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)

• — Day 6 : Record the weight of each animal. Inject 250 μL of sterile phosphate-buffered saline (PBS) containing 20 μCi (7,4 × 10⁵ Bq) of tritiated (³H)-methyl thymidine into all test and control mice via the tail vein. Alternatively, inject 250 μL sterile PBS containing 2 μCi (7,4 × 10⁴ Bq) of ¹²⁵I-iododeoxyuridine and 10^–5M fluorodeoxyuridine into all mice via the tail vein. Five hours (5 h) later, humanely kill the animals. Excise the draining auricular lymph nodes from each mouse ear and process together in PBS for each animal (individual animal approach); alternatively excise and pool the lymph nodes from each ear in PBS for each treatment group (pooled treatment group approach). Details and diagrams of the lymph node identification and dissection can be found in reference (12). 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.A single-cell suspension of lymph node cells (LNC) excised bilaterally using the individual animal approach or alternatively, the pooled treatment group approach is prepared by gentle mechanical disaggregation through 200 micron-mesh stainless steel gauze or another acceptable technique for generating a single-cell suspension. The LNC are washed twice with an excess of PBS and the DNA is precipitated with 5 % trichloroacetic acid (TCA) at 4 °C for 18h (3). Pellets are either resuspended in 1 mL TCA and transferred to scintillation vials containing 10 mL of scintillation fluid for ³H-counting, or transferred directly to gamma counting tubes for ¹²⁵I-counting.

Determination of cellular proliferation (incorporated radioactivity)

• 27.Incorporation of ³H-methyl thymidine is measured by β-scintillation counting as disintegrations per minute (DPM). Incorporation of ¹²⁵I-iododeoxyuridine is measured by ¹²⁵I-counting and also is expressed as DPM. Depending on the approach used, the incorporation is expressed as DPM/mouse (individual animal approach) or DPM/treatment group (pooled treatment group approach).

Reduced LLNA

• 28.In certain situations, when there is a regulatory need to confirm a negative prediction of skin sensitising potential, an optional rLLNA protocol (16) (17) (18) using fewer animals may be used, provided there is adherence to all other LLNA protocol specifications in this TM. Before applying the rLLNA approach, clear justifications and scientific rationale for its use should be provided. If a positive or equivocal result is obtained, additional testing may be needed in order to interpret or clarify the finding.

• 29.The reduction in number of dose groups is the only difference between the LLNA and the rLLNA test method protocols and for this reason the rLLNA does not provide dose-response information. Therefore, the rLLNA should not be used when dose-response information is needed. Like the multi-dose LLNA, the test substance concentration evaluated in the rLLNA should be the maximum concentration that does not induce overt systemic toxicity and/or excessive local skin irritation in the mouse (see paragraph 18).

OBSERVATIONS

Clinical observations

• 30.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 (37).

Body weights

• 31.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

• 32.Results for each treatment group are expressed as the SI. When using the individual animal approach, the SI is derived by dividing the mean DPM/mouse within each test substance group, and the PC group, by the mean DPM/mouse for the solvent/VC group. The average SI for the VCs is then one. When using the pooled treatment group approach, the SI is obtained by dividing the pooled radioactive incorporation for each treatment group by the incorporation of the pooled VC group; this yields a mean SI.

• 33.The decision process regards a result as positive when SI ≥ 3. However, the strength of the dose-response, the statistical significance and the consistency of the solvent/vehicle and PC responses may also be used when determining whether a borderline result is declared positive (4) (5) (6).

• 34.If it is necessary to clarify the results obtained, consideration should be given to various properties of the test substance, including whether it has a structural relationship to known skin sensitisers, whether it causes excessive local skin irritation in the mouse, and the nature of the dose-response relationship seen. These and other considerations are discussed in detail elsewhere (7).

• 35.Collecting radioactivity 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 VC 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

• 36.Data should be summarised in tabular form. When using the individual animal approach, show the individual animal DPM values, the group mean DPM/animal, its associated error term (e.g. SD, SEM), and the mean SI for each dose group compared against the concurrent VC group. When using the pooled treatment group approach, show the mean/median DPM and the mean SI for each dose group compared against the concurrent VC group.

Test report

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

Test and control substances:

• — identification data (e.g. CAS and EC numbers, 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:

• — 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:

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

• — concurrent and/or historical PC and concurrent NC 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;

• — a table of individual mouse (individual animal approach) or mean/median (pooled treatment group approach) DPM values and SI values for each treatment group;

• — mean and associated error term (e.g. SD, SEM) for DPM/mouse for each treatment group and the results of outlier analysis for each treatment group when using the individual animal approach;

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

• — 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 (2002), Skin Sensitisation: Local Lymph Node Assay. OECD Guideline for the Testing of Chemicals No 429, Paris. Available at: [http://www.oecd.org/env/testguidelines]

• (2) Kimber, I. and Basketter, D.A. (1992), The murine local lymph node assay; collaborative studies and new directions: A commentary, Food Chem. Toxicol., 30, 165-169.

• (3) 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.

• (4) 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.

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

• (6) 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.

• (7) Basketter, D.A., Gerberick, G.F. and Kimber, I. (1998), Strategies for identifying false positive responses in predictive sensitisation tests, Food Chem. Toxicol., 36, 327-33.

• (8) 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.

• (9) 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.

• (10) 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.

• (11) 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.

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

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

• (14) 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]

• (15) Dearman, R.J., Hilton, J., Evans, P., Harvey, P., Basketter, D.A. and Kimber, I. (1998), Temporal stability of local lymph node assay responses to hexyl cinnamic aldehyde, J. Appl. Toxicol., 18, 281-284.

• (16) Kimber, I., Dearman, R.J., Betts, C.J., Gerberick, G.F., Ryan, C.A., Kern, P.S., Patlewicz, G.Y. and Basketter, D.A. (2006), The local lymph node assay and skin sensitisation: a cut-down screen to reduce animal requirements? Contact Dermatitis, 54, 181-185.

