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Commission Regulation (EU) No 640/2012 of 6 July 2012 amending, for the purpose of its adaptation to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) (Text with EEA relevance)
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The Annex to Regulation (EC) No 440/2008 is amended as follows:
Chapter B.42 is replaced by the following:
structural and functional similarity to the class of the test substance being tested;
known physical/chemical characteristics;
supporting data from the LLNA;
supporting data from other animal models and/or from humans.
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 |
:
Individually identify and record the weight of each animal and any clinical observation. 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.
:
Repeat the application procedure carried out on Day 1.
:
No treatment.
:
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.
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.
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]
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.
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.
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.
Chamberlain, M. and Basketter, D.A. (1996), The local lymph node assay: status of validation, Food Chem. Toxicol., 34, 999-1002.
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.
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.
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.
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.
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.
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.
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]
OECD (1992), Skin Sensitisation. OECD Guideline for Testing of Chemicals No 406, OECD, Paris. Available at: [http://www.oecd.org/env/testguidelines]
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]
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.
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.
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]
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/]
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]
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.
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.
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/]
ILAR (1996), Institute of Laboratory Animal Research (ILAR) Guide for the Care and Use of Laboratory Animals, 7th ed. Washington, DC: National Academies Press.
McGarry, H.F. (2007), The murine local lymph node assay: regulatory and potency considerations under REACH, Toxicol., 238, 71-89.
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]
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.
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]
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.
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.
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.
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.
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.
Vohr, H.W. and Ahr, H.J. (2005), The local lymph node assay being too sensitive? Arch. Toxicol., 79, 721-728.
Patterson, R.M., Noga, E. and Germolec, D. (2007), Lack of evidence for contact sensitisation by Pfiesteria extract, Environ. Health Perspect., 115, 1023-1028.
OECD (1987), Acute Dermal Toxicity, OECD Guideline for Testing of Chemicals No 402, Paris, France. Available at: [http://www.oecd.org/env/testguidelines]
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]
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]
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.
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. | ||||||||||
a 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. | ||||||||||
b GP data not available. | ||||||||||
c Chemicals should be prepared daily unless stability data demonstrate the acceptability of storage. | ||||||||||
d 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). | ||||||||||
e 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). | ||||||||||
f Number of LLNA studies from which data were obtained. | ||||||||||
g 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). | ||||||||||
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. | ||||||||||
Number | Chemicalsc | CAS No | Form | Vehd | EC3 %e | Nf | 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)g | 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 | –/– | –/a |
15 | Isopropanol | 67-63-0 | Liq | AOO | 50 | 1 | NA | NA | –/– | –/+ |
16 | Lactic acid | 50-21-5 | Liq | DMSO | 25 | 1 | NA | NA | –/– | –/a |
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 | ||||||||||
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 | +/b | +/– |
22 | Nickel chloride | 7718-54-9 | Sol | DMSO | 5 | 2 | NA | NA | –/+ | –/+ |
:
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).
:
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 of a test substance needed to produce a stimulation index that is indicative of a positive response.
:
Estimated concentration of a test substance needed to produce a stimulation index of three.
:
A test substance incorrectly identified as negative or non-active by a test method, when in fact it is positive or active.
:
A test substance incorrectly identified as positive or active by a test, when in fact it is negative or non-active.
:
The potential for an adverse health or ecological effect. The adverse effect is manifested only if there is an exposure of sufficient level.
:
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).
:
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).
:
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).
:
An outlier is an observation that is markedly different from other values in a random sample from a population.
:
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).
:
A test method for which manufacture and distribution is restricted by patents, copyrights, trademarks, etc.
:
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.
:
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).
:
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).
:
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).
:
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.
:
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.
:
Any substance or mixture tested using this TM.
:
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).’
Chapter B.46 is replaced by the following:
Table 1 | ||||
Reference Chemicals a | ||||
a These Reference Chemicals are a subset of the Reference Chemicals used in the validation study. | ||||
b In vivo score in accordance with B.4 and OECD Test Guideline 404 (4). | ||||
c Under this Test Method, the UN GHS optional Category 3 (mild irritants) (1) is considered as No Category. | ||||
d The UN GHS optional Category3 is not applicable under the EU CLP. | ||||
Chemical | CAS NR | In vivo scoreb | Physical state | UN GHS/EU CLP Category |
---|---|---|---|---|
naphthalene acetic acid | 86-87-3 | 0 | Solid | No Cat. |
isopropanol | 67-63-0 | 0,3 | Liquid | No Cat. |
methyl stearate | 112-61-8 | 1 | Solid | No Cat. |
heptyl butyrate | 5870-93-9 | 1,7 | Liquid | No Cat. |
hexyl salicylate | 6259-76-3 | 2 | Liquid | No Cat. |
cyclamen aldehyde | 103-95-7 | 2,3 | Liquid | Cat. 2 |
1-bromohexane | 111-25-1 | 2,7 | Liquid | Cat. 2 |
potassium hydroxide (5 % aq.) | 1310-58-3 | 3 | Liquid | Cat. 2 |
1-methyl-3-phenyl-1-piperazine | 5271-27-2 | 3,3 | Solid | Cat. 2 |
Heptanal | 111-71-7 | 3,4 | Liquid | Cat. 2 |
Table 2 | ||
Acceptability ranges for negative control OD values | ||
Lower acceptance limit | Upper acceptance limit | |
---|---|---|
EpiSkinTM (SM) | ≥ 0,6 | ≤ 1,5 |
EpiDermTM SIT (EPI-200) | ≥ 1,0 | ≤ 2,5 |
SkinEthicTM RHE | ≥ 1,2 | ≤ 2,5 |
Table 3 | ||
Examples of QC batch release criteria | ||
Lower acceptance limit | Upper acceptance limit | |
---|---|---|
EpiSkinTM (SM)(18 hours treatment with SDS) (26) | IC50 = 1,0 mg/ml | IC50 = 3,0 mg/ml |
EpiDermTM SIT (EPI-200)(1 % Triton X-100) (27) | ET50 = 4,8 hr | ET50 = 8,7 hr |
SkinEthicTM RHE(1 % Triton X-100) (28) | ET50 = 4,0 hr | ET50 = 9,0 hr |
the test chemical is considered to be irritant to skin in accordance with UN GHS/EU CLP Category 2 if the tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50 %.
