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)

• 22.The pre-screen test is conducted under conditions identical to the main LLNA: DA study, except there is no assessment of lymph node proliferation and fewer animals per dose group can be used. One or two animals per dose group are suggested. All mice will be observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Body weights are recorded pre-test and prior to termination (Day 8). Both ears of each mouse are observed for erythema and scored using Table 1 (25). Ear thickness measurements are taken using a thickness gauge (e.g. digital micrometer or Peacock Dial thickness gauge) on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose), Day 7 (24 hours prior to termination) and Day 8. Additionally on Day 8, ear thickness could be determined by ear punch weight determinations, which should be performed after the animals are humanely killed. Excessive local irritation is indicated by an erythema score ≥ 3 and/or ear thickness of ≥ 25 % on any day of measurement (26) (27). The highest dose selected for the main LLNA: DA study will be the next lower dose in the pre-screen concentration series (see paragraph 18) that does not induce systemic toxicity and/or excessive local skin irritation

Table 1

Erythema Scores

Observation	Score
No erythema	0
Very slight erythema (barely perceptible)	1
Well-defined erythema	2
Moderate to severe erythema	3
Severe erythema (beet redness) to eschar formation preventing grading of erythema	4

• 23.In addition to a 25 % increase in ear thickness (26) (27), a statistically significant increase in ear thickness in the treated mice compared to control mice has also been used to identify irritants in the LLNA (28) (29) (30) (31) (32) (33) (34). However, while statistically significant increases can occur when ear thickness is less than 25 % they have not been associated specifically with excessive irritation (30) (31) (32) (33) (34).

• 24.The following clinical observations may indicate systemic toxicity (35) when used as part of an integrated assessment and therefore may indicate the maximum dose level to use in the main LLNA: DA: changes in nervous system function (e.g. pilo-erection, ataxia, tremors, and convulsions); changes in behaviour (e.g. aggressiveness, change in grooming activity, marked change in activity level); changes in respiratory patterns (i.e. changes in frequency and intensity of breathing such as dyspnea, gasping, and rales), and changes in food and water consumption. In addition, signs of lethargy and/or unresponsiveness and any clinical signs of more than slight or momentary pain and distress, or a > 5 % reduction in body weight from Day 1 to Day 8 and mortality, should be considered in the evaluation. Moribund animals or animals showing signs of severe pain and distress should be humanely killed (36).

Main study experimental schedule

• 25.The experimental schedule of the assay is as follows:

• — Day 1 : Individually identify and record the weight of each animal and any clinical observation. Apply 1 % sodium lauryl sulfate (SLS) aqueous solution to the dorsum of each ear by using a brush dipped in the SLS solution to cover the entire dorsum of each ear with four to five strokes. One hour after the SLS treatment, apply 25 μL of the appropriate dilution of the test substance, the vehicle alone, or the PC (concurrent or recent, based on laboratory policy in considering paragraphs 11-15), to the dorsum of each ear.

• — Days 2, 3 and 7 : Repeat the 1 % SLS aqueous solution pre-treatment and test substance application procedure carried out on Day 1.

• — Days 4, 5, and 6 : No treatment.

• — Day 8 : Record the weight of each animal and any clinical observation. Approximately 24 to 30 hours after the start of application on Day 7, humanely kill the animals. Excise the draining auricular lymph nodes from each mouse ear and process separately in phosphate buffered saline (PBS) for each animal. Details and diagrams of the lymph node identification and dissection can be found in reference (22). To further monitor the local skin response in the main study, additional parameters such as scoring of ear erythema or ear thickness measurements (obtained either by using a thickness gauge, or ear punch weight determinations at necropsy) may be included in the study protocol.

Preparation of cell suspensions

• 26.From each mouse, a single-cell suspension of lymph node cells (LNC) excised bilaterally is prepared by sandwiching the lymph nodes between two glass slides and applying light pressure to crush the nodes. After confirming that the tissue has spread out thinly pull the two slides apart. Suspend the tissue on both slides in PBS by holding each slide at an angle over the Petri dish and rinsing with PBS while concurrently scraping the tissue off of the slide with a cell scraper. Further, the lymph nodes in NC animals are small, so careful operation is important to avoid any artificial effects on SI values. A total volume of 1 mL PBS should be used for rinsing both slides. The LNC suspension in the Petri dish should be homogenised lightly with the cell scraper. A 20 μL aliquot of the LNC suspension is then collected with a micropipette, taking care not to take up the membrane that is visible to the eye, and subsequently mixed with 1,98 mL of PBS to yield a 2 mL sample. A second 2 mL sample is then prepared using the same procedure so that two samples are prepared for each animal.

Determination of cellular proliferation (measurement of ATP content of lymphocytes)

• 27.Increases in ATP content in the lymph nodes are measured by the luciferin/luciferase method using an ATP measurement kit, which measures bioluminescence in Relative Luminescence Units (RLU). The assay time from time of animal sacrifice to measurement of ATP content for each individual animal should be kept uniform, within approximately 30 minutes, because the ATP content is considered to gradually decrease with time after animal sacrifice (12) Thus, the series of procedures from excision of auricular lymph nodes to ATP measurement should be completed within 20 minutes by the pre-determined time schedule that is the same for each animal. ATP luminescence should be measured in each 2 mL sample so that a total of two ATP measurements are collected for each animal. The mean ATP luminescence is then determined and used in subsequent calculations (see paragraph 30).

OBSERVATIONS

Clinical observations

• 28.Each mouse should be carefully observed at least once daily for any clinical signs, either of local irritation at the application site or of systemic toxicity. All observations are systematically recorded with records being maintained for each mouse. Monitoring plans should include criteria to promptly identify those mice exhibiting systemic toxicity, excessive local skin irritation, or corrosion of skin for euthanasia (36).

Body weights

• 29.As stated in paragraph 25, individual animal body weights should be measured at the start of the test and at the scheduled humane kill.

CALCULATION OF RESULTS

• 30.Results for each treatment group are expressed as the mean SI. The SI is derived by dividing the mean RLU/mouse within each test substance group and the PC group by the mean RLU/mouse for the solvent/VC group. The average SI for the VCs is then one.

• 31.The decision process regards a result as positive when SI ≥ 1,8 (10) However, the strength of the dose-response relationship, the statistical significance and the consistency of the solvent/vehicle and PC responses may also be used when determining whether a borderline result (i.e. SI value between 1,8 and 2,5) is declared positive (2) (3) (37).

• 32.For a borderline positive response between an SI of 1,8 and 2,5, users may want to consider additional information such as dose-response relationship, evidence of systemic toxicity or excessive irritation, and where appropriate, statistical significance together with SI values to confirm that such results are positives (10). Consideration should also be given to various properties of the test substance, including whether it has a structural relationship to known skin sensitisers, whether it causes excessive skin irritation in the mouse, and the nature of the dose-response relationship observed. These and other considerations are discussed in detail elsewhere (4)

• 33.Collecting data at the level of the individual mouse will enable a statistical analysis for presence and degree of dose-response relationship in the data. Any statistical assessment could include an evaluation of the dose-response relationship as well as suitably adjusted comparisons of test groups (e.g. pair-wise dosed group versus concurrent solvent/vehicle control comparisons). Statistical analyses may include, e.g. linear regression or William’s test to assess dose-response trends, and Dunnett’s test for pair-wise comparisons. In choosing an appropriate method of statistical analysis, the investigator should maintain an awareness of possible inequalities of variances and other related problems that may necessitate a data transformation or a non-parametric statistical analysis. In any case, the investigator may need to carry out SI calculations and statistical analyses with and without certain data points (sometimes called ‘outliers’).