• (17) ESAC (2007), Statement on the Reduced Local Lymph Node Assay (rLLNA), European Commission Directorate-General, Joint Research Centre, Institute for Health and Consumer Protection, European Centre for the Validation of Alternative Methods, April 2007. Available at: [http://ecvam.jrc.it/ft_doc/ESAC26_statement_rLLNA_20070525-1.pdf]

• (18) ICCVAM (2009), The Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) Test Method Evaluation Report. The Reduced Murine Local Lymph Node Assay: An Alternative Test Method Using Fewer Animals to Assess the Allergic Contact Dermatitis Potential of Chemicals and Products, NIH Publication Number 09-6439, Research Triangle Park, NC: National Institute of Environmental Health Sciences. Available at: [http://iccvam.niehs.nih.gov/]

• (19) 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 Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM), NIH Publication No 99-4494, Research Triangle Park, NC: National Institute of Environmental Health Sciences. Available at: [http://iccvam.niehs.nih.gov/docs/immunotox_docs/llna/llnarep.pdf]

• (20) 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.

• (21) Basketter, D., Ball, N., Cagen, S., Carrilo, 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.

• (22) ICCVAM (2009), ICCVAM Test Method Evaluation Report. Assessment of the Validity of the LLNA for Testing Pesticide Formulations and Other Products, Metals, and Substances in Aqueous Solutions, NIH Publication Number 10-7512, Research Triangle Park, NC: National Institute of Environmental Health Sciences, Available at: [http://iccvam.niehs.nih.gov/]

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

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

• (25) OECD (2002), Acute Dermal Irritation/Corrosion. OECD Guideline for Testing of Chemicals No 404, Paris, France. Available at: [http://www.oecd.org/document/40/0,3343,en_2649_34377_37051368_1_1_1_1,00.html]

• (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), Non-radioactive 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) OECD (1987), Acute Dermal Toxicity, OECD Guideline for Testing of Chemicals No 402, Paris, France. Available at: [http://www.oecd.org/env/testguidelines]

• (36) 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]

• (37) 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]

Appendix 1

Performance standards for assessment of proposed similar or modified llna test methods for skin sensitisation

INTRODUCTION

• 1.The purpose of Performance Standards (PS) is to communicate the basis by which new test methods, both proprietary (i.e. copyrighted, trademarked, registered) and non-proprietary can be determined to have sufficient accuracy and reliability for specific testing purposes. These PS, based on validated and accepted test methods, can be used to evaluate the reliability and accuracy of other similar methods (colloquially referred to as ‘me-too’ tests) that are based on similar scientific principles and measure or predict the same biological or toxic effect (14).

• 2.Prior to adoption of modified methods (i.e. proposed potential improvements to an approved test method), there should be an evaluation to determine the effect of the proposed changes on the test’s performance and the extent to which such changes affect the information available for the other components of the validation process. Depending on the number and nature of the proposed changes, the generated data and supporting documentation for those changes, they should either be subjected to the same validation process as described for a new test, or, if appropriate, to a limited assessment of reliability and relevance using established PS (14).

• 3.Similar or modified methods proposed for use under this TM should be evaluated to determine their reliability and accuracy using chemicals representing the full range of the LLNA scores. To avoid unwarranted animal use, it is strongly recommended that model developers consult the appropriate authorities before starting validation studies in accordance with the PS and guidance provided in this TM.

• 4.These PS are based on the US-ICCVAM, EC-ECVAM and Japanese-JaCVAM harmonised PS (12), for evaluating the validity of similar or modified versions of the LLNA. The PS consists of essential test method components, recommended reference chemicals and standards for accuracy and reliability that the proposed method should meet or exceed.

I. Essential test method components

• 5.To ensure that a similar or modified LLNA method is functionally and mechanistically analogous to the LLNA and measures the same biological effect, the following components should be included in the test method protocol:

• — The test substance should be applied topically to both ears of the mouse;

• — Lymphocyte proliferation should be measured in the lymph nodes draining from the site of test substance application;

• — Lymphocyte proliferation should be measured during the induction phase of skin sensitisation;

• — For test substances, the highest dose selected should be the maximum concentration that does not induce systemic toxicity and/or excessive local skin irritation in the mouse. For positive reference chemicals, the highest dose should be at least as high as the LLNA EC3 values of the corresponding reference chemicals (see Table 1) without producing systemic toxicity and/or excessive local skin irritation in the mouse;

• — A concurrent VC should be included in each study and, where appropriate, a concurrent PC should also be used;

• — A minimum of four animals per dose group should be used;

• — Either individual or pooled animal data may be collected.

• If any of these criteria are not met, then these PS cannot be used for validation of the similar or modified method.

II.   Minimum list of reference chemicals

• 6.The US-ICCVAM, EC-ECVAM and Japanese-JaCVAM harmonised PS (12) identified 18 minimum reference chemicals that should be used and four optional reference chemicals (i.e. substances that produced either false positive or false negative results in the LLNA, when compared to human and guinea pig results (B.6, or OECD Test Guideline 406) (13), and therefore provide the opportunity to demonstrate equal to or better performance than the LLNA) that are included in the LLNA PS. The selection criteria for identifying these chemicals were:

• — The list of reference chemicals represented the types of substances typically tested for skin sensitisation potential and the range of responses that the LLNA is capable of measuring or predicting;

• — The substances had well-defined chemical structures;

• — LLNA data from guinea pig tests (i.e. B.6; OECD Test Guideline 406) (13) and (where possible) data from humans were available for each substance; and

• — The substances were readily available from a commercial source.

• The recommended reference chemicals are listed in Table 1. Studies using the proposed reference chemicals should be evaluated in the vehicle with which they are listed in Table 1. In situations where a listed substance may not be available, other substances that meet the selection criteria mentioned may be used, with adequate justification.

Table 1

Recommended Reference chemicals for the LLNA PS.