depending on the regulatory framework in which the results of this TM are used, the test chemical may be considered to be non-irritant to skin in accordance with UN GHS/EU CLP No Category if the tissue viability after exposure and post-treatment incubation is more than (>) 50 %.
Test and Control Chemicals:
Chemical name(s) such as CAS name and number, EC name and number, if known;
Purity and composition of the chemical (in percentage(s) by weight);
Physical/chemical properties relevant to the conduct of the study (e.g. physical state, stability, volatility, pH and water solubility if known);
Treatment of the test/control chemicals prior to testing, if applicable (e.g. warming, grinding);
Storage conditions;
Justification of the RhE model and protocol used
Test Conditions:
Cell system used;
Complete supporting information for the specific RhE model used including its performance. This should include, but is not limited to;
viability
barrier function
morphology
reproducibility and predictivity
Quality controls (QC) of the model
Details of the test procedure used;
Test doses used, duration of exposure and post treatment incubation period;
Description of any modifications of the test procedure;
Reference to historical data of the model. This should include, but is not limited to:
acceptability of the QC data with reference to historical batch data
acceptability of the positive and negative control values with reference to positive and negative control means and ranges
Description of evaluation criteria used including the justification for the selection of the cut-off point(s) for the prediction model;
Reference to historical control data;
Results:
Tabulation of data from individual test chemicals for each run and each replicate measurement;
Indication of controls used for direct MTT-reducers and/or colouring test chemicals;
Description of other effects observed;
Discussion of the results
Conclusion
UN (2009), United Nations Globally Harmonised System of Classification and Labelling of Chemicals (GHS), Third revised edition, UN New York and Geneva. Available at: [http://www.unece.org/trans/danger/publi/ghs/ghs_rev03/03files_e.html]
EC-ECVAM (2009), Statement on the “Performance under UN GHS of three in vitro assays for skin irritation testing and the adaptation of the Reference Chemicals and Defined Accuracy Values of the ECVAM skin irritation Performance Standards”, issued by the ECVAM Scientific Advisory Committee (ESAC30), 9 April 2009. Available at: [http://ecvam.jrc.ec.europa.eu]
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.
OECD (2004), Acute Dermal Irritation/Corrosion, OECD Guideline for the Testing of Chemicals No 404, OECD, Paris. Available at: [http://www.oecd.org/env/testguidelines]
OECD (2004), In Vitro Skin Corrosion: Transcutaneous Electrical Resistance (TER), OECD Guideline for the Testing of Chemicals No 430, OECD, Paris. Available at: [http://www.oecd.org/env/testguidelines]
OECD (2004), In Vitro Skin Corrosion: Human Skin Model Test, OECD Guideline for the Testing of Chemicals No 431, OECD, Paris. Available at: [http://www.oecd.org/env/testguidelines]
OECD (2006), In Vitro Membrane Barrier Test Method for Skin Corrosion, OECD Guideline for the Testing of Chemicals No 435, OECD, Paris. Available at: [http://www.oecd.org/env/testguidelines]
EC-ECVAM (2009), Performance Standards for in vitro skin irritation test methods based on Reconstructed human Epidermis (RhE)? Available at: [http://ecvam.jrc.ec.europa.eu]
OECD (2005), Guidance Document on the Validation and International Acceptance of New or Updated Test Methods for Hazard Assessment, OECD Series on Testing and Assessment No 34, OECD, Paris. Available at: [http://www.oecd.org/env/testguidelines]
Fentem, J.H., Briggs, D., Chesné, C., Elliot, G.R., Harbell, J.W., Heylings, J.R., Portes, P., Roguet, R., van de Sandt, J.J. M. and Botham, P. (2001), A prevalidation study on in vitro tests for acute skin irritation, Results and evaluation by the Management Team, Toxicol. in Vitro 15, 57-93.
Portes, P., Grandidier, M.-H., Cohen, C. and Roguet, R. (2002), Refinement of the EPISKIN protocol for the assessment of acute skin irritation of chemicals: follow-up to the ECVAM prevalidation study, Toxicol. in Vitro 16, 765-770.
Kandárová, H., Liebsch, M., Genschow, E., Gerner, I., Traue, D., Slawik, B. and Spielmann, H. (2004), Optimisation of the EpiDerm test protocol for the upcoming ECVAM validation study on in vitro skin irritation tests, ALTEX 21, 107-114.
