 animals

2000 mg/kg 3 animals

GHS

Category 1

0 - 5

Category 2

5 - 50

Category 3

50 - 300

Category 4

300 - 2000

Category 5

2000 - 5000

∞

3 (at 50) at the 1^st step

other

3 (at 50) at the 1^st step

other

LD50 cut-off mg/kg b.w.

• per step 3 animals of a single sex (normally females) are used

• 0, 1, 2, 3: Number of moribund or dead animals at each step

• GHS: Globally Harmonised Classification System (mg/kg b.w.)

• ∞: unclassified

• Testing at 5000 mg/kg b.w.: see Appendix 2

Appendix 1D

TEST PROCEDURE WITH A STARTING DOSE OF 2 000 MG/KG BODY WEIGHT

Image omitted; see official source

Text of image

Start

5 mg/kg 3 animals

50 mg/kg 3 animals

300 mg/kg 3 animals

2000 mg/kg 3 animals

5 mg/kg 3 animals

50 mg/kg 3 animals

300 mg/kg 3 animals

2000 mg/kg 3 animals

GHS

Category 1

0 - 5

Category 2

5 - 50

Category 3

50 - 300

Category 4

300 - 2000

Category 5

2000 - 5000

∞

3 (at 50) at 1^st step

other

3 (at 300) at 1^st step

other

3 (at 2000) at 1^st step

2 (at 2000) at 1^st step

other

LD50 cut-off mg/kg b.w.

• per step 3 animals of a single sex (normally females) are used

• 0, 1, 2, 3: Number of moribund or dead animals at each step

• GHS: Globally Harmonised Classification System (mg/kg b.w.)

• ∞: unclassified

• Testing at 5000 mg/kg b.w.: see Appendix 2

Appendix 2

CRITERIA FOR CLASSIFICATION OF TEST SUBSTANCES WITH EXPECTED LD50 VALUES EXCEEDING 2 000 MG/KG WITHOUT THE NEED FOR TESTING

Criteria for hazard Category 5 are intended to enable the identification of test substances which are of relatively low acute toxicity hazard but which, under certain circumstances may present a danger to vulnerable populations. These substances are anticipated to have an oral or dermal LD50 in the range of 2 000 -5 000 mg/kg or equivalent doses for other routes. The test substance should be classified in the hazard category defined by: 2 000 mg/kg < LD50 < 5 000 mg/kg (Category 5 in the GHS) in the following cases:

• (a) If directed to this category by any of the testing schemes of Appendix 1a-1d, based on mortality incidences;

• (b) if reliable evidence is already available that indicates the LD50 to be in the range of Category 5 values; or other animal studies or toxic effects in humans indicate a concern for human health of an acute nature;

• (c) through extrapolation, estimation or measurement of data if assignment to a more hazardous class is not warranted; and
• — reliable information is available indicating significant toxic effects in humans, or
• — any mortality is observed when tested up to Category 4 values by the oral route, or
• — where expert judgement confirms significant clinical signs of toxicity, when tested up to Category 4 values, except for diarrhoea, piloerection or an ungroomed appearance, or
• — where expert judgement confirms reliable information indicating the potential for significant acute effects from the other animal studies.

TESTING AT DOSES ABOVE 2 000 MG/KG

Recognising the need to protect animal welfare, testing of animals in Category 5 (5 000 mg/kg) ranges is discouraged and should only be considered when there is a strong likelihood that results of such a test have a direct relevance for protecting human or animal health (10). No further testing should be conducted at higher dose levels.

When testing is required a dose of 5 000 mg/kg, only one step (i.e. three animals) is required. If the first animal dosed dies, then dosing proceeds at 2 000 mg/kg in accordance with the flowcharts in Appendix 1. If the first animal survives, two further animals are dosed. If only one of the three animals dies, the LD50 value is expected to exceed 5 000 mg/kg. If both animals die, then dosing proceeds at 2 000 mg/kg.

Appendix 3

TEST METHOD B.1 tris: Guidance on classififcation according to EU scheme to cover the transition period until full implementation of the Globally Harmonised Classification System (GHS) (taken from reference (8))

Image omitted; see official source

Text of image

Start

5 mg/kg

3 animals

50 mg/kg

3 animals

300 mg/kg

3 animals

2000 mg/kg

3 animals

5 mg/kg

3 animals

50 mg/kg

3 animals

300 mg/kg

3 animals

2000 mg/kg

3 animals

GHS

Category 1

0 - 5

Category 2

5 - 50

Category 3

50 - 300

Category 4

300 - 2000

Category 5

2000 - 5000

∞

LD50 cut-off

mg/kg b. w.