DATA AND REPORTING

Data

• 34.Data should be summarised in tabular form showing the individual animal RLU values, the group mean RLU/animal, its associated error term (e.g. SD, SEM), and the mean SI for each dose group compared against the concurrent solvent/vehicle control group.

Test report

• 35.The test report should contain the following information:

Test and control chemicals:

• — identification data (e.g. CAS number and EC number, if available; source; purity; known impurities; lot number);

• — physical nature and physicochemical properties (e.g. volatility, stability, solubility);

• — if mixture, composition and relative percentages of components;

Solvent/vehicle:

• — identification data (purity; concentration, where appropriate; volume used);

• — justification for choice of vehicle;

Test animals:

• — source of CBA mice;

• — microbiological status of the animals, when known;

• — number and age of animals;

• — source of animals, housing conditions, diet, etc.;

Test conditions:

• — the source, lot number and manufacturer’s quality assurance/quality control data for the ATP kit;

• — details of test substance preparation and application;

• — justification for dose selection (including results from pre-screen test, if conducted);

• — vehicle and test substance concentrations used, and total amount of test substance applied;

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

• — details of treatment and sampling schedules;

• — methods for measurement of toxicity;

• — criteria for considering studies as positive or negative;

• — details of any protocol deviations and an explanation on how the deviation affects the study design and results;

Reliability check:

• — a summary of results of latest reliability check, including information on test substance, concentration and vehicle used;

• — concurrent and/or historical PC and concurrent negative (solvent/vehicle) control data for testing laboratory;

• — if a concurrent PC was not included, the date and laboratory report for the most recent periodic PC and a report detailing the historical PC data for the laboratory justifying the basis for not conducting a concurrent PC;

Results:

• — individual weights of mice at start of dosing and at scheduled kill; as well as mean and associated error term (e.g. SD, SEM) for each treatment group;

• — time course of onset and signs of toxicity, including dermal irritation at site of administration, if any, for each animal;

• — time of animal termination and time of ATP measurement for each animal;

• — a table of individual mouse RLU values and SI values for each dose treatment group;

• — mean and associated error term (e.g. SD, SEM) for RLU/mouse for each treatment group and the results of outlier analysis for each treatment group;

• — calculated SI and an appropriate measure of variability that takes into account the inter-animal variability in both the test substance and control groups;

• — dose response relationship;

• — statistical analyses, where appropriate;

Discussion of results:

• — a brief commentary on the results, the dose-response analysis, and statistical analyses, where appropriate, with a conclusion as to whether the test substance should be considered a skin sensitiser.

LITERATURE

• (1) OECD (2010), Skin Sensitisation: Local Lymph Node Assay, Test Guideline No 429, Guidelines for the Testing of Chemicals, OECD, Paris. Available at: [http://www.oecd.org/env/testguidelines]

• (2) Chamberlain, M. and Basketter, D.A. (1996), The local lymph node assay: status of validation. Food Chem, Toxicol., 34, 999-1002.

• (3) Basketter, D.A., Gerberick, G.F., Kimber, I. and Loveless, S.E. (1996), The local lymph node assay: A viable alternative to currently accepted skin sensitisation tests. Food Chem, Toxicol., 34, 985-997.

• (4) Basketter, D.A., Gerberick, G.F. and Kimber, I. (1998), Strategies for identifying false positive responses in predictive sensitisation tests. Food Chem. Toxicol., 36, 327-333.

• (5) Van Och, F.M.M., Slob, W., De Jong, W.H., Vandebriel, R.J. and Van Loveren, H. (2000), A quantitative method for assessing the sensitising potency of low molecular weight chemicals using a local lymph node assay: employment of a regression method that includes determination of uncertainty margins. Toxicol., 146, 49-59.

• (6) ICCVAM (1999), The murine local lymph node Assay: A test method for assessing the allergic contact dermatitis potential of chemicals/compounds: The results of an independent peer review evaluation coordinated by the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the National Toxicology Program Center for the Evaluation of Alternative Toxicological Methods (NICETAM). NIH Publication No: 99-4494. Research Triangle Park, N.C. Available at: [http://iccvam.niehs.nih.gov/docs/immunotox_docs/llna/llnarep.pdf]

• (7) Dean, J.H., Twerdok, L.E., Tice, R.R., Sailstad, D.M., Hattan, D.G., Stokes, W.S. (2001), ICCVAM evaluation of the murine local lymph node assay: II. Conclusions and recommendations of an independent scientific peer review panel. Reg. Toxicol. Pharmacol. 34, 258-273.

• (8) Haneke, K.E., Tice, R.R., Carson, B.L., Margolin, B.H., Stokes, W.S. (2001), ICCVAM evaluation of the murine local lymph node assay: III. Data analyses completed by the national toxicology program interagency center for the evaluation of alternative toxicological methods. Reg. Toxicol. Pharmacol. 34, 274-286.

• (9) Sailstad, D.M., Hattan, D., Hill, R.N., Stokes, W.S. (2001), ICCVAM evaluation of the murine local lymph node assay: I. The ICCVAM review process. Reg. Toxicol. Pharmacol. 34, 249-257.

• (10) ICCVAM (2010), ICCVAM Test Method Evaluation Report. Nonradioactive local lymph node assay: modified by Daicel Chemical Industries, Ltd, based on ATP content test method protocol (LLNA: DA). NIH Publication No 10-7551A/B. Research Triangle Park, NC: National Institute of Environmental Health Sciences. Available at: [http://iccvam.niehs.nih.gov/methods/immunotox/llna-DA/TMER.htm]

• (11) ICCVAM (2009), Independent Scientific Peer Review Panel Report: Updated validation status of new versions and applications of the murine local lymph node assay: a test method for assessing the allergic contact dermatitis potential of chemicals and products. Research Triangle Park, NC: National Institute of Environmental Health Sciences. Available at: [http://iccvam.niehs.nih.gov/docs/immunotox_docs/LLNAPRPRept2009.pdf].

• (12) Idehara, K., Yamagishi, G., Yamashita, K. and Ito, M. (2008), Characterization and evaluation of a modified local lymph node assay using ATP content as a non-radio isotopic endpoint. J. Pharmacol. Toxicol. Meth., 58, 1-10.

• (13) Omori, T., Idehara, K., Kojima, H., Sozu, T., Arima, K., Goto, H., Hanada, T., Ikarashi, Y., Inoda, T., Kanazawa, Y., Kosaka, T., Maki, E., Morimoto, T., Shinoda, S., Shinoda, N., Takeyoshi, M., Tanaka, M., Uratani, M., Usami, M., Yamanaka, A., Yoneda, T., Yoshimura, I. and Yuasa, A. (2008), Interlaboratory validation of the modified murine local lymph node assay based on adenosine triphosphate measurement. J. Pharmacol. Toxicol. Meth., 58, 11-26.