Kandárová, H., Liebsch, M., Gerner, I., Schmidt, E., Genschow, E., Traue, D. and Spielmann, H. (2005), The EpiDerm test protocol for the upcoming ECVAM validation study on in vitro skin irritation tests — An assessment of the performance of the optimised test, ATLA 33, 351-367.
Cotovio, J., Grandidier, M.-H., Portes, P., Roguet, R. and Rubinsteen, G. (2005), The in vitro acute skin irritation of chemicals: optimisation of the EPISKIN prediction model within the framework of the ECVAM validation process, ATLA 33, 329-349.
Zuang, V., Balls, M., Botham, P.A., Coquette, A., Corsini, E., Curren, R.D., Elliot, G.R., Fentem, J.H., Heylings, J.R., Liebsch, M., Medina, J., Roguet, R., van De Sandt, J.J.M., Wiemann, C. and Worth, A. (2002), Follow-up to the ECVAM prevalidation study on in vitro tests for acute skin irritation, The European Centre for the Validation of Alternative Methods Skin Irritation Task Force report 2, ATLA 30, 109-129.
Spielmann, H., mailto:Hoffmann, S., Liebsch, M., Botham, P., Fentem, J., Eskes, C., Roguet, R., Cotovio, J., Cole, T., Worth, A., Heylings, J., Jones, P., Robles, C., Kandárová, H., mailto:Gamer, A., Remmele, M., Curren, R., Raabe, H., Cockshott, A., Gerner, I. and Zuang, V. (2007), The ECVAM international validation study on in vitro tests for acute skin irritation: Report on the validity of the EPISKIN and EpiDerm assays and on the skin integrity function test, ATLA 35, 559-601.
Hoffmann, S. (2006), ECVAM skin irritation validation study phase II: Analysis of the primary endpoint MTT and the secondary endpoint IL1-α. Available at: [http://ecvam.jrc.ec.europa.eu]
Eskes, C., Cole, T., Hoffmann, S., Worth, A., Cockshott, A., Gerner, I. and Zuang, V. (2007), The ECVAM international validation study on in vitro tests for acute skin irritation: selection of test chemicals, ATLA 35, 603-619.
Cotovio, J., Grandidier, M.-H., Lelièvre, D., Roguet, R., Tinois-Tessonneaud, E. and Leclaire, J. (2007), In vitro acute skin irritancy of chemicals using the validated EPISKIN model in a tiered strategy — Results and performances with 184 cosmetic ingredients, AATEX, 14, 351-358.
EC-ECVAM (2007), Statement on the validity of in vitro tests for skin irritation, issued by the ECVAM Scientific Advisory Committee (ESAC26), 27 April 2007. Available at: [http://ecvam.jrc.ec.europa.eu]
EC-ECVAM (2007), Performance Standards for applying human skin models to in vitro skin irritation testing. Available at: [http://ecvam.jrc.ec.europa.eu]
EC-ECVAM (2008), Statement on the scientific validity of in vitro tests for skin irritation testing, issued by the ECVAM Scientific Advisory Committee (ESAC29), 5 November 2008. Available at: [http://ecvam.jrc.ec.europa.eu]
OECD (2010), Explanatory background document to the OECD draft Test Guideline on in vitro skin irritation testing. OECD Series on Testing and Assessment, No 137, OECD, Paris. Available at: [http://www.oecd.org/document/24/0,3746,en_2649_34377_47858904_1_1_1_1,00.html]
Welss, T., Basketter, D.A. and Schröder, K.R. (2004), In vitro skin irritation: fact and future. State of the art review of mechanisms and models, Toxicol. in Vitro 18, 231-243.
Mosmann, T. (1983), Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays, J. Immunol. Methods 65, 55-63.
EpiSkinTM SOP, Version 1.8 (February 2009), ECVAM Skin Irritation Validation Study: Validation of the EpiSkinTM test method 15 min-42 hours for the prediction of acute skin irritation of chemicals. Available at: [http://ecvam.jrc.ec.europa.eu]
EpiDermTM SOP, Version 7.0 (Revised March 2009), Protocol for: In vitro EpiDermTM skin irritation test (EPI-200-SIT), For use with MatTek Corporation’s reconstructed human epidermal model EpiDerm (EPI-200). Available at: [http://ecvam.jrc.ec.europa.eu]
SkinEthicTM RHE SOP, Version 2.0 (February 2009), SkinEthic skin irritation test-42a test method for the prediction of acute skin irritation of chemicals: 42 minutes application + 42 hours post-incubation. Available at: [http://ecvam.jrc.ec.europa.eu]
Harvell, J.D., Lamminstausta, K., and Maibach, H.I. (1995), Irritant contact dermatitis, In: Practical Contact Dermatitis, pp 7-18, (Ed. Guin J. D.). Mc Graw-Hill, New York.
Commission Directive 2001/59/EC of 6 August 2001 adapting to technical progress for the 28th time Council Directive 67/548/EEC on the approximation of laws, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous substances, OJ L 225, 21.8.2001, p. 1.
Basketter, D.A., York, M., McFadden, J.P. and Robinson, M.K. (2004), Determination of skin irritation potential in the human 4-h patch test. Contact Dermatitis 51, 1-4.
Jirova, D., Liebsch, M., Basketter, D., Spiller, E., Kejlova, K., Bendova, H., Marriott, M. and Kandarova, H. (2007), Comparison of human skin irritation and photo-irritation patch test data with cellular in vitro assays and animal in vivo data, ALTEX, 14, 359-365.