EU/chemicals Liquid pesticides

EU solid pesticides

UN liquids

UN solids

Switzerland

US EPA crk

Japan PDSCA

Canada/WHMIS/US OSHA

US EPA pesticides

US CPSC

Canada pesticides

• per step 3 animals of a single sex (normally female) are used

• 0,1, 2, 3: Number of moribund or dead animals at each step

• ∞: unclassified

• GHS: Globally Harmonised Classification System (mg/kg b. w.)

Image omitted; see official source

Text of image

Start

5 mg/kg 3 animals

50 mg/kg 3 animals

300 mg/kg 3 animals

2000 mg/kg 3 animals

5 mg/kg 3 animals

50 mg/kg 3 animals

300 mg/kg 3 animals

2000 mg/kg 3 animals

GHS

Category 1 > 0 - 5

Category 2 > 5 - 50

Category 3 > 50 - 300

Category 4 > 300 - 2000

Category 5 > 2000 - 5000

3*

(at 50)

other

LD50 cut-off mg/kg b. w.

EU/chemicals Liquid pesticides

EU solid pesticides

UN liquids

UN solids

Switzerland

US EPA crk

Japan PDSCA

Canada/WHMIS/US OSHA

US EPA pesticides

US CPSC

Canada pesticides

• per step 3 animals of a single sex (normally female) are used

• 0,1, 2, 3: Number of moribund or dead animals at each step

• ∞: unclassified

• *: at first step

• GHS: Globally Harmonised Classification System (mg/kg b. w.)

Image omitted; see official source

Text of image

Start

5 mg/kg

3 animals

50 mg/kg

3 animals

300 mg/kg

3 animals

2000 mg/kg

3 animals

5 mg/kg

3 animals

50 mg/kg

3 animals

300 mg/kg

3 animals

2000 mg/kg

3 animals

GHS

Category 1

0 - 5

Category 2

5 - 50

Category 3

50 - 300

Category 4

300 - 2000

Category 5

2000 - 5000

3*

(at 50)

other

3*

(at 300)

other

LD50 cut-off

mg/kg b. w.

EU/chemicals Liquid pesticides

EU solid pesticides

UN liquids

UN solids

Switzerland

US EPA crk

Japan PDSCA

Canada/WHMIS/US OSHA

US EPA pesticides

US CPSC

Canada pesticides

• per step 3 animals of a single sex (normally female) are used

• 0,1,2,3: Number of moribund or dead animals at each step

• ∞: unclassified

• *: at first step

• GHS: Globally Harmonised Classification System (mg/kg b. w.)

Image omitted; see official source

Text of image

Start

5 mg/kg

3 animals

50 mg/kg

3 animals

300 mg/kg

3 animals

2000 mg/kg

3 animals

5 mg/kg

3 animals

50 mg/kg

3 animals

300 mg/kg

3 animals

2000 mg/kg

3 animals

GHS

Category 1

0 - 5

Category 2

5 - 50

Category 3

50 - 300

Category 4

300 - 2000

Category 5

2000 - 5000

3*

(at 50)

other

3*

(at 300)

other

3*

(at 2000)

2*

(at 2000)

other

LD50 cut-off

mg/kg b. w.

EU/chemicals Liquid pesticides

EU solid pesticides

UN liquids

UN solids

Switzerland

US EPA crk

Japan PDSCA

Canada/WHMIS/US OSHA

US EPA pesticides

US CPSC

Canada pesticides

• per step 3 animals of a single sex (normally female) are used

• 0, 1, 2, 3: Number of moribund or dead animals at each step

• ∞: unclassified

• *: at first step

• GHS: Globally Harmonised Classification System (mg/kg b. w.)

B.2.   ACUTE INHALATION TOXICITY

The full description of this test method has been deleted. The equivalent international test method appears in Part 0, Table 2.

B.3.   ACUTE TOXICITY (DERMAL)

The full description of this test method has been deleted. The equivalent international test method appears in Table 2 of Part 0.