• (14) OECD (1992), Skin Sensitisation, Test Guideline No 406, Guidelines for Testing of Chemicals, OECD, Paris. Available at: [http://www.oecd.org/env/testguidelines]

• (15) Kreiling, R., Hollnagel, H.M., Hareng, L., Eigler, L., Lee, M.S., Griem, P., Dreessen, B., Kleber, M., Albrecht, A., Garcia, C. and Wendel, A. (2008), Comparison of the skin sensitising potential of unsaturated compounds as assessed by the murine local lymph node assay (LLNA) and the guinea pig maximization test (GPMT). Food Chem. Toxicol., 46, 1896-1904.

• (16) Basketter, D., Ball, N., Cagen, S., Carrillo, J.C., Certa, H., Eigler, D., Garcia, C., Esch, H., Graham, C., Haux, C., Kreiling, R. and Mehling, A. (2009), Application of a weight of evidence approach to assessing discordant sensitisation datasets: implications for REACH. Reg. Toxicol. Pharmacol., 55, 90-96.

• (17) Crouch, S.P., Kozlowski, R., Slater, K.J. and Fletcher J. (1993), The use of ATP bioluminescence as a measure of cell proliferation and cytotoxicity. J. Immunol. Meth., 160, 81-88.

• (18) Ishizaka, A., Tono-oka, T. and Matsumoto, S. (1984), Evaluation of the proliferative response of lymphocytes by measurement of intracellular ATP. J. Immunol. Meth., 72, 127-132.

• (19) Dexter, S.J., Cámara, M., Davies, M. and Shakesheff, K.M. (2003), Development of a bioluminescent ATP assay to quantify mammalian and bacterial cell number from a mixed population. Biomat., 24, 27-34.

• (20) Lundin A. (2000), Use of firefly luciferase in ATP-related assays of biomass, enzymes, and metabolites. Meth. Enzymol., 305, 346-370.

• (21) ILAR (1996), Institute of Laboratory Animal Research (ILAR) Guide for the Care and Use of Laboratory Animals. 7th ed. Washington, DC: National Academies Press.

• (22) ICCVAM (2009), Recommended Performance Standards: Murine Local Lymph Node Assay, NIH Publication Number 09-7357, Research Triangle Park, NC: National Institute of Environmental Health Science. Available at: [http://iccvam.niehs.nih.gov/docs/immunotox_docs/llna-ps/LLNAPerfStds.pdf]

• (23) McGarry, H.F. (2007), The murine local lymph node assay: regulatory and potency considerations under REACH. Toxicol., 238, 71-89.

• (24) Kimber, I., Dearman, R.J., Scholes E.W. and Basketter, D.A. (1994), The local lymph node assay: developments and applications. Toxicol., 93, 13-31.

• (25) OECD (2002), Acute Dermal Irritation/Corrosion, Test Guideline No 404, Guidelines for Testing of Chemicals, OECD, Paris. Available at: [http://www.oecd.org/env/testguidelines]

• (26) Reeder, M.K., Broomhead, Y.L., DiDonato, L. and DeGeorge, G.L. (2007), Use of an enhanced local lymph node assay to correctly classify irritants and false positive substances. Toxicologist, 96, 235.

• (27) ICCVAM (2009), Nonradioactive Murine Local Lymph Node Assay: Flow Cytometry Test Method Protocol (LLNA: BrdU-FC) Revised Draft Background Review Document. Research Triangle Park, NC: National Institute of Environmental Health Sciences. Available at: [http://iccvam.niehs.nih.gov/methods/immunotox/fcLLNA/BRDcomplete.pdf].

• (28) Hayes, B.B., Gerber, P.C., Griffey, S.S. and Meade, B.J. (1998), Contact hypersensitivity to dicyclohexylcarbodiimide and diisopropylcarbodiimide in female B6C3F1 mice. Drug. Chem. Toxicol., 21, 195-206.

• (29) Homey, B., von Schilling, C., Blumel, J., Schuppe, H.C., Ruzicka, T., Ahr, H.J., Lehmann, P. and Vohr, V.W. (1998), An integrated model for the differentiation of chemical-induced allergic and irritant skin reactions. Toxicol. Appl. Pharmacol., 153, 83-94.

• (30) Woolhiser, M.R., Hayes, B.B. and Meade, B.J. (1998), A combined murine local lymph node and irritancy assay to predict sensitisation and irritancy potential of chemicals. Toxicol. Meth., 8, 245-256.

• (31) Hayes, B.B. and Meade, B.J. (1999), Contact sensitivity to selected acrylate compounds in B6C3F1 mice: relative potency, cross reactivity, and comparison of test methods. Drug Chem. Toxicol., 22, 491-506.

• (32) Ehling, G., Hecht, M., Heusener, A., Huesler, J., Gamer, A.O., van Loveren, H., Maurer, T., Riecke, K., Ullmann, L., Ulrich, P., Vandebriel, R. and Vohr, H.W. (2005), A European inter-laboratory validation of alternative endpoints of the murine local lymph node assay: first round. Toxicol., 212, 60-68.

• (33) Vohr, H.W. and Ahr, H.J. (2005), The local lymph node assay being too sensitive? Arch. Toxicol., 79, 721-728.

• (34) Patterson, R.M., Noga, E. and Germolec, D. (2007), Lack of evidence for contact sensitisation by Pfiesteria extract. Environ. Health Perspect., 115, 1023-1028.

• (35) ICCVAM (2009), Report on the ICCVAM-NICEATM/ECVAM/JaCVAM Scientific Workshop on Acute Chemical Safety Testing: Advancing In Vitro Approaches and Humane Endpoints for Systemic Toxicity Evaluations. Research Triangle Park, NC: National Institute of Environmental Health Sciences. Available at: [http://iccvam.niehs.nih.gov/methods/acutetox/Tox_workshop.htm]

• (36) 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, ENV/JM/MONO(2000)7, OECD, Paris. Available at: [http://www.oecd.org/env/testguidelines]

• (37) Kimber, I., Hilton, J., Dearman, R.J., Gerberick, G.F., Ryan, C.A., Basketter, D.A., Lea, L., House, R.V., Ladies, G.S., Loveless, S.E. and Hastings, K.L. (1998), Assessment of the skin sensitisation potential of topical medicaments using the local lymph node assay: An interlaboratory exercise. J. Toxicol. Environ. Health, 53 563-79.

• (38) OECD (2005), Guidance Document on the Validation and International Acceptance of New or Updated Test Methods for Hazard Assessment, Environment, Health and Safety Monograph Series on Testing and Assessment No 34, ENV/JM/MONO (2005)14, OECD, Paris. Available at: [http://www.oecd.org/env/testguidelines]

Appendix 1

DEFINITIONS

Accuracy : The closeness of agreement between test method results and accepted reference values. It is a measure of test method performance and one aspect of relevance. The term is often used interchangeably with ‘concordance’ to mean the proportion of correct outcomes of a test method (38).

Benchmark substance : A sensitising or non-sensitising substance used as a standard for comparison to a test substance. A benchmark substance should have the following properties; (i) a consistent and reliable source(s); (ii) structural and functional similarity to the class of substances being tested; (iii) known physicochemical characteristics; (iv) supporting data on known effects, and (v) known potency in the range of the desired response.

False negative : A substance incorrectly identified as negative or non-active by a test method, when in fact it is positive or active.

False positive : A substance incorrectly identified as positive or active by a test, when in fact it is negative or non-active.