Jírová, D., Basketter, D., Liebsch, M., Bendová, H., Kejlová, K., Marriott, M. and Kandárová, H. (2010), Comparison of human skin irritation patch test data with in vitro skin irritation assays and animal data, Contact Dermatitis, 62, 109-116.
:
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 (9).
:
Parameter measuring total activity of a cell population e.g. as ability of cellular mitochondrial dehydrogenases to reduce the vital dye MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue), which depending on the endpoint measured and the test design used, correlates with the total number and/or vitality of living cells.
:
This is a measure of test method performance for test methods that give a categorical result, and is one aspect of relevance. The term is used interchangeably with accuracy, and is defined as the proportion of all chemicals tested that are correctly classified as positive or negative. (9).
:
Can be estimated by determination of the exposure time required to reduce cell viability by 50 % upon application of the marker chemical at a specified, fixed concentration, see also IC50.
:
Implements in the European Union (EU) the UN GHS system for the classification and labelling of chemicals (substances and mixtures) (3).
:
A system proposing the classification of chemicals (substances and mixtures) according to standardised types and levels of physical, health and environmental hazards, and addressing corresponding communication elements, such as pictograms, signal words, hazard statements, precautionary statements and safety data sheets, so that to convey information on their adverse effects with a view to protect people (including employers, workers, transporters, consumers and emergency responders) and the environment (1).
:
Can be estimated by determination of the concentration at which a marker chemical reduces the viability of the tissues by 50 % (IC50) after a fixed exposure time, see also ET50.
:
Amount of test chemical applied to the epidermis exceeding the amount required to completely and uniformly cover the epidermis surface.
:
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 (9).
:
Standards, based on a validated test method, that provide a basis for evaluating the comparability of a proposed test method that is mechanistically and functionally 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 comparable 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 (9).
:
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 (9).
:
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 (9).
:
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 (9).
:
A test which is designed to substitute for a test that is in routine use and accepted for hazard identification and/or risk assessment, and which has been determined to provide equivalent or improved protection of human or animal health or the environment, as applicable, compared to the accepted test, for all possible testing situations and chemicals (9).
:
The proportion of all positive/active test chemicals that are correctly classified by the test. It is a measure of accuracy for a test method that produces categorical results, and is an important consideration in assessing the relevance of a test method (9).
:
The production of reversible damage to the skin following the application of a test chemical for up to 4 hours. Skin irritation is a locally arising, non-immunogenic reaction, which appears shortly after stimulation (29). Its main characteristic is its reversible nature involving inflammatory reactions and most of the clinical characteristic signs of irritation (erythema, oedema, itching and pain) related to an inflammatory process.
:
The proportion of all negative/inactive test chemicals that are correctly classified by the test. It is a measure of accuracy for a test method that produces categorical results and is an important consideration in assessing the relevance of a test method (9).
:
Testing which uses test methods in a sequential manner; the test methods selected in each succeeding level are decided based on the results in the previous level of testing (9).
:
Any substance or mixture tested using this TM.
Essential Test Method Components
Minimum List of Reference Chemicals
Defined Reliability and Accuracy Values
The general conditions (paragraph 16)
The functional conditions, which include:
viability (paragraph 17);
barrier function (paragraph 18);
morphology (paragraph 19);
reproducibility (paragraph 20); and,
quality control (paragraph 21)
Table 1 | |||||
Minimum List of Reference Chemicals for Determination of Accuracy and Reliability Values for Similar or Modified RhE Skin Irritation Methods a | |||||
a The chemical selection is based on the following criteria: (i) the chemicals are commercially available; (ii) they are representative of the full range of Draize irritancy scores (from non-irritant to strong irritant); (iii) they have a well-defined chemical structure; (iv) they are representative of the chemical functionality used in the validation process; and (v) they are not associated with an extremely toxic profile (e.g. carcinogenic or toxic to the reproductive system) and they are not associated with prohibitive disposal costs. | |||||
b Chemicals that are irritant in the rabbit but for which there is reliable evidence that they are non-irritant in humans (31) (32) (33). | |||||
c Under the UN GHS, not in the EU CLP | |||||
Chemical | CAS Number | Physical state | In vivo score | VRM in vitro Cat. | UN GHS/EU CLP in vivo Cat. |
---|---|---|---|---|---|
1-bromo-4-chlorobutane | 6940-78-9 | Liquid | 0 | Cat. 2 | No Cat. |
diethyl phthalate | 84-66-2 | Liquid | 0 | No Cat. | No Cat. |
naphthalene acetic acid | 86-87-3 | Solid | 0 | No Cat. | No Cat. |
allyl phenoxy-acetate | 7493-74-5 | Liquid | 0,3 | No Cat. | No Cat. |
isopropanol | 67-63-0 | Liquid | 0,3 | No Cat. | No Cat. |
4-methyl-thio-benzaldehyde | 3446-89-7 | Liquid | 1 | Cat. 2 | No Cat. |
methyl stearate | 112-61-8 | Solid | 1 | No Cat. | No Cat. |
heptyl butyrate | 5870-93-9 | Liquid | 1,7 | No Cat. | No Cat. |
hexyl salicylate | 6259-76-3 | Liquid | 2 | No Cat. | No Cat. |
Cinnamaldehyde | 104-55-2 | Liquid | 2 | Cat. 2 | No Cat. (Optional Cat. 3)c |
1-decanol b | 112-30-1 | Liquid | 2,3 | Cat. 2 | Cat. 2 |
cyclamen aldehyde | 103-95-7 | Liquid | 2,3 | Cat. 2 | Cat. 2 |
1-bromohexane | 111-25-1 | Liquid | 2,7 | Cat. 2 | Cat. 2 |
2-chloromethyl-3,5-dimethyl-4-methoxypyridine HCl | 86604-75-3 | Solid | 2,7 | Cat. 2 | Cat. 2 |
di-n-propyl disulphide b | 629-19-6 | Liquid | 3 | No Cat. | Cat. 2 |
potassium hydroxide (5 % aq.) | 1310-58-3 | Liquid | 3 | Cat. 2 | Cat. 2 |
benzenethiol, 5-(1,1-dimethylethyl)-2-methyl | 7340-90-1 | Liquid | 3,3 | Cat. 2 | Cat. 2 |
1-methyl-3-phenyl-1-piperazine | 5271-27-2 | Solid | 3,3 | Cat. 2 | Cat. 2 |
Heptanal | 111-71-7 | Liquid | 3,4 | Cat. 2 | Cat. 2 |
Tetrachloroethylene | 127-18-4 | Liquid | 4 | Cat. 2 | Cat. 2 |
Only the data of runs from complete run sequences qualify for the calculation of the method within, and between-laboratory variability and predictive capacity (accuracy).