Number	Chemicals [^E0024]	CAS No	Form	Veh [^E0025]	EC3 % [^E0026]	N [^E0027]	0,5x-2,0x EC3	Actual EC3 Range	LLNA vs GP	LLNA vs Human
1	5-Chloro-2-methyl-4-isothiazolin-3-one (CMI)/2-methyl-4-isothiazolin-3-one (MI) [^E0028]	26172-55-4/ 2682-20-4	Liq	DMF	0,009	1	0,0045-0,018	NC	+/+	+/+
2	DNCB	97-00-7	Sol	AOO	0,049	15	0,025-0,099	0,02-0,094	+/+	+/+
3	4-Phenylenediamine	106-50-3	Sol	AOO	0,11	6	0,055-0,22	0,07-0,16	+/+	+/+
4	Cobalt chloride	7646-79-9	Sol	DMSO	0,6	2	0,3-1,2	0,4-0,8	+/+	+/+
5	Isoeugenol	97-54-1	Liq	AOO	1,5	47	0,77-3,1	0,5-3,3	+/+	+/+
6	2-Mercaptobenzothiazole	149-30-4	Sol	DMF	1,7	1	0,85-3,4	NC	+/+	+/+
7	Citral	5392-40-5	Liq	AOO	9,2	6	4,6-18,3	5,1-13	+/+	+/+
8	HCA	101-86-0	Liq	AOO	9,7	21	4,8-19,5	4,4-14,7	+/+	+/+
9	Eugenol	97-53-0	Liq	AOO	10,1	11	5,05-20,2	4,9-15	+/+	+/+
10	Phenyl benzoate	93-99-2	Sol	AOO	13,6	3	6,8-27,2	1,2-20	+/+	+/+
11	Cinnamic alcohol	104-54-1	Sol	AOO	21	1	10,5-42	NC	+/+	+/+
12	Imidazolidinyl urea	39236-46-9	Sol	DMF	24	1	12-48	NC	+/+	+/+
13	Methyl methacrylate	80-62-6	Liq	AOO	90	1	45-100	NC	+/+	+/+
14	Chlorobenzene	108-90-7	Liq	AOO	25	1	NA	NA	–/–	–/ [^E0022]
15	Isopropanol	67-63-0	Liq	AOO	50	1	NA	NA	–/–	–/+
16	Lactic acid	50-21-5	Liq	DMSO	25	1	NA	NA	–/–	–/ [^E0022]
17	Methyl salicylate	119-36-8	Liq	AOO	20	9	NA	NA	–/–	–/–
18	Salicylic acid	69-72-7	Sol	AOO	25	1	NA	NA	–/–	–/–
Optional Substances to Demonstrate Improved Performance Relative to the LLNA	Optional Substances to Demonstrate Improved Performance Relative to the LLNA	Optional Substances to Demonstrate Improved Performance Relative to the LLNA	Optional Substances to Demonstrate Improved Performance Relative to the LLNA	Optional Substances to Demonstrate Improved Performance Relative to the LLNA	Optional Substances to Demonstrate Improved Performance Relative to the LLNA	Optional Substances to Demonstrate Improved Performance Relative to the LLNA	Optional Substances to Demonstrate Improved Performance Relative to the LLNA	Optional Substances to Demonstrate Improved Performance Relative to the LLNA	Optional Substances to Demonstrate Improved Performance Relative to the LLNA	Optional Substances to Demonstrate Improved Performance Relative to the LLNA
19	Sodium lauryl sulphate	151-21-3	Sol	DMF	8,1	5	4,05-16,2	1,5-17,1	+/–	+/–
20	Ethylene glycol dimethacrylate	97-90-5	Liq	MEK	28	1	14-56	NC	+/–	+/+
21	Xylene	1330-20-7	Liq	AOO	95,8	1	47,9-100	NC	+/ [^E0023]	+/–
22	Nickel chloride	7718-54-9	Sol	DMSO	5	2	NA	NA	–/+	–/+
Abbreviations: AOO = acetone: olive oil (4:1, v/v); CAS No = Chemical Abstracts Service Number; DMF = N,N-dimethylformamide; DMSO = dimethyl sulfoxide; DNCB = 2,4-dinitrochlorobenzene; EC3 = estimated concentration needed to produce a stimulation index of 3; GP = guinea pig test result (i.e. B.6 or OECD Test Guideline 406) (13); HCA = hexyl cinnamic aldehyde; Liq = liquid; LLNA = murine local lymph node assay result (i.e. B.42 or OECD Test Guideline 429) (1); MEK = methyl ethyl ketone; NA = not applicable since stimulation index < 3; NC = not calculated since data was obtained from a single study; Sol = solid; Veh = test vehicle.	Abbreviations: AOO = acetone: olive oil (4:1, v/v); CAS No = Chemical Abstracts Service Number; DMF = N,N-dimethylformamide; DMSO = dimethyl sulfoxide; DNCB = 2,4-dinitrochlorobenzene; EC3 = estimated concentration needed to produce a stimulation index of 3; GP = guinea pig test result (i.e. B.6 or OECD Test Guideline 406) (13); HCA = hexyl cinnamic aldehyde; Liq = liquid; LLNA = murine local lymph node assay result (i.e. B.42 or OECD Test Guideline 429) (1); MEK = methyl ethyl ketone; NA = not applicable since stimulation index < 3; NC = not calculated since data was obtained from a single study; Sol = solid; Veh = test vehicle.	Abbreviations: AOO = acetone: olive oil (4:1, v/v); CAS No = Chemical Abstracts Service Number; DMF = N,N-dimethylformamide; DMSO = dimethyl sulfoxide; DNCB = 2,4-dinitrochlorobenzene; EC3 = estimated concentration needed to produce a stimulation index of 3; GP = guinea pig test result (i.e. B.6 or OECD Test Guideline 406) (13); HCA = hexyl cinnamic aldehyde; Liq = liquid; LLNA = murine local lymph node assay result (i.e. B.42 or OECD Test Guideline 429) (1); MEK = methyl ethyl ketone; NA = not applicable since stimulation index < 3; NC = not calculated since data was obtained from a single study; Sol = solid; Veh = test vehicle.	Abbreviations: AOO = acetone: olive oil (4:1, v/v); CAS No = Chemical Abstracts Service Number; DMF = N,N-dimethylformamide; DMSO = dimethyl sulfoxide; DNCB = 2,4-dinitrochlorobenzene; EC3 = estimated concentration needed to produce a stimulation index of 3; GP = guinea pig test result (i.e. B.6 or OECD Test Guideline 406) (13); HCA = hexyl cinnamic aldehyde; Liq = liquid; LLNA = murine local lymph node assay result (i.e. B.42 or OECD Test Guideline 429) (1); MEK = methyl ethyl ketone; NA = not applicable since stimulation index < 3; NC = not calculated since data was obtained from a single study; Sol = solid; Veh = test vehicle.	