Hazard : The potential for an adverse health or ecological effect. The adverse effect is manifested only if there is an exposure of sufficient level.

Inter-laboratory reproducibility : A measure of the extent to which different qualified laboratories, using the same protocol and testing the same test substances, can produce qualitatively and quantitatively similar results. Inter-laboratory reproducibility is determined during the pre-validation and validation processes, and indicates the extent to which a test can be successfully transferred between laboratories, also referred to as between-laboratory reproducibility (38).

Intra-laboratory reproducibility : A determination of the extent that qualified people within the same laboratory can successfully replicate results using a specific protocol at different times. Also referred to as within-laboratory reproducibility (38).

Outlier : An outlier is an observation that is markedly different from other values in a random sample from a population.

Quality assurance : A management process by which adherence to laboratory testing standards, requirements, and record keeping procedures, and the accuracy of data transfer, are assessed by individuals who are independent from those performing the testing.

Reliability : Measures of the extent that a test method can be performed reproducibly within and between laboratories over time, when performed using the same protocol. It is assessed by calculating intra- and inter-laboratory reproducibility (38).

Skin sensitisation : An immunological process that results when a susceptible individual is exposed topically to an inducing chemical allergen, which provokes a cutaneous immune response that can lead to the development of contact sensitisation.

Stimulation Index (SI) : A value calculated to assess the skin sensitisation potential of a test substance that is the ratio of the proliferation in treated groups to that in the concurrent vehicle control group.

Test substance (also referred to as test chemical) : Any substance or mixture tested using this TM.

B.51.   SKIN SENSITISATION: LOCAL LYMPH NODE ASSAY: BrdU-ELISA

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

B.52.   ACUTE INHALATION TOXICITY — ACUTE TOXIC CLASS METHOD

INTRODUCTION

• 1.This Test Method is equivalent to OECD Test Guideline (TG) 436 (2009). The first acute inhalation TG 403 was adopted in 1981, and has since been revised (see chapter B.2 of this Annex (1)). Development of an Inhalation Acute Toxic Class (ATC) method (2) (3) (4) was considered appropriate following the adoption of the revised oral ATC method (chapter B.1 tris of this Annex) (5). A retrospective performance assessment of the ATC test method for acute inhalation toxicity showed that the method is suitable for being used for Classification and Labelling purposes (6). The inhalation ATC Test Method will allow the use of serial steps of fixed target concentrations to provide a ranking of test chemical toxicity. Lethality is used as key endpoint, however, animals in severe pain or distress, suffering or impending death should be humanely killed to minimise suffering. Guidance on humane endpoints is available in the OECD Guidance Document No 19 (7).

• 2.Guidance on the conduct and interpretation of this Test Method can be found in the Guidance Document No 39 on Acute Inhalation Toxicity Testing (GD 39) (8).

• 3.Definitions used in the context of this Test Method are provided in Appendix 1 and in GD 39 (8).

• 4.The Test Method provides information on the hazardous properties and allows the test chemical to be ranked and classified according to the Regulation (EC) No 1272/2008 for the classification of chemicals that cause acute toxicity (9). In case point estimates of LC50-values or concentration-response analyses are required, chapter B.2 of this Annex (1) is the appropriate Test Method to use. Further guidance on Test Method selection can be found in GD 39 (8). This Test Method is not specially intended for the testing of specialized materials, such as poorly soluble isometric or fibrous materials or manufactured nanomaterials.

INITIAL CONSIDERATIONS

• 5.Before considering testing in accordance with this Test Method, all available information on the test chemical, including existing studies whose data would support not doing additional testing should be considered by the testing laboratory in order to minimize animal usage. Information that may assist in the selection of the most appropriate species, strain, sex, mode of exposure and appropriate test concentrations include the identity, chemical structure, and physico-chemical properties of the test chemical; results of any in vitro or in vivo toxicity tests; anticipated use(s) and potential for human exposure; available (Q)SAR data and toxicological data on structurally related chemicals. Concentrations that are expected to cause severe pain and distress, due to corrosive ([^E0030]) or severely irritant actions, should not be tested with this Test Method [see GD 39 (8)].

PRINCIPLE OF THE TEST

• 6.It is the principle of the test that based on a stepwise procedure, sufficient information is obtained on the acute inhalation toxicity of the test chemical during an exposure period of 4 hours to enable its classification. Other durations of exposure may apply to serve specific regulatory purposes. At any of the defined concentration steps, 3 animals of each sex are tested. Depending on the mortality and/or the moribund status of the animals, 2 steps may be sufficient to allow judgement on the acute toxicity of the test chemical. If evidence is provided that one sex is more susceptible than the other, then the test may be continued with the more susceptible sex only. The outcome of the previous step will determine the following step such that:

• a) No further testing is needed,

• b) Testing of three animals per sex, or

• c) Testing with 6 animals of the more susceptible sex only i.e. the lower boundary estimates of the toxic class should be based on 6 animals per test concentration group, regardless of sex.

• 7.Moribund animals or animals obviously in pain or showing signs of severe and enduring distress should 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 Guidance Document No 19 on Humane Endpoints (7).

DESCRIPTION OF THE METHOD

Selection of animal species

• 8.Healthy young adult animals of commonly used laboratory strains should be used. The preferred species is the rat and justifications should be provided if other species are used.

Preparation of animals

• 9.Females should be nulliparous and non-pregnant. On the exposure day, animals should be young adults 8 to 12 weeks of age, and body weights should be within ± 20 % of the mean weight for each sex of any previously exposed animals at the same age. The animals are randomly selected, marked for individual identification. The animals are kept in their cages for at least 5 days prior to the start of the test to allow for acclimatisation to laboratory conditions. Animals should also be acclimatised to the test apparatus for a short period prior to testing, as this will lessen the stress caused by introduction to the new environment.

Animal husbandry

• 10.The temperature of the experimental animal maintenance room should be 22 ± 3 °C. The relative humidity should ideally be maintained in the range of 30 to 70 %, though this may not be possible when using water as a vehicle. Before and after exposures, animals generally should be caged in groups by sex and concentration, but the number of animals per cage should not interfere with clear observation of each animal and should minimise losses due to cannibalism and fighting. When animals are to be exposed nose-only, it may be necessary for them to be acclimated to the restraining tubes. The restraining tubes should not impose undue physical, thermal, or immobilisation stress on the animals. Restraint may affect physiological endpoints such as body temperature (hyperthermia) and/or respiratory minute volume. If generic data are available to show that no such changes occur to any appreciable extent, then pre-adaptation to the restraining tubes is not necessary. Animals exposed whole-body to an aerosol should be housed individually during exposure to prevent them from filtering the test aerosol through the fur of their cage mates. Conventional and certified laboratory diets may be used, except during exposure, accompanied with an unlimited supply of municipal drinking water. Lighting should be artificial, the sequence being 12 hours light/12 hours dark.

Inhalation chambers

• 11.The nature of the test chemical and the objective of the test should be considered when selecting an inhalation chamber. The preferred mode of exposure is nose-only (which term includes head-only, nose-only or snout-only). Nose-only exposure is generally preferred for studies of liquid or solid aerosols and for vapours that may condense to form aerosols. Special objectives of the study may be better achieved by using a whole-body mode of exposure, but this should be justified in the study report. To ensure atmosphere stability when using a whole-body chamber, the total volume of the test animals should not exceed 5 % of the chamber volume. Principles of the nose-only and whole body exposure techniques and their particular advantages and disadvantages are described in GD 39 (8).