The final classification for each Reference Chemicals in each participating laboratory should be obtained by using the mean value of viability over the different runs of a complete run sequence.
Only the data obtained for chemicals that have complete run sequences in all participating laboratories qualify for the calculation of the method between-laboratory variability.
The calculation of the accuracy values should be done on the basis of the individual laboratory predictions obtained for the 20 Reference Chemicals by the different participating laboratories.
In this context, a run sequence consists of three independent runs from one laboratory for one test chemical. A complete run sequence is a run sequence from one laboratory for one test chemical where all three runs are valid. This means that any single invalid run invalidates an entire run sequence of three runs.
Table 2 | ||
Required predictive values for sensitivity, specificity and overall accuracy for any similar or modified method to be considered valid | ||
Sensitivity | Specificity | Overall Accuracy |
---|---|---|
≥ 80 % | ≥ 70 % | ≥ 75 % |
All 20 Reference Chemicals should have at least one complete run sequence.
In each of at least three participating laboratories, a minimum of 85 % of the run sequences need to be complete (for 20 chemicals; i.e. 3 invalid run sequences are allowed in a single laboratory).
A minimum of 90 % of all possible run sequences from at least three laboratories need to be complete (for 20 chemicals tested in 3 laboratories; i.e. 6 invalid run sequences are allowed in total).’
The following Chapters are added:
Table 1 | ||
Cell treatment and harvest times for the MNvit assay | ||
Lymphocytes, primary cells and cell lines treated with cytoB | + S9 | Treat for 3-6 hrs in the presence of S9; remove the S9 and treatment medium; add fresh medium and cytoB; harvest 1,5-2,0 normal cell cycles later. |
– S9 Short exposure | Treat for 3-6 hrs; remove the treatment medium; add fresh medium and cytoB; harvest 1,5-2,0 normal cell cycles later. | |
– S9 Extended exposure | Option A: Treat for 1,5-2 normal cell cycles in the presence of cytoB; harvest at the end of the exposure period. Option B: Treat for 1,5-2,0 normal cell cycles; remove the test substance; add fresh medium and cytoB; harvest 1,5-2,0 normal cell cycles later. | |
Cell lines treated without cytoB (Identical to the treatment schedules outlined above with the exception that no cytoB is added) |
Option A: The cells are treated with the test substance for 1,5-2,0 normal cell cycles, and harvested at the end of the treatment time.
Option B: The cells are treated with the test substance for 1,5-2,0 normal cell cycles. The treatment medium is removed and replaced with fresh medium, and the cells are harvested after additional 1,5-2,0 normal cell cycles.
Option A: The cells are treated with the test substance for 1,5-2,0 normal cell cycles, and harvested at the end of the treatment time.
Option B: The cells are treated with the test substance for 1,5-2,0 normal cell cycles. The treatment medium is removed and replaced with fresh medium, and the cells are harvested after additional 1,5-2,0 normal cell cycles.