Abbreviations: AOO = acetone: olive oil (4:1, v/v); CAS No = Chemical Abstracts Service Number; DMF = N,N-dimethylformamide; DMSO = dimethyl sulfoxide; DNCB = 2,4-dinitrochlorobenzene; EC3 = estimated concentration needed to produce a stimulation index of 3; GP = guinea pig test result (i.e. B.6 or OECD Test Guideline 406) (13); HCA = hexyl cinnamic aldehyde; Liq = liquid; LLNA = murine local lymph node assay result (i.e. B.42 or OECD Test Guideline 429) (1); MEK = methyl ethyl ketone; NA = not applicable since stimulation index < 3; NC = not calculated since data was obtained from a single study; Sol = solid; Veh = test vehicle.	Abbreviations: AOO = acetone: olive oil (4:1, v/v); CAS No = Chemical Abstracts Service Number; DMF = N,N-dimethylformamide; DMSO = dimethyl sulfoxide; DNCB = 2,4-dinitrochlorobenzene; EC3 = estimated concentration needed to produce a stimulation index of 3; GP = guinea pig test result (i.e. B.6 or OECD Test Guideline 406) (13); HCA = hexyl cinnamic aldehyde; Liq = liquid; LLNA = murine local lymph node assay result (i.e. B.42 or OECD Test Guideline 429) (1); MEK = methyl ethyl ketone; NA = not applicable since stimulation index < 3; NC = not calculated since data was obtained from a single study; Sol = solid; Veh = test vehicle.	Abbreviations: AOO = acetone: olive oil (4:1, v/v); CAS No = Chemical Abstracts Service Number; DMF = N,N-dimethylformamide; DMSO = dimethyl sulfoxide; DNCB = 2,4-dinitrochlorobenzene; EC3 = estimated concentration needed to produce a stimulation index of 3; GP = guinea pig test result (i.e. B.6 or OECD Test Guideline 406) (13); HCA = hexyl cinnamic aldehyde; Liq = liquid; LLNA = murine local lymph node assay result (i.e. B.42 or OECD Test Guideline 429) (1); MEK = methyl ethyl ketone; NA = not applicable since stimulation index < 3; NC = not calculated since data was obtained from a single study; Sol = solid; Veh = test vehicle.	Abbreviations: AOO = acetone: olive oil (4:1, v/v); CAS No = Chemical Abstracts Service Number; DMF = N,N-dimethylformamide; DMSO = dimethyl sulfoxide; DNCB = 2,4-dinitrochlorobenzene; EC3 = estimated concentration needed to produce a stimulation index of 3; GP = guinea pig test result (i.e. B.6 or OECD Test Guideline 406) (13); HCA = hexyl cinnamic aldehyde; Liq = liquid; LLNA = murine local lymph node assay result (i.e. B.42 or OECD Test Guideline 429) (1); MEK = methyl ethyl ketone; NA = not applicable since stimulation index < 3; NC = not calculated since data was obtained from a single study; Sol = solid; Veh = test vehicle.	Abbreviations: AOO = acetone: olive oil (4:1, v/v); CAS No = Chemical Abstracts Service Number; DMF = N,N-dimethylformamide; DMSO = dimethyl sulfoxide; DNCB = 2,4-dinitrochlorobenzene; EC3 = estimated concentration needed to produce a stimulation index of 3; GP = guinea pig test result (i.e. B.6 or OECD Test Guideline 406) (13); HCA = hexyl cinnamic aldehyde; Liq = liquid; LLNA = murine local lymph node assay result (i.e. B.42 or OECD Test Guideline 429) (1); MEK = methyl ethyl ketone; NA = not applicable since stimulation index < 3; NC = not calculated since data was obtained from a single study; Sol = solid; Veh = test vehicle.	Abbreviations: AOO = acetone: olive oil (4:1, v/v); CAS No = Chemical Abstracts Service Number; DMF = N,N-dimethylformamide; DMSO = dimethyl sulfoxide; DNCB = 2,4-dinitrochlorobenzene; EC3 = estimated concentration needed to produce a stimulation index of 3; GP = guinea pig test result (i.e. B.6 or OECD Test Guideline 406) (13); HCA = hexyl cinnamic aldehyde; Liq = liquid; LLNA = murine local lymph node assay result (i.e. B.42 or OECD Test Guideline 429) (1); MEK = methyl ethyl ketone; NA = not applicable since stimulation index < 3; NC = not calculated since data was obtained from a single study; Sol = solid; Veh = test vehicle.	Abbreviations: AOO = acetone: olive oil (4:1, v/v); CAS No = Chemical Abstracts Service Number; DMF = N,N-dimethylformamide; DMSO = dimethyl sulfoxide; DNCB = 2,4-dinitrochlorobenzene; EC3 = estimated concentration needed to produce a stimulation index of 3; GP = guinea pig test result (i.e. B.6 or OECD Test Guideline 406) (13); HCA = hexyl cinnamic aldehyde; Liq = liquid; LLNA = murine local lymph node assay result (i.e. B.42 or OECD Test Guideline 429) (1); MEK = methyl ethyl ketone; NA = not applicable since stimulation index < 3; NC = not calculated since data was obtained from a single study; Sol = solid; Veh = test vehicle.