B.4.   ACUTE DERMAL IRRITATION/CORROSION

INTRODUCTION

• 1.This test method is equivalent to OECD test guideline (TG) 404 (2015). OECD guidelines for testing of Chemicals are periodically reviewed to ensure that they reflect the best available science. In the review of OECD TG 404, special attention was given to possible improvements in relation to animal welfare concerns and to the evaluation of all existing information on the test chemical in order to avoid unnecessary testing in laboratory animals. The updated version of OECD TG 404 (originally adopted in 1981, revised in 1992, 2002 and 2015) includes reference to the Guidance Document on Integrated Approaches to Testing and Assessment (IATA) for Skin Irritation/Corrosion (1), proposing a modular approach for skin irritation and skin corrosion testing. The IATA describes several modules which group information sources and analysis tools, and (i) provides guidance on how to integrate and use existing testing and non-testing data for the assessment of the skin irritation and skin corrosion potentials of chemicals and (ii) proposes an approach when further testing is needed (1). In addition, where needed, the successive, instead of simultaneous, application of the three test patches to the animal in the initial in vivo test is recommended in that Guideline.

• 2.Definitions of dermal irritation and corrosion are set out in the Appendix to this test method.

INITIAL CONSIDERATIONS

• 3.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 test chemical have been evaluated in a weight-of-the-evidence (WoE) analysis as presented in the Guidance Document on Integrated Approaches to Testing and Assessment for Skin Corrosion and Irritation, i.e. over the three Parts of this guidance and their corresponding modules (1). Briefly, under Part 1 existing data is addressed over seven modules covering human data, in vivo data, in vitro data, physico-chemical properties data (e.g. pH, in particular strong acidity or alkalinity) and non-testing methods. Under Part 2, WoE analysis is performed. If this WoE is still inconclusive, Part 3 should be conducted with additional testing, starting with in vitro methods, and in vivo testing is used as last resort. This analysis should therefore decrease the need for in vivo testing for dermal corrosivity/irritation of test chemicals for which sufficient evidence already exists from other studies as to those two endpoints.

PRINCIPLE OF THE IN VIVO TEST

• 4.The test chemical 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.

• 5.Animals showing continuing signs of severe distress and/or pain at any stage of the test should be humanely killed, and the test chemical assessed accordingly. Criteria for making the decision to humanely kill moribund and severely suffering animals are the subject of a separate Guidance Document (2).

PREPARATIONS FOR THE IN VIVO TEST

Selection of animal species

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

Preparation of the animals

• 7.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.

• 8.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.

Housing and feeding conditions

• 9.Animals should be individually housed. The temperature of the experimental animal room should be 20 °C (± 3 °C) 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

TEST PROCEDURE

Application of the test chemical

• 10.The test chemical 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 chemical 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 chemical 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 test chemical on the skin. Access by the animal to the patch and ingestion or inhalation of the test chemical should be prevented.

• 11.Liquid test chemicals are generally used undiluted. When testing solids (which may be pulverised, if considered necessary), the test chemical 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 chemical should be minimal, if any.

• 12.At the end of the exposure period, which is normally 4 hours, residual test chemical should be removed, where practicable, using water or an appropriate solvent without altering the existing response or the integrity of the epidermis.

Dose level

• 13.A dose of 0,5 ml of liquid or 0,5 g of solid or paste is applied to the test site.

Initial test (In vivo dermal irritation/corrosion test using one animal)

• 14.When a test chemical has been judged to be corrosive, irritant or non-classified on the basis of a weight of evidence analyses or of previous in vitro testing, further in vivo testing is normally not necessary. However, in the cases where additional data are felt warranted, the in vivo test is performed initially using one animal and applying the following approach. Up to three test 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 at a different site 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.

• 15.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.

• 16.In those cases in which the test chemical is not expected to produce corrosion but may be irritating, a single patch should be applied to one animal for four hours.

Confirmatory test (In vivo dermal irritation test with additional animals)

• 17.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.

Observation period

• 18.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.

Clinical observations and grading of skin reactions

• 19.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.

• 20.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 (3).

DATA AND REPORTING

• 21.Study results should be summarised in tabular form in the final test report and should cover all items listed in paragraph 24.

Evaluation of results

• 22.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.

• 23.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 chemical should be considered an irritant.

Test report

• 24.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 chemical:

• — Mono-constituent substance: chemical identification, such as IUPAC or CAS name, CAS number, SMILES or InChI code, structural formula, purity, chemical identity of impurities as appropriate and practically feasible, etc;

• — Multi-constituent substance, mixture and substances of unknown or variable composition, complex reaction products or biological materials (UVCB): characterised as far as possible by chemical identity (see above), quantitative occurrence and relevant physico-chemical properties of the constituents;

• — Physical appearance, water solubility, and additional relevant physico-chemical properties;

• — Source, lot number if available;

• — Treatment of the test chemical/control substance prior to testing, if applicable (e.g. warming, grinding);

• — Stability of the test chemical, limit date for use, or date for re-analysis if known;

• — Storage conditions.