EXPOSURE CONDITIONS

Administrations of concentrations

• 12.A fixed duration of exposure for four hours, excluding equilibration time, is recommended. Other durations may be needed to meet specific requirements, however, justification should be provided in the study report [see GD 39 (8)]. Animals exposed in whole-body chambers should be housed individually to prevent ingestion of test chemical due to grooming of cage mates. Feed should be withheld during the exposure period. Water may be provided throughout a whole-body exposure.

• 13.Animals are exposed to the test chemical as a gas, vapour, aerosol, or a mixture thereof. The physical state to be tested depends on the physico-chemical properties of the test chemical, the selected concentration, and/or the physical form most likely present during the handling and use of the test chemical. Hygroscopic and chemically reactive test chemicals should be tested under dry air conditions. Care should be taken to avoid generating explosive concentrations.

Particle-size distribution

• 14.Particle sizing should be performed for all aerosols and for vapours that may condense to form aerosols. To allow for exposure of all relevant regions of the respiratory tract, aerosols with mass median aerodynamic diameters (MMAD) ranging from 1 to 4 μm with a geometric standard deviation (σg) in the range of 1,5 to 3,0 are recommended (8) (13) (14). Although a reasonable effort should be made to meet this standard, expert judgment should be provided if it cannot be achieved. For example, metal fumes may be smaller than this standard, and charged particles, fibres, and hygroscopic materials (which increase in size in the moist environment of the respiratory tract) may exceed this standard.

Test chemical preparation in a vehicle

• 15.A vehicle may be used to generate an appropriate concentration and particle size of the test chemical in the atmosphere. As a rule, water should be given preference. Particulate material may be subjected to mechanical processes to achieve the required particle size distribution, however, care should be taken not to decompose or alter the test chemical. In cases where mechanical processes are believed to have altered test chemical composition (e.g. extreme temperature from excessive milling due to friction), the composition of the test chemical should be verified analytically. Adequate care should be taken to not contaminate the test chemical. It is not necessary to test non-friable granular materials which are purposefully formulated to be un-inhalable. An attrition test should be used to demonstrate that respirable particles are not produced when the granular material is handled. If an attrition test produces respirable particles, an inhalation toxicity test should be performed.

Control animals

• 16.A concurrent negative (air) control group is not necessary. When a vehicle other than water is used to assist in generating the test atmosphere, a vehicle control group should only be used when historical inhalation toxicity data are not available. If a toxicity study of a test chemical formulated in a vehicle reveals no toxicity, it follows that the vehicle is non-toxic at the concentration tested; thus, there is no need for a vehicle control.

MONITORING OF EXPOSURE CONDITIONS

Chamber airflow

• 17.The flow of air through the chamber should be carefully controlled, continuously monitored, and recorded at least hourly during each exposure. The monitoring of test atmosphere concentration (or stability) is an integral measurement of all dynamic parameters and provides an indirect means to control all relevant dynamic atmosphere generation parameters. Special consideration should be given to avoiding re-breathing in nose-only chambers in cases where airflow through the exposure system are inadequate to provide dynamic flow of test chemical atmosphere. There are prescribed methodologies that can be used to demonstrate that re-breathing does not occur under the selected operation conditions (8) (15). Oxygen concentration should be at least 19 % and carbon dioxide concentration should not exceed 1 %. If there is reason to believe that these standards cannot be met, oxygen and carbon dioxide concentrations should be measured.

Chamber temperature and relative humidity

• 18.Chamber temperature should be maintained at 22 ± 3 °C. Relative humidity in the animals’ breathing zone, for both nose-only and whole-body exposures, should be monitored and recorded at least three times for durations up to 4 hrs, and hourly for shorter durations. The relative humidity should ideally be maintained in the range of 30 to 70 %, but this may either be unattainable (e.g. when testing water based mixtures) or not measurable due to test chemical interference with the test method.

Test chemical: nominal concentration

• 19.Whenever feasible, the nominal exposure chamber concentration should be calculated and recorded. The nominal concentration is the mass of generated test chemical divided by the total volume of air passed through the chamber system. The nominal concentration is not used to characterise the animals’ exposure, but a comparison of the nominal and the actual concentration gives an indication of the generation efficiency of the test system, and thus may be used to discover generation problems.

Test chemical: actual concentration

• 20.The actual concentration is the test chemical concentration at the animals’ breathing zone in an inhalation chamber. Actual concentrations can be obtained either by specific methods (e.g. direct sampling, adsorptive or chemical reactive methods, and subsequent analytical characterisation) or by non-specific methods such as gravimetric filter analysis. The use of gravimetric analysis is acceptable only for single component powder aerosols or aerosols of low volatility liquids and should be supported by appropriate pre-study test chemical-specific characterisations. Multi-component powder aerosol concentration may also be determined by gravimetric analysis. However, this requires analytical data which demonstrate that the composition of airborne material is similar to the starting material. If this information is not available, a reanalysis of the test chemical (ideally in its airborne state) at regular intervals during the course of the study may be necessary. For aerosolised agents that may evaporate or sublimate, it should be shown that all phases were collected by the method chosen. The target, nominal, and actual concentrations should be provided in the study report, but only actual concentrations are used in statistical analyses to calculate lethal concentration values.

• 21.One lot of the test chemical should be used, if possible, and the test sample should be stored under conditions that maintain its purity, homogeneity, and stability. Prior to the start of the study, there should be a characterisation of the test chemical, including its purity and, if technically feasible, the identity, and quantities of identified contaminants and impurities. This can be demonstrated by, but is not limited to, the following data: retention time and relative peak area, molecular weight from mass spectroscopy or gas chromatography analyses, or other estimates. Although the test sample’s identity is not the responsibility of the test laboratory, it may be prudent for the test laboratory to confirm the sponsor’s characterisation at least in a limited way (e.g. colour, physical nature, etc.).

• 22.The exposure atmosphere shall be held as constant as practicable and monitored continuously and/or intermittently depending on the method of analysis. When intermittent sampling is used, chamber atmosphere samples should be taken at least twice in a four hour study. If not feasible due to limited air flow rates or low concentrations, one sample may be collected over the entire exposure period. If marked sample-to-sample fluctuations occur, the next concentrations tested should use four samples per exposure. Individual chamber concentration samples should not deviate from the mean chamber concentration by more than ± 10 % for gases and vapours, and by no more than ± 20 % for liquid or solid aerosols. Time to chamber equilibration (t95) should be calculated and recorded. The duration of an exposure spans the time that the test chemical is generated and this takes into account the times required to attain t95. Guidance for estimating t95 can be found in GD 39 (8).

• 23.For very complex mixtures consisting of vapours/gases, and aerosols (e.g. combustion atmospheres and test chemicals propelled from purpose-driven end-use products/devices), each phase may behave differently in an inhalation chamber so at least one indicator substance (analyte), normally the principal active substance in the mixture, of each phase (vapour/gas and aerosol) should be selected. When the test chemical is a mixture, the analytical concentration should be reported for the total mixture and not just for the active ingredient or the component (analyte). Additional information regarding actual concentrations can be found in GD 39 (8).