Presumed to be a non-sensitiser in humans based on the fact that no clinical patch test results were located, it is not included as a patch test kit allergen, and no case reports of human sensitisation were located.

GP data not available.

Chemicals should be prepared daily unless stability data demonstrate the acceptability of storage.

Because of the potential impact of different vehicles on the performance of the LLNA, the recommended vehicle for each reference chemical should be used (24) (32).

Mean value where more than one EC3 value was available. For negative substances (i.e. with stimulation index < 3, the highest concentration tested is provided).

Number of LLNA studies from which data were obtained.

Commercially available as Kathon CG (CAS No 55965-84-9), which is a 3:1 mixture of CMI and MI. The relative concentrations of each component range from 1,1 % to 1,25 % (CMI) and 0,3 % to 0,45 % (MI). The inactive components are magnesium salts (21,5 % to 24 %) and copper nitrate (0,15 % to 0,17 %), with the remaining formulation 74 % to 77 % water. Kathon CG is readily available through Sigma-Aldrich and Rohm and Haas (now Dow Chemical Corporation).

III.   Defined reliability and accuracy standards

• 7.The accuracy of a similar or modified LLNA method should meet or exceed that of the LLNA PS when it is evaluated using the 18 minimum reference chemicals that should be used. The new or modified method should result in the correct classification based on a ‘yes/no’ decision. However, the new or modified method might not correctly classify all of the minimum reference chemicals that should be used. If, for example, one of the weak sensitisers were misclassified, a rationale for the misclassification and appropriate additional data (e.g. test results that provide correct classifications for other substances with physical, chemical, and sensitising properties similar to those of the misclassified reference chemical) could be considered to demonstrate equivalent performance. Under such circumstances, the validation status of the new or modified LLNA test method would be evaluated on a case-by-case basis.

Intra-laboratory reproducibility

• 8.To determine intra-laboratory reproducibility, a new or modified LLNA method should be assessed using a sensitising substance that is well characterised in the LLNA. Therefore, the LLNA PS are based on the variability of results from repeated tests of hexyl cinnamic aldehyde (HCA). To assess intra-laboratory reliability, threshold estimated concentration (ECt) values for HCA should be derived on four separate occasions with at least one week between tests. Acceptable intra-laboratory reproducibility is indicated by a laboratory’s ability to obtain, in each HCA test, ECt values between 5 % and 20 %, which represents the range of 0,5-2,0 times the mean EC3 specified for HCA (10 %) in the LLNA (see Table 1).

Inter-laboratory reproducibility

• 9.Inter-laboratory reproducibility of a new or modified LLNA method should be assessed using two sensitising substances that are well characterised in the LLNA. The LLNA PS are based on the variability of results from tests of HCA and 2,4-dinitrochlorobenzene (DNCB) in different laboratories. ECt values should be derived independently from a single study conducted in at least three separate laboratories. To demonstrate acceptable inter-laboratory reproducibility, each laboratory should obtain ECt values of 5 % to 20 % for HCA and 0,025 % to 0,1 % for DNCB, which represents the range of 0,5-2,0 times the mean EC3 concentrations specified for HCA (10 %) and DNCB (0,05 %), respectively, in the LLNA (see Table 1).

Appendix 2

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 (14).

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) 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.

Estimated concentration threshold (ECt) : Estimated concentration of a test substance needed to produce a stimulation index that is indicative of a positive response.

Estimated concentration three (EC3) : Estimated concentration of a test substance needed to produce a stimulation index of three.

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

False positive : A test 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 (14).

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 (14).

Me-too test : A colloquial expression for a test method that is structurally and functionally similar to a validated and accepted reference test method. Such a test method would be a candidate for catch-up validation. Interchangeably used with similar test method (14).

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

Performance standards (PS) : Standards, based on a validated test method, that provide a basis for evaluating the comparability of a proposed test method that is functionally and mechanistically similar. Included are: (i) essential test method components; (ii) a minimum list of Reference Chemicals selected from among the chemicals used to demonstrate the acceptable performance of the validated test method; and (iii) the similar levels of accuracy and reliability, based on what was obtained for the validated test method, that the proposed test method should demonstrate when evaluated using the minimum list of Reference Chemicals (14).

Proprietary test method : A test method for which manufacture and distribution is restricted by patents, copyrights, trademarks, etc.

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.

Reference chemicals : Chemicals selected for use in the validation process, for which responses in the in vitro or in vivo reference test system or the species of interest are already known. These chemicals should be representative of the classes of chemicals for which the test method is expected to be used, and should represent the full range of responses that may be expected from the chemicals for which it may be used, from strong, to weak, to negative. Different sets of reference chemicals may be required for the different stages of the validation process, and for different test methods and test uses (14).

Relevance : Description of relationship of the test to the effect of interest and whether it is meaningful and useful for a particular purpose. It is the extent to which the test correctly measures or predicts the biological effect of interest. Relevance incorporates consideration of the accuracy (concordance) of a test method (14).

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 (14).

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.

Validated test method : A test method for which validation studies have been completed to determine the relevance (including accuracy) and reliability for a specific purpose. It is important to note that a validated test method may not have sufficient performance in terms of accuracy and reliability to be found acceptable for the proposed purpose (14).

B.43.   NEUROTOXICITY STUDY IN RODENTS

1. METHOD

This method is equivalent of OECD TG 424 (1997).

This test method has been designed to obtain the information necessary to confirm or to further characterise the potential neurotoxicity of chemicals in adult animals. It can either be combined with existing test methods for repeated dose toxicity studies or to be carried out as a separate study. It is recommended that the OECD Guidance Document on Neurotoxicity Testing Strategies and Methods (1) be consulted to assist in the design of studies based on this test method. This is particularly important when modifications of the observations and test procedures as recommended for routine use of this method are considered. The guidance document has been prepared to facilitate the selection of other test procedures for use in specific circumstances.