Test chemical:
identification data and Chemical Abstract Services Registry Number and EC Number;
physical nature and purity;
physico-chemical properties relevant to the conduct of the study;
reactivity of the test chemical with the solvent/vehicle or cell culture media;
Solvent/Vehicle:
justification for choice of solvent/vehicle;
solubility and stability of the test substance in solvent/vehicle;
Cells:
type and source of cells used;
suitability of the cell type used;
absence of mycoplasma, if applicable;
information on cell cycle length, doubling time or proliferation index;
where lymphocytes are used, sex, age and number of blood donors, if applicable;
where lymphocytes are used, whether whole blood or separated lymphocytes are exposed;
number of passages, if applicable;
methods for maintenance of cell cultures, if applicable;
modal number of chromosomes;
normal (negative control) cell cycle time;
Test Conditions:
identity of cytokinesis blocking substance (e.g. cytoB), if used, and its concentration and duration of cell exposure;
rationale for selection of concentrations and number of cultures, including cytotoxicity data and solubility limitations, if available;
composition of media, CO2 concentration, if applicable;
concentrations of test substance;
concentration (and/or volume) of vehicle and test substance added;
incubation temperature and time;
duration of treatment;
harvest time after treatment;
cell density at seeding, if applicable;
type and composition of metabolic activation system, including acceptability criteria;
positive control chemicals and negative controls;
methods of slide preparation and staining technique used;
criteria for micronucleus identification;
numbers of cells analysed;
methods for the measurements of cytotoxicity;
any supplementary information relevant to cytotoxicity;
criteria for considering studies as positive, negative, or equivocal;
method(s) of statistical analysis used;
methods, such as use of kinetochore antibody, to characterise whether micronuclei contain whole or fragmented chromosomes, if applicable;
Results:
measurement of cytotoxicity used, e.g. CBPI or RI in the case of cytokinesis-block method; RICC, RPD or PI when cytokinesis-block methods are not used; other observations when applicable, e.g. cell confluency, apoptosis, necrosis, metaphase counting, frequency of binucleated cells;
signs of precipitation;
data on pH and osmolality of the treatment medium, if determined;
definition of acceptable cells for analysis;
distribution of mono-, bi-, and multi-nucleated cells if a cytokinesis block method is used;
number of cells with micronuclei given separately for each treated and control culture, and defining whether from binucleate or mononucleate cells, where appropriate;
concentration-response relationship, where possible;
concurrent negative (solvent/vehicle) and positive control chemical data (concentrations and solvents);
historical negative (solvent/vehicle) and positive control chemical data, with ranges, means and standard deviation and confidence interval (e.g. 95 %);
statistical analysis; p-values if any;
Discussion of the results
Conclusions
Kirsch-Volders, M. (1997), Towards a validation of the micronucleus test. Mutation Res., 392, 1-4.
Parry, J.M. and Sors, A. (1993), The detection and assessment of the aneugenic potential of environmental chemicals: the European Community aneuploidy project, Mutation Res., 287, 3-15.
Fenech, M. and Morley, A.A. (1985), Solutions to the kinetic problem in the micronucleus assay, Cytobios., 43, 233-246.
Kirsch-Volders, M., Sofuni, T., Aardema, M., Albertini, S., Eastmond, D., Fenech, M., Ishidate, M. Jr, Lorge, E., Norppa, H., Surralles, J., von der Hude, W. and Wakata, A. (2000), Report from the In Vitro Micronucleus Assay Working Group, Environ. Mol. Mutagen., 35, 167-172.
Fenech, M. (2007), Cytokinesis-block micronucleus cytome assay, Nature Protocols, 2(5), 1084-1104.
Fenech, M. and Morley, A.A. (1986), Cytokinesis-block micronucleus method in human lymphocytes: effect of in-vivo ageing and low dose X-irradiation, Mutation Res., 161, 193-198.
Eastmond, D.A. and Tucker, J.D. (1989), Identification of aneuploidy-inducing agents using cytokinesis-blocked human lymphocytes and an antikinetochore antibody, Environ. Mol. Mutagen., 13, 34-43.
Eastmond, D.A. and Pinkel, D. (1990), Detection of aneuploidy and aneuploidy-inducing agents in human lymphocytes using fluorescence in-situ hybridisation with chromosome-specific DNA probes, Mutation Res., 234, 9-20.
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Migliore, L., Bocciardi, R., Macri, C. and Lo Jacono, F. (1993), Cytogenetic damage induced in human lymphocytes by four vanadium compounds and micronucleus analysis by fluorescence in situ hybridization with a centromeric probe, Mutation Res., 319, 205-213.
Norppa, H., Renzi, L. and Lindholm, C. (1993), Detection of whole chromosomes in micronuclei of cytokinesis-blocked human lymphocytes by antikinetochore staining and in situ hybridization, Mutagenesis, 8, 519-525.
Eastmond, D.A, Rupa, D.S. and Hasegawa, L.S. (1994), Detection of hyperdiploidy and chromosome breakage in interphase human lymphocytes following exposure to the benzene metabolite hydroquinone using multicolor fluorescence in situ hybridization with DNA probes, Mutation Res., 322, 9-20.
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Kalweit, S., Utesch, U., von der Hude, W. and Madle, S. (1999), Chemically induced micronucleus formation in V79 cells — comparison of three different test approaches, Mutation Res. 439, 183-190.
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ESAC (2006), ECVAM Scientific Advisory Committee (ESAC) Peer Review, Retrospective Validation of the In Vitro Micronucleus Test, Summary and Conclusions of the Peer Review Panel, Available at: [http://ecvam.jrc.it/index.htm]
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Morita, T., Nagaki, T., Fukuda, I. and Okumura, K. (1992), Clastogenicity of low pH to various cultured mammalian cells, Mutation Res., 268, 297-305.
Brusick, D. (1986), Genotoxic effects in cultured mammalian cells produced by low pH treatment conditions and increased ion concentrations, Environ. Mutagen., 8, 789-886.
Fenech, M. and Morley, A.A. (1985), Measurement of micronuclei in lymphocytes, Mutation Res., 147, 29-36.
Fenech, M. (1997), The advantages and disadvantages of cytokinesis-blood micronucleus method, Mutation Res., 392, 11-18.