Test chemical: particle size distribution

• 24.The particle size distribution of aerosols should be determined at least twice during each 4 hour exposure by using a cascade impactor or an alternative instrument such as an aerodynamic particle sizer. If equivalence of the results obtained by a cascade impactor or an alternative instrument can be shown, then the alternative instrument may be used throughout the study. A second device, such as a gravimetric filter or an impinger/gas bubbler, should be used in parallel to the primary instrument to confirm the collection efficiency of the primary instrument. The mass concentration obtained by particle size analysis should be within reasonable limits of the mass concentration obtained by filter analysis [see GD 39 (8)]. If equivalence can be demonstrated in the early phase of the study, then further confirmatory measurements may be omitted. For animal welfare reasons, measures should be taken to minimize inconclusive data which may lead to a need to repeat an exposure. Particle sizing should be performed for vapours if there is any possibility that vapour condensation may result in the formation of an aerosol, or if particles are detected in a vapour atmosphere with potential for mixed phases (see paragraph 14).

PROCEDURE

Main test

• 25.Three animals per sex, or six animals of the more susceptible sex, are used for each step. If rodent species other than rats are exposed nose-only, maximum exposure durations may be adjusted to minimise species-specific distress. The concentration level to be used as the starting dose is selected from one of four fixed levels and the starting concentration level should be that which is most likely to produce toxicity in some of the dosed animals. The testing schemes for gases, vapours and aerosols (included in Appendixes2-4) represent the testing with the cut-off values of the CLP categories 1-4 (9) for gases (100, 500, 2 500 , 20 000 ppm/4h) (Appendix 2), for vapours (0,5, 2, 10, 20 mg/l/4h) (Appendix 3) and for aerosols (0,05, 0,5, 1, 5 mg/l/4h) (Appendix 4). Category 5, which is not implemented in Regulation (EC) No 1272/2008 (9) relates to concentrations above the respective limit concentrations. For each starting concentration, the respective testing scheme applies. Depending on the number of humanely killed or dead animals, the test procedure follows the indicated arrows until a categorisation can be made.

• 26.The time interval between exposure groups is determined by the onset, duration, and severity of toxic signs. Exposure of animals at the next concentration level should be delayed until there is reasonable confidence in the survival of the previously tested animals. A period of three or four days between the exposures at each concentration level is recommended to allow for the observation of delayed toxicity. The time interval may be adjusted as appropriate, e.g. in case of inconclusive responses.

Limit test

• 27.The limit test is used when the test chemical is known or expected to be virtually non-toxic, i.e. eliciting a toxic response only above the regulatory limit concentration. Information about the toxicity of the test chemical can be gained from knowledge about similar tested substances or similar mixtures, 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 the test chemical is expected to be toxic, the main test should be performed [further guidance can be found in GD 39 (8)].

• 28.Using the normal procedure, three animals per sex, or six animals of the more susceptible sex, are exposed at concentrations of 20 000 ppm for gases, 20 mg/l for vapours and 5 mg/l for dusts/mists, respectively (if achievable), which serves as the limit test for this Test Method. When testing aerosols, the primary goal should be to achieve a respirable particle size (i.e. an MMAD of 1-4 μm). This is possible with most test chemicals at a concentration of 2 mg/l Aerosol testing at greater than 2 mg/l should only be attempted if a respirable particle size can be achieved [see GD 39 (8)]. In accordance with GHS (16), testing in excess of a limit concentration is discouraged for animal welfare reasons. Testing in GHS Category 5 (16), which is not implemented in Regulation (EC) No 1272/2008 (9), should only be considered when there is a strong likelihood that results of such a test would have direct relevance for protecting human health, and justification provided in the study report. In the case of potentially explosive test chemicals, care should be taken to avoid conditions favourable for an explosion. To avoid an unnecessary use of animals, a test run without animals should be conducted prior to the limit test to ensure that the chamber conditions for a limit test can be achieved.

OBSERVATIONS

• 29.The animals should be clinically observed frequently during the exposure period. Following exposure, clinical observations should be made at least twice on the day of exposure, or more frequently when indicated by the response of the animals to treatment, and at least once daily thereafter for a total of 14 days. The length of the observation period is not fixed, but should be determined by the nature and time of onset of clinical signs and length of the recovery period. The times at which signs of toxicity appear and disappear are important, especially if there is a tendency for signs of toxicity to be delayed. All observations are systematically recorded with individual records being maintained for each animal. Animals found in a moribund condition and animals showing severe pain and/or enduring signs of severe distress should be humanely killed for animal welfare reasons. Care should be taken when conducting examinations for clinical signs of toxicity that initial poor appearance and transient respiratory changes, resulting from the exposure procedure, are not mistaken for treatment-related effects. The principles and criteria summarised in the Humane Endpoints Guidance Document should be taken into consideration (7). When animals are killed for humane reasons or found dead, the time of death should be recorded as precisely as possible.

• 30.Cage-side observations should include changes in the skin and fur, eyes and mucous membranes, and also respiratory, circulatory, autonomic and central nervous systems, and somato-motor activity and behaviour patterns. When possible, any differentiation between local and systemic effects should be noted. Attention should be directed to observations of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma. The measurement of rectal temperatures may provide supportive evidence of reflex bradypnea or hypo/hyperthermia related to treatment or confinement.

Body weights

• 31.Individual animal weights should be recorded once during the acclimatisation period, on the day of exposure prior to exposure (day 0) and at least on days 1, 3 and 7 (and weekly thereafter), and at the time of death or euthanasia if exceeding day 1. Body weight is recognised as a critical indicator of toxicity and animals exhibiting a sustained decrement of ≥ 20 %, compared to pre-study values, should be closely monitored. Surviving animals are weighed and humanely killed at the end of the post-exposure period.

Pathology

• 32.All test animals, including those which die during the test or are euthanised and removed from the study for animal welfare reasons, should be subjected to gross necropsy. If necropsy cannot be performed immediately after a dead animal is discovered, the animal should be refrigerated (not frozen) at temperatures low enough to minimize autolysis. Necropsies should be performed as soon as possible, normally within a day or two. All gross pathological changes should be recorded for each animal with particular attention to any changes in the respiratory tract.

• 33.Additional examinations included a priori by design may be considered to extend the interpretive value of the study, such as measuring lung weight of surviving rats and/or providing evidence of irritation by microscope examination of the respiratory tract. Examined organs may include those showing evidence of gross pathology in animals surviving 24 or more hours, and organs known or expected to be affected. Microscopic examination of the entire respiratory tract may provide useful information for test chemicals that are reactive with water, such as acids and hygroscopic test chemicals.

DATA AND REPORTING

Data

• 34.Individual animal data on body weights and necropsy findings should be provided. Clinical observation data should be summarised in tabular form, showing for each test group the number of animals used, the number of animals displaying specific 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 time course of toxic effects and reversibility, and necropsy findings.