The assessment of developmental neurotoxicity is not the subject of this method.

1.1.   INTRODUCTION

In the assessment and evaluation of the toxic characteristics of chemicals, it is important to consider the potential for neurotoxic effects. Already the test method for repeated dose systemic toxicity includes observations that screen for potential neurotoxicity. This test method can be used to design a study to obtain further information on, or to confirm, the neurotoxic effects observed in the repeated dose systemic toxicity studies. However, consideration of the potential neurotoxicity of certain classes of chemicals may suggest that they may be more appropriately evaluated using this Method without prior indications of the potential neurotoxicity from repeated dose systemic toxicity studies. Such considerations include, for example:

• — observation of neurological signs or neuropathological lesions in toxicity studies other than repeated dose systemic toxicity studies, or

• — structural relationship or other information linking them to known neurotoxicants.

In addition there may be other instances when use of this test method is appropriate; for further details see (1).

This method has been developed so that it can be tailored to meet particular needs to confirm the specific histopathological and behavioural neurotoxicity of a chemical as well as provide a characterisation and quantification of the neurotoxic responses.

In the past, neurotoxicity was equated with neuropathy involving neuropathological lesions or neurological dysfunctions, such as seizure, paralysis or tremor. Although neuropathy is an important manifestation of neurotoxicity, it is now clear that there are many other signs of nervous system toxicity (e.g. loss of motor co-ordination, sensory deficits, learning and memory dysfunctions) that may not be reflected in neuropathy or other types of studies.

This neurotoxicity test method is designed to detect major neurobehavioural and neuropathological effects in adult rodents. While behavioural effects, even in the absence of morphological changes, can reflect an adverse impact on the organism, not all behavioural changes are specific to the nervous system. Therefore, any changes observed should be evaluated in conjunction with correlative histopathological, haematological or biochemical data as well as data on other types of systemic toxicity. The testing called for in this method to provide a characterisation and quantification of the neurotoxic responses includes specific histopathological and behavioural procedures that may be further supported by electrophysiological and/or biochemical investigations (1)(2)(3)(4).

Neurotoxicants may act on a number of targets within the nervous system and by a variety of mechanisms. Since no single array of tests is capable of thoroughly assessing the neurotoxic potential of all substances, it may be necessary to utilise other in vivo or in vitro tests specific to the type of neurotoxicity observed or anticipated.

This test method can also be used, in conjunction with the guidance set out in the OECD Guidance Document on Neurotoxicity Testing Strategies and Methods (1) to design studies intended to further characterise or increase the sensitivity of the dose-response quantification in order or better estimate a no-observed-adverse effect level or to substantiate known or suspected hazards of the chemical. For example, studies may be designed to identify and evaluate the neurotoxic mechanism(s) or supplement the data already available from the use of basic neurobehavioural and neuropathological observation procedures. Such studies need not replicate data that would be generated from the use of the standard procedures recommended in this Method, if such data are already available and are not considered necessary for the interpretation of the results of the study.

This neurotoxicity study, when used alone or in combination, provides information that can:

• — identify whether the nervous system is permanently or reversibly affected by the chemical tested;

• — contribute to the characterisation of the nervous system alterations associated with exposure to the chemical, and to understanding the underlying mechanism.

• — determine dose-and time-response relationships in order to estimate a no-observed-adverse-effect level (which can be used to establish safety criteria for the chemical).

This test method uses oral administration of the test substance. Other routes of administration (e.g. dermal or inhalation) may be more appropriate, and may require modification of the procedures recommended. Considerations of the choice of the route of administration depend on the human exposure profile and available toxicological or kinetic information.

1.2.   DEFINITIONS

Adverse effect: is any treatment-related alteration from baseline that diminishes an organism's ability to survive, reproduce or adapt to the environment.

Dose: is the amount of test substance administered. Dose is expressed as weight (g, mg) or as weight of test substance per unit weight of the test animal (e.g. mg/Kg), or as constant dietary concentrations (ppm).

Dosage: is a general term comprising of dose, its frequency and the duration of dosing.

Neurotoxicity: is an adverse change in the structure or function of the nervous system that results from exposure to a chemical, biological or physical agent.

Neurotoxicant: is any chemical, biological or physical agent having the potential to cause neurotoxicity.

NOAEL: is the abbreviation for no-observed-adverse effect level and is the highest dose level where no adverse treatment-related findings are observed.

1.3.   PRINCIPLE OF THE TEST METHOD

The test chemical is administered by the oral route across a range of doses to several groups of laboratory rodents. Repeated doses are normally required, and the dosing regimen may be 28 days, subchronic (90 days) or chronic (1 year or longer). The procedures set out in this test method may also be used for an acute neurotoxicity study. The animals are tested to allow the detection or the characterisation of behavioural and/or neurological abnormalities. A range of behaviours that could be affected by neurotoxicants is assessed during each observation period. At the end of the test, a subset of animals of each sex from each group are perfused in situ and sections of the brain, spinal cord, and peripheral nerves are prepared and examined.

When the study is conducted as a stand-alone study to screen for neurotoxicity or to characterise neurotoxic effects, the animals in each group not used for perfusion and subsequent histopathology (see Table 1) can be used for specific neurobehavioural, neuropathological, neurochemical or electrophysiological procedures that may supplement the data obtained from the standard examinations required by this method (1). These supplemental procedures can be particularly useful when empirical observations or anticipated effects indicate a specific type or target of a chemical's neurotoxicity. Alternatively, the remaining animals can be used for evaluations such as those called for in test methods for repeated dose toxicity studies in rodents.

When the procedures of this test method are combined with those of other test methods, a sufficient number of animals is needed to satisfy the requirements for the observations of both studies.