Vehicle:

• — Identification, concentration (where appropriate), volume used;

• — Justification for choice of vehicle.

Test animal(s):

• — Species/strain used, rationale for using animal(s) other than albino rabbit;

• — Number of animal(s) of each sex;

• — Individual animal weight(s) at start and conclusion of test;

• — Age at start of study;

• — Source of animal(s), housing conditions, diet, etc.

Test conditions:

• — Technique of patch site preparation;

• — Details of patch materials used and patching technique;

• — Details of test chemical 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

Conclusions

LITERATURE

• (1) OECD (2014). Guidance document on Integrated Approaches to Testing and Assessment for Skin Irritation/Corrosion. Environmental Health and Safety Publications, Series on Testing and Assessment, (No 203), Organisation for Economic Cooperation and Development, Paris.

• (2) OECD (1998) Harmonized Integrated Hazard Classification System for Human Health and Environmental Effects of Chemical Substances, as endorsed by the 28^th Joint Meeting of the Chemicals Committee and the Working Party on Chemicals, November 1998.

• (3) 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 Publications, Series on Testing and Assessment (No 19), Organistion for Economic Cooperation and Development, Paris. Table Grading of Skin Reactions Erythema and Eschar Formation 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 Oedema Formation 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.

Appendix

DEFINITIONS

Chemical is a substance or a mixture.

Dermal irritation is the production of reversible damage of the skin following the application of a test chemical for up to 4 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 chemical for up to four hours. Corrosive reactions are typified by ulcers, bleeding, bloody scabs, and, by the end of observation at 14 days, by discolouration due to blanching of the skin, complete areas of alopecia, and scars. Histopathology should be considered to evaluate questionable lesions.

Test chemical is any substance or mixture tested using this test method.

B.5.   ACUTE EYE IRRITATION/CORROSION

The full description of this test method has been deleted. The equivalent international test method appears in Table 2 of Part 0.

B.6.   SKIN SENSITISATION

The full description of this test method has been deleted. The equivalent international test method appears in Table 2 of Part 0.

B.7.   REPEATED DOSE 28-DAY ORAL TOXICITY STUDY IN RODENTS

INTRODUCTION

• 1.This Test Method is equivalent to OECD Test Guideline 407 (2008). The original Test Guideline 407 was adopted in 1981. In 1995 a revised version was adopted, to obtain additional information from the animal used in the study, in particular on neurotoxicity and immunotoxicity.

• 2.In 1998, the OECD initiated a high-priority activity, to revise existing Test Guidelines and to develop new Test Guidelines for the screening and testing of potential endocrine disruptors (8). One element of the activity was to update the existing OECD guideline for ‘repeated dose 28-day oral toxicity study in rodents’ (TG 407) by parameters suitable to detect endocrine activity of test chemicals. This procedure underwent an extensive international program to test for the relevance and practicability of the additional parameters, the performance of these parameters for chemicals with (anti)oestrogenic, (anti)androgenic, and (anti)thyroid activity, the intra- and inter-laboratory reproducibility, and the interference of the new parameters with those required by the prior TG 407. The large amount of data thereby obtained has been compiled and evaluated in detail in a comprehensive OECD report (9). This updated Test Method B.7 (as equivalent to TG 407) is the outcome of the experience and results gained during the international test program. This Test Method allows certain endocrine mediated effects to be put into context with other toxicological effects.

INITIAL CONSIDERATIONS AND LIMITATIONS

• 3.In the assessment and evaluation of the toxic characteristics of a chemical, the determination of oral toxicity using repeated doses may be carried out after initial information on toxicity has been obtained by acute toxicity testing. This Test Method is intended to investigate effects on a very broad variety of potential targets of toxicity. It provides information on the possible health hazards likely to arise from repeated exposure over a relatively limited period of time, including effects on the nervous, immune and endocrine systems. Regarding these particular endpoints, it should identify chemicals with neurotoxic potential, which may warrant further in-depth investigation of this aspect, and chemicals that interfere with thyroid physiology. It may also provide data on chemicals that affect the male and/or female reproductive organs in young adult animals and may give an indication of immunological effects.

• 4.The results from this Test Method B.7 should be used for hazard identification and risk assessment. The results obtained by the endocrine related parameters should be seen in the context of the ‘OECD Conceptual Framework for Testing and Assessment of Endocrine Disrupting Chemicals’ (11). The method comprises the basic repeated dose toxicity study that may be used for chemicals on which a 90-day study is not warranted (e.g. when the production volume does not exceed certain limits) or as a preliminary to a long-term study. The duration of exposure should be 28 days.