Bonassi, S., Fenech, M., Lando, C., Lin, Y.P., Ceppi, M., Chang, W.P., Holland, N., Kirsch-Volders, M., Zeiger, E., Ban, S., Barale, R., Bigatti, M.P., Bolognesi, C., Jia, C., Di Giorgio, M., Ferguson, L.R., Fucic, A., Lima, O.G., Hrelia, P., Krishnaja, A.P., Lee, T.K., Migliore, L., Mikhalevich, L., Mirkova, E., Mosesso, P., Muller, W.U., Odagiri, Y., Scarffi, M.R., Szabova, E., Vorobtsova, I., Vral, A. and Zijno, A. (2001), HUman MicroNucleus Project: international database comparison for results with the cytokinesis-block micronucleus assay in human lymphocytes, I. Effect of laboratory protocol, scoring criteria and host factors on the frequency of micronuclei, Environ. Mol. Mutagen. 37, 31-45.
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Regulation (EC) No 850/2004 of the European Parliament and of the Council of 29 April 2004 on persistent organic pollutants and amending Directive 79/117/EEC, OJ L 229, 30.4.2004, p. 5.
:
any substance or process that, by interacting with the components of the mitotic and meiotic cell division cycle, leads to aneuploidy in cells or organisms.
:
any deviation from the normal diploid (or haploid) number of chromosomes by a single chromosome or more than one, but not by entire set(s) of chromosomes (polyploidy).
:
programmed cell death characterised by a series of steps leading to a disintegration of cells into membrane-bound particles that are then eliminated by phagocytosis or by shedding.
:
increase in cell number as a result of mitotic cell division.
:
DNA region of a chromosome where both chromatids are held together and on which both kinetochores are attached side-to-side.
:
any substance or process which causes structural chromosomal aberrations in populations of cells or organisms.
:
the process of cell division immediately following mitosis to form two daughter cells, each containing a single nucleus.
:
the proportion of second-division cells in the treated population relative to the untreated control (see Appendix 2 for formula).
:
inhibition of cell growth (see Appendix 2 for formula).
:
harmful effects to cell structure or function ultimately causing cell death.
:
a general term encompassing all types of DNA or chromosome damage, including breaks, adducts rearrangements, mutations, chromosome aberrations, and aneuploidy. Not all types of genotoxic effects result in mutations or stable chromosome damage.
:
cells not in the mitotic stage.
:
a protein-containing structure that assembles at the centromere of a chromosome to which spindle fibres associate during cell division, allowing orderly movement of daughter chromosomes to the poles of the daughter cells.
:
small nuclei, separate from and additional to the main nuclei of cells, produced during telophase of mitosis or meiosis by lagging chromosome fragments or whole chromosomes.
:
division of the cell nucleus usually divided into prophase, prometaphase, metaphase, anaphase and telophase.
:
the ratio of cells in metaphase divided by the total number of cells observed in a population of cells; an indication of the degree of cell proliferation of that population.
:
produces a heritable change of DNA base-pair sequences(s) in genes or of the structure of chromosomes (chromosome aberrations).
:
failure of paired chromatids to disjoin and properly segregate to the developing daughter cells, resulting in daughter cells with abnormal numbers of chromosomes.
:
numerical chromosome aberrations in cells or organisms involving entire set(s) of chromosomes, as opposed to an individual chromosome or chromosomes (aneuploidy).
:
method for cytotoxicity measurement when cytoB is not used (see Appendix 2 for formula).
:
method for cytotoxicity measurement when cytoB is not used (see Appendix 2 for formula).
:
method for cytotoxicity measurement when cytoB is not used (see Appendix 2 for formula).
:
the proportion of cell division cycles completed in a treated culture, relative to the untreated control, during the exposure period and recovery (see Appendix 2 for formula).
:
Any substance or mixture tested using this TM.
% Cytostasis = 100 – 100{(CBPIT – 1) ÷ (CBPIC – 1)}
And:
=
test chemical treatment culture
=
vehicle control culture
Where:
Thus, a CBPI of 1 (all cells are mononucleate) is equivalent to 100 % cytostasis.
Cytostasis = 100 – RI
=
treated cultures
=
control cultures
where:
Population Doubling = [log (Post-treatment cell number ÷ Initial cell number)] ÷ log 2
a The reference chemicals are the recommended chemicals for use. Substitution or adding of chemicals to the list of reference chemicals can be done if their activity is known and if they induce micronuclei by the same mechanisms of action, and if they are shown to be relevant to the chemicals that will be tested using the MNvit procedure. Depending on the purpose, justification could also include a validation study employing a broad variety of substances or focused on a narrower spectrum based on the chemical class of the test substance or the mechanism of damage being studied. | |||
Category | Chemical | CAS No | EC No |
---|---|---|---|
1. Clastogens active without metabolic activation | |||
Cytosine arabinoside | 147-94-4 | 205-705-9 | |
Mitomycin C | 50-07-7 | 200-008-6 | |
2. Clastogens requiring metabolic activation | |||
Benzo(a)pyrene | 50-32-8 | 200-028-5 | |
Cyclophosphamide | 50-18-0 | 200-015-4 | |
3. Aneugens | |||
Colchicine | 64-86-8 | 200-598-5 | |
Vinblastine | 143-67-9 | 205-606-0 | |
4. Negative substances | |||
Di(2-ethylhexyl)phthalate | 117-81-7 | 204-211-0 | |
Nalidixic acid | 389-08-2 | 206-864-7 | |
Pyrene | 129-00-0 | 204-927-3 | |
Sodium chloride | 7647-14-5 | 231-598-3 |
ATP + Luciferin + O 2 Oxyluciferin + AMP + PPi + CO 2 + Light
The emitted light intensity is linearly related to the ATP concentration and is measured using a luminometer. The luciferin-luciferase assay is a sensitive method for ATP quantitation used in a wide variety of applications (20).
structural and functional similarity to the class of the test substance being tested;
known physical chemical characteristics;
supporting data from the LLNA: DA;
supporting data from other animal models and/or from humans.