1.4.   DESCRIPTION OF THE TEST METHOD

1.4.1.   Selection of animal species

The preferred rodent species is the rat, although other rodent species, with justification, may be used. Commonly used laboratory strains of young adult healthy animals should be employed. The females should be nulliparous and non-pregnant. Dosing should normally begin as soon as possible after weaning, preferably not later than when animals are six weeks, and, in any case, before the animals are nine weeks age. However, when this study is combined with other studies this age requirement may need adjustment. At the commencement of the study the weight variation of animals used should not exceed ± 20 % of the mean weight of each sex. Where a repeated dose study of short duration is conducted as a preliminary to a long term study, animals from the same strain and source should be used in both studies.

1.4.2.   Housing and feeding conditions

The temperature in the experimental animal room should be 22 °C (± 3 °C). 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. Loud intermittent noise should be kept to a minimum. For feeding, conventional laboratory diets may be used with an unlimited supply of drinking water. The choice of diet may be influenced by the need to ensure a suitable admixture of a test substance when administered by this method. Animals may be housed individually, or be caged in small groups of the same sex.

1.4.3.   Preparation of animals

Healthy young animals are randomly assigned to the treatment and control groups. Cages should be arranged in such a way that possible effects due to cage placement are minimised. The animals are identified uniquely and kept in their cages for at least (5) five days prior the start of the study to allow for acclimatisation to the laboratory conditions.

1.4.4.   Route of administration and preparation of doses

This test method specifically addresses the oral administration of the test substance. Oral administration may be by gavage, in the diet, in drinking water or by capsules. Other routes of administration (e.g. dermal or inhalation) can be used but may require modification of the procedures recommended. Considerations of the choice of the route of administration depend on the human exposure profile and available toxicological or kinetic information. The rationale for choosing the route of administration as well as resulting modifications to the procedures of this test method should be indicated.

Where necessary, the test substance may be dissolved or suspended in a suitable vehicle. It is recommended that the use of an aqueous solution/suspension be considered first, followed by consideration of a solution/suspension in oil (e.g. corn oil) and then by possible solution/suspension in other vehicle. The toxic characteristics of the vehicle must be known. In addition, consideration should be given to the following characteristics of the vehicle: effects of the vehicle on absorption, distribution, metabolism, or retention of the test substance which may alter its toxic characteristics; and effects on the food or water consumption or the nutritional status of the animals.

1.5.   PROCEDURES

1.5.1.   Number and sex animals

When the study is conducted as a separate study, at least 20 animals (10 females and 10 males) should be used in each dose and control group for the evaluation of detailed clinical and functional observations. At least five males and five females, selected from these 10 males and 10 females, should be perfused in situ and used for detailed neurohistopathology at the end of the study. In cases where only a limited number of animals in a given dose group are observed for signs of neurotoxic effects, consideration should be given to the inclusion of these animals in those selected for perfusion. When the study is conducted in combination with a repeated dose toxicity study, adequate numbers of animals should be used to meet the objectives of both studies. The minimum numbers of animals per group for various combinations of studies are given in Table 1. If interim kills or recovery groups for observation of reversibility, persistence or delayed occurrence of toxic effects post treatment are planned or when supplemental observations are considered, then the number of animals should be increased to ensure that the number of animals required for observation and histopathology are available.

1.5.2.   Treatment and control group

At least three dose groups and a control group should generally be used, but if from the assessment of other data, no effects would be expected at a repeated dose of 1 000 mg/kg body weight/day, a limit test may be performed. If there are no suitable data available, a range finding study may be performed to aid in the determination of the doses to be used. Except for treatment with the test substance, animals in the control group should be handled in an identical manner to the test group subjects. If a vehicle is used in administering the test substance, the control group should receive the vehicle at the highest volume used.

1.5.3.   Reliability check

The laboratory performing the study should present data demonstrating its capability to carry out the study and the sensitivity of the procedures used. Such data should provide evidence of the ability to detect and quantify, as appropriate, changes in the different end points recommended for observation, such as autonomic signs, sensory reactivity, limb grip strength and motor activity. Information on chemicals that cause different types of neurotoxic responses and could be used as positive control substances can be found in references 2 to 9. Historical data may be used if the essential aspects of the experimental procedures remain the same. Periodic updating of historical data is recommended. New data that demonstrate the continuing sensitivity of the procedures should be developed when some essential element of the conduct of the test or procedures has been changed by the performing laboratory.

1.5.4.   Dose selection

Dose levels should be selected by taking into account any previously observed toxicity and kinetic data available for the test compound or related materials. The highest dose level should be chosen with the aim of inducing neurotoxic effects or clear systemic toxic effects. Thereafter, a descending sequence of dose levels should be selected with a view to demonstrating any dose-related response and no-observed-adverse effect (NOAEL) at the lowest dose level. In principle, dose levels should be set so that primary toxic effects on the nervous system can be distinguished from effects related to systemic toxicity. Two to three intervals are frequently optimum and addition of a fourth test group is often preferable to using very large intervals (e.g. more than a factor of 10) between dosages. Where there is a reasonable estimation of human exposure this should also be taken into account.

1.5.5.   Limit test

If a study at one dose level of at least 1 000 mg/kg body weight/day, using the procedures described, produces no observable neurotoxic effects and if toxicity would not be expected based upon data from structurally related compounds, then a full study using three dose levels may not be considered necessary. Expected human exposure may indicate the need for a higher oral dose level to be used in the limit test. For other types of administration, such as inhalation or dermal application, the physical chemical properties of the test substance often may dictate the maximum attainable level of exposure. For the conduct of an oral acute study, the dose for a limit test should be at least 2 000 mg/kg.

1.5.6.   Administration of doses

The animals are dosed with the test substance daily, seven days each week, for a period at least 28 days; use of a five-day dosing regime or a shorter exposure period needs to be justified. When the test substance is administered by gavage, this should be done in a single dose using a stomach tube or a suitable intubation cannula. The maximum volume of a liquid that can be administered at one time depends on the size of the test animals. The volume should not exceed 1 ml/100 g body weight. However in the case of aqueous solutions, the use of up to 2 ml/100 g body weight can be considered. Except for irritating or corrosive substances, which will normally reveal exacerbated effects with higher concentrations, variability in test volume should be minimised by adjusting the concentration to ensure a constant volume at all dose levels.