Test report

• 35.The test report should include the following information, as appropriate:

Test animals and husbandry

• — Description of caging conditions, including: number (or change in number) of animals per cage, bedding material, ambient temperature and relative humidity, photoperiod, and identification of diet;

• — Species/strain used and justification for using a species other than the rat;

• — Number, age, and sex of animals;

• — Method of randomisation;

• — Details of food and water quality (including diet type/source, water source);

• — Description of any pre-test conditioning including diet, quarantine, and treatment for disease;

Test chemical

• — Physical nature, purity, and, where relevant, physico-chemical properties (including isomerisation);

• — Identification data and Chemical Abstract Services (CAS) Registry Number, if known;

Vehicle

• — Justification for use of vehicle and justification for choice of vehicle (if other than water);

• — Historical or concurrent data demonstrating that the vehicle does not interfere with the outcome of the study;

Inhalation chamber

• — Description of the inhalation chamber including dimensions and volume;

• — Source and description of equipment used for the exposure of animals as well as generation of atmosphere;

• — Equipment for measuring temperature, humidity, particle-size, and actual concentration;

• — Source of air, treatment of air supplied/extracted and system used for conditioning;

• — Methods used for calibration of equipment to ensure a homogeneous test atmosphere;

• — Pressure difference (positive or negative);

• — Exposure ports per chamber (nose-only); location of animals in the system (whole-body);

• — Temporal homogeneity/stability of test atmosphere;

• — Location of temperature and humidity sensors and sampling of test atmosphere in the chamber;

• — Air flow rates, air flow rate/exposure port (nose-only), or animal load/chamber (whole-body);

• — Information about the equipment used to measure oxygen and carbon dioxide, if applicable;

• — Time required to reach inhalation chamber equilibrium (t95);

• — Number of volume changes per hour;

• — Metering devices (if applicable);

Exposure data

• — Rationale for target concentration selection in the main study;

• — Nominal concentrations (total mass of test chemical generated into the inhalation chamber divided by the volume of air passed through the chamber);

• — Actual test chemical concentrations collected from the animals’ breathing zone; for test mixtures that produce heterogeneous physical forms (gases, vapours, aerosols), each may be analysed separately;

• — All air concentrations should be reported in units of mass (e.g. mg/l, mg/m³, etc.), units of volume (e.g. ppm, ppb) may also be reported parenthetically;

• — Particle size distribution, mass median aerodynamic diameter (MMAD), and geometric standard deviation (σg), including their methods of calculation. Individual particle size analyses should be reported;

Test conditions

• — Details of test chemical preparation, including details of any procedures used to reduce the particle size of solid substances or to prepare solutions of the test chemical. In cases where mechanical processes may have altered test chemical composition, include the results of analyses to verify the composition of the test chemical;

• — A description (preferably including a diagram) of the equipment used to generate the test atmosphere and to expose the animals to the test atmosphere;

• — Details of the chemical analytical method used and method validation (including efficiency of recovery of test chemical from the sampling medium);

• — The rationale for the selection of test concentrations;

Results

• — Tabulation of chamber temperature, humidity, and airflow;

• — Tabulation of chamber nominal and actual concentration data;

• — Tabulation of particle size data including analytical sample collection data, particle size distribution, and calculations of the MMAD and σg;

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

• — Individual body weights of animals collected on study days, date and time of death if prior to scheduled euthanasia; 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;

• — The CLP category classification and the LC50 cut-off value;

Discussion and interpretation of results

• — Particular emphasis should be made to the description of methods used to meet this Test Method’s criteria, e.g. the limit concentration or the particle size;

• — The respirability of particles in light of the overall findings should be addressed, especially if the particle-size criteria could not be met;

• — The consistency of methods used to determine nominal and actual concentrations, and the relation of actual concentration to nominal concentration should be included in the overall assessment of the study;

• — The likely cause of death and predominant mode of action (systemic versus local) should be addressed;

• — An explanation should be provided if there was a need to humanely sacrifice animals in pain or showing signs of severe and enduring distress, based on the criteria in the OECD Guidance Document on Humane Endpoints (7).

LITERATURE:

• (1) Chapter B.2 of this Annex, Acute Toxicity (Inhalation).

• (2) Holzhütter H-G, Genschow E, Diener W, and Schlede E (2003). Dermal and Inhalation Acute Toxicity Class Methods: Test Procedures and Biometric Evaluations for the Globally Harmonized Classification System. Arch. Toxicol. 77: 243-254.

• (3) Diener W, Kayser D and Schlede E (1997). The Inhalation Acute-Toxic-Class Method; Test Procedures and Biometric Evaluations. Arch. Toxicol. 71: 537-549.

• (4) Diener W and Schlede E (1999). Acute Toxic Class Methods: Alternatives to LD/LC50 Tests. ALTEX 1: 129-134.

• (5) Chapter B.1 tris of this Annex, Acute Oral Toxicity — Acute Toxic Class Method.

• (6) OECD (2009). Report on Biostatistical Performance Assessment of the Draft TG 436 Acute Toxic Class Testing Method for Acute Inhalation Toxicity. Environmental Health and Safety Monograph Series on Testing and Assessment No 105, OECD, Paris. Available at: [http://www.oecd.org/env/testguidelines]

• (7) 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. Available at: [http://www.oecd.org/env/testguidelines]

• (8) OECD (2009). Guidance Document on Acute Inhalation Toxicity Testing. Environmental Health and Safety Monograph Series on Testing and Assessment No 39, OECD, Paris. Available at: [http://www.oecd.org/env/testguidelines]

• (9) Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, amending and repealing Directives 67/548/EEC and 1999/45/EC, and amending Regulation (EC) No 1907/2006 (OJ L 353, 31.12.2008, p. 1).

• (10) Chapter B.40 of this Annex, In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER).

• (11) Chapter B.40 bis of this Annex, In Vitro Skin Corrosion: Human Skin Model Test.

• (12) OECD (2005). In Vitro Membrane Barrier Test Method for Skin Corrosion. OECD Guideline for testing of chemicals No 435, OECD, Paris. Available at: [http://www.oecd.org/env/testguidelines]

• (13) Phalen RF (2009). Inhalation Studies: Foundations and Techniques. (2^nd Edition) Informa Healthcare, New York.

• (14) SOT (1992). Technical Committee of the Inhalation Specialty Section, Society of Toxicology (SOT). Recommendations for the Conduct of Acute Inhalation Limit Tests. Fund. Appl. Toxicol. 18: 321-327.

• (15) Pauluhn J and Thiel A (2007). A Simple Approach to Validation of Directed-Flow Nose-Only Inhalation Chambers. J. Appl. Toxicol. 27: 160-167

• (16) UN (2007), United Nations Globally Harmonized System of Classification and Labelling of Chemicals (GHS), ST/SG/AC.10/30, UN New York and Geneva. Available: [http://www.unece.org/trans/danger/publi/ghs/ghs_welcome_e.html]

Appendix 1

DEFINITION

Test chemical : Any substance or mixture tested using this Test Method.