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 |
:
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.
:
Repeat the 1 % SLS aqueous solution pre-treatment and test substance application procedure carried out on Day 1.
:
No treatment.
:
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.
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.
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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]
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.
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].
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.
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.
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.
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.
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.
Vohr, H.W. and Ahr, H.J. (2005), The local lymph node assay being too sensitive? Arch. Toxicol., 79, 721-728.
Patterson, R.M., Noga, E. and Germolec, D. (2007), Lack of evidence for contact sensitisation by Pfiesteria extract. Environ. Health Perspect., 115, 1023-1028.
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]
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]
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.
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]
:
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).
:
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.
:
A substance incorrectly identified as negative or non-active by a test method, when in fact it is positive or active.
:
A substance incorrectly identified as positive or active by a test, when in fact it is negative or non-active.
:
The potential for an adverse health or ecological effect. The adverse effect is manifested only if there is an exposure of sufficient level.
:
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).
:
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).
:
An outlier is an observation that is markedly different from other values in a random sample from a population.
:
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.
:
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).
:
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.
:
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.
:
Any substance or mixture tested using this TM.
structural and functional similarity to the class of the test substance being tested;
known physical chemical characteristics;
supporting data from the LLNA: BrdU-ELISA;
supporting data from other animal models and/or from humans.
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 |
:
Individually identify and record the weight of each animal and any clinical observation. 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.
:
Repeat the application procedure carried out on Day 1.
:
No treatment.
:
Inject 0,5 mL (5 mg/mouse) of BrdU (10 mg/mL) solution intra-peritoneally.
:
Record the weight of each animal and any clinical observation. Approximately 24 hours (24 h) after BrdU injection, 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 (17). 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 into the study protocol.
The BrdU labelling index is defined as:
BrdU labelling index = (ABSem – ABS blankem) – (ABSref – ABS blankref)
Where: em = emission wavelength; and ref = reference wavelength.
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:
source, lot number, and manufacturer’s quality assurance/quality control data (antibody sensitivity and specificity and the limit of detection) for the ELISA 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 humane 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 BrdU labelling indices and SI values for each treatment group;
mean and associated error term (e.g. SD, SEM) for BrdU labelling index/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.
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]
Chamberlain, M. and Basketter, D.A. (1996), The local lymph node assay: status of validation. Food Chem. Toxicol., 34, 999-1002.
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.
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.
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.
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]
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(3), 258-273.
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(3), 274-286.
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(3), 249-257.
ICCVAM (2010), ICCVAM Test Method Evaluation Report. Nonradioactive local lymph node assay: BrdU-ELISA Test Method Protocol (LLNA: BrdU-ELISA). NIH Publication No 10-7552A/B. Research Triangle Park, NC: National Institute of Environmental Health Sciences. Available at: [http://iccvam.niehs.nih.gov/methods/immunotox/llna-ELISA/TMER.htm]
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]
Takeyoshi, M., Iida, K., Shiraishi, K. and Hoshuyama, S. (2005), Novel approach for classifying chemicals according to skin sensitising potency by non-radioisotopic modification of the local lymph node assay. J. Appl. Toxicol., 25, 129-134.
OECD (1992), Skin Sensitisation, Test Guideline No 406, Guidelines for Testing of Chemicals, OECD, Paris. Available at: [http://www.oecd.org/env/testguidelines]
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.
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.
ILAR (1996), Institute of Laboratory Animal Research (ILAR) Guide for the Care and Use of Laboratory Animals. 7th ed. Washington, DC: National Academies Press.
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]
McGarry, H.F. (2007), The murine local lymph node assay: regulatory and potency considerations under REACH. Toxicol., 238, 71-89.
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.
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]
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.
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].
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.
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.
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.
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.
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.
Vohr, H.W. and Ahr, H.J. (2005), The local lymph node assay being too sensitive? Arch. Toxicol., 79, 721-728.
Patterson, R.M., Noga, E. and Germolec D. (2007), Lack of evidence for contact sensitisation by Pfiesteria extract. Environ. Health Perspect., 115, 1023-1028.
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].
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]
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.l Health, 53, 563-79.
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]
:
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 (33).
:
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 physical/chemical characteristics; (iv) supporting data on known effects; and (v) known potency in the range of the desired response.
:
A test substance incorrectly identified as negative or non-active by a test method, when in fact it is positive or active (33).
:
A test substance incorrectly identified as positive or active by a test, when in fact it is negative or non-active (33).
:
The potential for an adverse health or ecological effect. The adverse effect is manifested only if there is an exposure of sufficient level.
:
A measure of the extent to which different qualified laboratories, using the same protocol and testing the same test substance, 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 (33).
:
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 (33).
:
An outlier is an observation that is markedly different from other values in a random sample from a population.
:
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.
:
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 (33).
:
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.
:
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.
:
Any substance or mixture tested using this TM.’
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