For substances administered via the diet or drinking water, it is important to ensure that the quantities of the test substance involved do not interfere with normal nutrition or water balance. When the test substance is administered in the diet either a constant dietary concentration (ppm) or a constant dose level in terms of the animals' body weight may be used; the alternative used must be specified. For a substance administered by gavage, the dose should be given at similar times each day, and adjusted as necessary to maintain a constant dose level in terms of animal body weight. Where a repeat dose study is used as a preliminary to a long term study, a similar diet should be used in both studies. For acute studies, if a single dose is not possible, the dose may be given in smaller fractions over a period not exceeding 24 hours.

1.6.   OBSERVATION

1.6.1.   Frequency of observations and tests

In repeated dose studies, the observation period should cover the dosage period. In acute studies, 14-day post-treatment period should be observed. For animals in satellite groups which are kept without exposure during a post-treatment period, observations should cover this period as well.

Observations should be made with sufficient frequency to maximise the probability of detection of any behavioural and/or neurological abnormalities. Observations should be made preferably at the same times each day with consideration given to the peak period of anticipated effects after dosing. The frequency of clinical observations and functional tests is summarised in Table 2. If kinetic or other data generated from previous studies indicates the need to use different time points for observations, tests or post-observation periods, an alternative schedule should be adopted in order to achieve maximum information. The rationale for changes to the schedule should be provided.

1.6.1.1.   Observations of general health condition and mortality/morbidity

All animals should be carefully observed at least once daily with respect to their health condition as well as at least twice daily for morbidity and mortality.

1.6.1.2.   Detailed clinical observations

Detailed clinical observations should be made on all animals selected for this purpose (see Table 1) once before the first exposure (to allow for within-subject comparisons) and at different intervals thereafter, dependant on the duration of the study (see Table 2). Detailed clinical observations on satellite recovery groups should be made at the end of the recovery period. Detailed clinical observations should be made outside the home cage in a standard arena. They should be carefully recorded using scoring systems that include criteria or scoring scales for each measurement in the observations. The criteria or scales used should be explicitly defined by the testing laboratory. Effort should be made to ensure that variations in the test conditions are minimal (not systematically related to treatment) and that observations are conducted by trained observers unaware of the actual treatment.

It is recommended that the observations be carried out in a structured fashion in which well-defined criteria (including the definition of the normal ‘range’) are systematically applied to each animal at each observation time. The ‘normal range’ should be adequately documented. All observed signs should be recorded. Whenever feasible, the magnitude of the observed signs should also be recorded. Clinical observations should include, but not be limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern and/or mouth breathing, any unusual signs of urination or defecation, and discoloured urine).

Any unusual responses with respect to body position, activity level (e.g. decreased or increased exploration of the standard arena) and co-ordination of movement should also be noted. Changes in gait (e.g. waddling, ataxia), posture (e.g. hunched-back) and reactivity to handling, placing or other environmental stimuli, as well as the presence of clonic or tonic movements, convulsions or tremors, stereotypes (e.g. excessive grooming, unusual head movements, repetitive circling) or bizarre behaviour (e.g. biting or excessive licking, self mutilation, walking backwards, vocalisation) or aggression should be recorded.

1.6.1.3.   Functional tests

Similar to the detailed clinical observations, functional tests should also be conducted once prior to exposure and frequently thereafter in all animals selected for this purpose (see Table 1). The frequency of functional testing is also dependent on the study duration (see Table 2). In addition to the observation periods as set out in Table 2, functional observations on satellite recovery groups should also be made as close as possible to the terminal kill. Functional tests should include sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli (5)(6)(7)), assessment of limb grip strength (8) and assessment of motor activity (9). Motor activity should be measured with an automated device capable of detecting both decreases and increases in activity. If another defined system is used it should be quantitative and its sensitivity and reliability should be demonstrated. Each device should be tested to ensure reliability across time and consistency between devices. Further details of the procedures that can be followed are given in the respective references. If there are no data (e.g. structure-activity, epidemiological data, other toxicology studies) to indicate the potential neurotoxic effects, the inclusion of more specialised tests of sensory and motor function or learning and memory to examine these possible effects in greater details should be considered. More information on more specialised tests and their use is provided in (1).

Exceptionally, animals that reveal signs of toxicity to an extent that would significantly interfere with the functional test may be omitted from that test. Justification for the elimination of animals from a functional test should be provided.

1.6.2.   Body weight and food/water consumption

For studies up to 90 days duration, all animals should be weighed at least once a week and measurements should be made of food consumption (water consumption, when the test substance is administered by that medium) at least weekly. For long term studies, all animals should be weighed at least once at week for the first 13 weeks and at least once every four weeks thereafter. Measurements should be made of food consumption (water consumption, when the test substance is administered by that medium) at least weekly for the first 13 weeks and then at approximately three-month intervals unless the health status or body weight changes dictate otherwise.

1.6.3.   Ophthalmology

For studies longer than 28 days duration, ophthalmologic examination, using an ophthalmoscope or an equivalent suitable instrument, should be made prior to the administration of the test substance and at the termination of the study, preferably on all animals, but at least on animals in the high dose and control groups. If changes in the eyes are detected or, if clinical signs indicate the need, all animals should be examined. For long term studies, an ophthalmologic examination should also be carried out at 13 weeks. Ophthalmologic examinations need not to be conducted if this data is already available from others studies of similar duration and at similar dose levels.

1.6.4.   Haematology and clinical biochemistry

When the neurotoxicity study is carried out in combination with a repeated dose systemic toxicity study, haematological examinations and clinical biochemistry determinations should be carried out as set out in the respective method of the systemic toxicity study. Collection of samples should be carried out in such a way that any potential effects on neurobehaviour are minimised.

1.6.5.   Histopathology