Appendix 2

Procedure to be followed by each of the starting concentrations for gases (ppm/4h)

General remarks ([^E0031])

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

Appendix 2a : Starting concentration is 100 ppm

Appendix 2b : Starting concentration is 500 ppm

Appendix 2c : Starting concentration is 2 500 ppm

Appendix 2d : Starting concentration is 20 000 ppm

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

Appendix 2a

Acute Inhalation Toxicity:

Test Procedure with a starting concentration of 100 ppm/4h for gases

Image omitted; see official source

Text of image

3 ♂ + 3 ♀, or 6 animals of the more susceptible sex are used per step

0-6: Number of moribund or dead animals/tested concentraion

GHS: Globally Harmonized Classification System

∞: unclassified

Testing at ≥ 20000 ppm/4h: see Guidance Document 39 (8)

Appendix 2b

Acute Inhalation Toxicity:

Test Procedure with a starting concentration of 500 ppm/4h for gases

Image omitted; see official source

Text of image

3 ♂ + 3 ♀, or 6 animals of the more susceptible sex are used per step

0-6: Number of moribund or dead animals/tested concentraion

GHS: Globally Harmonized Classification System

∞: unclassified

Testing at ≥ 20000 ppm/4h: see Guidance Document 39 (8)

Appendix 2c

Acute Inhalation Toxicity:

Test Procedure with a starting concentration of 2 500 ppm/4h for gases

Image omitted; see official source

Text of image

3 ♂ + 3 ♀, or 6 animals of the more susceptible sex are used per step

0-6: Number of moribund or dead animals/tested concentraion

GHS: Globally Harmonized Classification System

∞: unclassified

Testing at ≥ 20000 ppm/4h: see Guidance Document 39 (8)

Appendix 2d

Acute Inhalation Toxicity:

Test Procedure with a starting concentration of 20 000 ppm/4h for gases

Image omitted; see official source

Text of image

3 ♂ + 3 ♀, or 6 animals of the more susceptible sex are used per step

0-6: Number of moribund or dead animals/tested concentraion

GHS: Globally Harmonized Classification System

∞: unclassified

Testing at ≥ 20000 ppm/4h: see Guidance Document 39 (8)

Appendix 3

Procedure to be followed by each of the starting concentrations for vapour (mg/l/4h)

General remarks ([^E0032])

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

Appendix 3a : Starting concentration is 0,5 mg/l

Appendix 3b : Starting concentration is 2,0 mg/l

Appendix 3c : Starting concentration is 10 mg/l

Appendix 3d : Starting concentration is 20 mg/l

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

Appendix 3a

Acute Inhalation Toxicity:

Test procedure with a starting concentration of 0,5 mg/L/4h for vapours

Image omitted; see official source

Text of image

• 3 ♂ + 3 ♀, or 6 animals of the more susceptible sex are used per step

• 0-6: Number of moribund or dead animals/tested concentraion

• GHS: Globally Harmonized Classification System

• ∞: unclassified

• Testing at 50 mg/L/4h: see Guidance Document 39 (8)

Appendix 3b

Acute Inhalation Toxicity:

Test procedure with a starting concentration of 2 mg/L/4h for vapours

Image omitted; see official source

Text of image

3 ♂ + 3 ♀, or 6 animals of the more susceptible sex are used per step

0-6: Number of moribund or dead animals/tested concentraion

GHS: Globally Harmonized Classification System

∞: unclassified

Testing at 50 mg/L4h: see Guidance Document 39 (8)

Appendix 3c

Acute Inhalation Toxicity:

Test procedure with a starting concentration of 10 mg/L/4h for vapours

Image omitted; see official source

Text of image

3 ♂ + 3 ♀, or 6 animals of the more susceptible sex are used pers step

0-6: Number of moribund or dead animals/tested concentraion

GHS: Globally Harmonized Classification System

∞: unclassified

Testing at 50 mg/L/4h: see Guidance Document 39 (8)

Appendix 3d

Acute Inhalation Toxicity:

Test procedure with a starting concentration of 20 mg/L/4h for vapours

Image omitted; see official source

Text of image

3 ♂ + 3 ♀, or 6 animals of the more susceptible sex are used per step

0-6: Number of moribund or dead animals/tested concentraion

GHS: Globally Harmonized Classification System

∞: unclassified

Testing at 50 mg/L/4h: see Guidance Document 39(8)

Appendix 4

Procedure to be followed by each of the starting concentrations for aerosols (mg/l/4h)

General remarks ([^E0033])

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

Appendix 4a : Starting concentration is 0,05 mg/l

Appendix 4b : Starting concentration is 0,5 mg/l

Appendix 4c : Starting concentration is 1 mg/l

Appendix 4d : Starting concentration is 5 mg/l

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

Appendix 4a

Acute Inhalation Toxicity:

Test procedure with a starting concentration of 0,05 mg/L/4h for aerosols

Image omitted; see official source

Text of image

3 ♂ + 3 ♀, or 6 animals of the more susceptible sex are used per step

0-6: Number of moribund or dead animals/tested concentraion

GHS: Globally Harmonized Classification System

∞: unclassified

Testing at 12.5 mg/L4h: see Guidance Document 39 (8)

Appendix 4b

Acute Inhalation Toxicity:

Test procedure with a starting concentration of 0,5 mg/L/4h for aerosols

Image omitted; see official source

Text of image

3 ♂ + 3 ♀, or 6 animals of the more susceptible sex are used per step

0-6: Number of moribund or dead animals/tested concentraion

GHS: Globally Harmonized Classification System

∞: unclassified

Testing at 12.5 mg/L4h: see Guidance Document 39 (8)

Appendix 4c

Acute Inhalation Toxicity:

Test procedure with a starting concentration of 1 mg/L/4h for aerosols

Image omitted; see official source

Text of image

3 ♂ + 3 ♀, or 6 animals of the more susceptible sex are used per step

0-6: Number of moribund or dead animals/tested concentraion

GHS: Globally Harmonized Classification System

∞: unclassified

Testing at 12.5 mg/L4h: see Guidance Document 39 (8)

Appendix 4d

Acute Inhalation Toxicity:

Test procedure with a starting concentration of 5 mg/L/4h for_aerosols

Image omitted; see official source

Text of image

3 ♂ + 3 ♀, or 6 animals of the more susceptible sex are used per step

0-6 : Number of moribund or dead animals/tested concentraion

GHS: Globally Harmonized Classification System

∞: unclassified

Testing at 12.5 mg/L/4h: see Guidance Document 39 (8)

B.53   DEVELOPMENTAL NEUROTOXICITY STUDY

INTRODUCTION

• 1. This test method is equivalent to OECD Test Guideline (TG) 426 (2007). In Copenhagen in June 1995, an OECD Working Group on Reproduction and Developmental Toxicity discussed the need to update existing OECD test guidelines for reproduction and developmental toxicity, and the development of new guidelines for endpoints not yet covered (1). The working group recommended that a test guideline for developmental neurotoxicity should be written based on a US EPA guideline, which has since been revised (2). In June 1996, a second consultation meeting was held in Copenhagen to provide the Secretariat with guidance on the outline of a new test guideline on developmental neurotoxicity, including the major elements, e.g. details concerning choice of animal species, dosing period, testing period, endpoints to be assessed, and criteria for evaluating results. A US neurotoxicity risk assessment guideline was published in 1998 (3). An OECD Expert Consultation Meeting and an ILSI Risk Science Institute Workshop were held back-to-back in October 2000 and an expert consultation meeting was held in Tokyo 2005. These meetings were held to discuss the scientific and technical issues related to the current test guideline and the recommendations from the meetings (4)(5)(6)(7) were considered in the development of this test method. Additional information on the conduct, interpretation and terminology used for this test method can be found in OECD Guidance Documents No 43 on ‘Reproductive Toxicity Testing and Assessment’ (8) and No 20 on ‘Neurotoxicity Testing’ (9).

INITIAL CONSIDERATIONS