3.

The following Chapters are added: ‘B.49. IN VITRO MAMMALIAN CELL MICRONUCLEUS TEST INTRODUCTION 1. The in vitro micronucleus (MNvit) assay is a genotoxicity test for the detection of micronuclei (MN) in the cytoplasm of interphase cells. Micronuclei may originate from acentric chromosome fragments (i.e. lacking a centromere), or whole chromosomes that are unable to migrate to the poles during the anaphase stage of cell division. The assay detects the activity of clastogenic and aneugenic chemicals (substances and mixtures) (1) (2) in cells that have undergone cell division during or after exposure to the test substance. This Test Method (TM) allows the use of protocols with and without the actin polymerisation inhibitor cytochalasin B (cytoB). The addition of cytoB prior to the targeted mitosis allows for the identification and selective analysis of micronucleus frequency in cells that have completed one mitosis because such cells are binucleate (3) (4). This TM also allows the use of protocols without cytokinesis block, provided there is evidence that the cell population analysed has undergone mitosis. 2. In addition to using the MNvit assay to identify chemicals (substances and mixtures) that induce micronuclei, the use of a cytokinesis block, immunochemical labelling of kinetochores, or hybridisation with centromeric/telomeric probes (fluorescence in situ hybridisation (FISH)), also can provide information on the mechanisms of chromosome damage and micronucleus formation (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16). The labelling and hybridisation procedures can be used when there is an increase in micronucleus formation and the investigator wishes to determine if the increase was the result of clastogenic and/or aneugenic events. 3. Micronuclei represent damage that has been transmitted to daughter cells, whereas chromosome aberrations scored in metaphase cells may not be transmitted. Because micronuclei in interphase cells can be assessed relatively objectively, laboratory personnel need only determine whether or not the cells have undergone division and how many cells contain a micronucleus. As a result, the preparations can be scored relatively quickly and analysis can be automated. This makes it practical to score thousands instead of hundreds of cells per treatment, increasing the power of the assay. Finally, as micronuclei may arise from lagging chromosomes, there is the potential to detect aneuploidy-inducing agents that are difficult to study in conventional chromosomal aberration tests, e.g. OECD Test Guideline 473 (Chapter B.10 of this Annex) (17). However, the MNvit assay does not allow for the differentiation of chemicals inducing polyploidy from those inducing clastogenicity without special techniques such as FISH described under paragraph 2. 4. The MNvit assay is an in vitro method that typically uses cultured human or rodent cells. It provides a comprehensive basis for investigating chromosome damaging potential in vitro because both aneugens and clastogens can be detected. 5. The MNvit assay is robust and effective in a variety of cell types, and in the presence or absence of cytoB. There are extensive data to support the validity of the MNvit assay using various rodent cell lines (CHO, V79, CHL/IU, and L5178Y) and human lymphocytes (18) (19) (20) (21) (22) (23) (24) (25) (26) (27) (28) (29) (30) (31). These include, in particular, the international validation studies coordinated by the Société Française de Toxicologie Génétique (SFTG) (18) (19) (20) (21) (22) and the reports of the International Workshop on Genotoxicity Testing (4) (16). The available data have also been re-evaluated in a weight-of-evidence retrospective validation study by the European Centre for the Validation of Alternative Methods (ECVAM) of the European Commission, and the test method has been endorsed as scientifically valid by the ECVAM Scientific Advisory Committee (ESAC) (32) (33) (34). The use of the human TK6 lymphoblastoid cell line (35), HepG2 cells (36) (37) and primary Syrian Hamster Embryo cells (38) has been described, although they have not been used in validation studies. DEFINITIONS 6. Definitions used are provided in Appendix 1. INITIAL CONSIDERATIONS 7. Tests conducted in vitro generally require the use of an exogenous source of metabolic activation unless the cells are metabolically competent with respect to the substances being tested. The exogenous metabolic activation system does not entirely mimic in vivo conditions. Care should also be taken to avoid conditions that would lead to artefactual positive results which do not reflect intrinsic mutagenicity, and may arise from such factors as marked changes in pH or osmolality, or by high levels of cytotoxicity (39) (40) (41). If the test chemical causes a change in the pH of the medium at the time of addition, the pH should be adjusted, preferably by buffering the stock solution so that all the volumes at all test concentrations, and for all controls, remain the same. 8. To analyse the induction of micronuclei, it is essential that mitosis has occurred in both treated and untreated cultures. The most informative stage for scoring micronuclei is in cells that have completed one mitosis during or after treatment with the test substance. PRINCIPLE OF THE TEST 9. Cell cultures of human or mammalian origin are exposed to the test substance both with and without an exogenous source of metabolic activation unless cells with an adequate metabolising capability are used. Concurrent solvent/vehicle (VC) and positive control chemicals (PC) are included in all tests. 10. During or after exposure to the test substance, the cells are grown for a period sufficient to allow chromosome or spindle damage to lead to the formation of micronuclei in interphase cells. For induction of aneuploidy, the test substance should ordinarily be present during mitosis. Harvested and stained interphase cells are analysed for the presence of micronuclei. Ideally, micronuclei should only be scored in those cells that have completed mitosis during exposure to the test substance or during the post-exposure period, if one is used. In cultures that have been treated with a cytokinesis blocker, this is achieved by scoring only binucleate cells. In the absence of a cytokinesis blocker, it is important to demonstrate that the cells analysed are likely to have undergone cell division during or after exposure to the test substance. For all protocols, it is important to demonstrate that cell proliferation has occurred in both the control and treated cultures, and the extent of test substance-induced cytotoxicity or cytostasis should be assessed in the cultures (or in parallel cultures) that are scored for micronuclei. DESCRIPTION OF THE ASSAY Preparations 11. Cultured primary human peripheral blood lymphocytes (5) (19) (42) (43) and a number of rodent cell lines such as CHO, V79, CHL/IU, and L5178Y cells may be used (18) (19) (20) (21) (22) (25) (26) (27) (28) (30). The use of other cell lines and types should be justified based on their demonstrated performance in the assay, as described in the Acceptability Criteria section. Because the background frequency of micronuclei will influence the sensitivity of the assay, it is recommended that cell types with a low, stable background frequency of micronucleus formation be used. 12. Human peripheral blood lymphocytes should be obtained from young (approximately 18-35 years of age), healthy, non-smoking individuals with no known recent exposures to genotoxic chemicals or radiation. If cells from more than one donor are pooled for use, the number of donors should be specified. The micronucleus frequency increases with age and this trend is more marked in females than in males (44) and this should be taken into account in the selection of donor cells for pooling. Media and culture conditions 13. Appropriate culture medium and incubation conditions (culture vessels, CO2 concentration, temperature, and humidity) should be used for maintaining cultures. Established cell lines and strains should be checked routinely for the stability of the modal chromosome number and the absence of mycoplasma contamination, and should not be used if contaminated or if the modal chromosome number has changed. The normal cell cycle time for the culture conditions used in the testing laboratory should be known. If the cytokinesis-block method is used then the concentration of the cytokinesis inhibitor should be optimised for the particular cell type and should be shown to produce a good yield of binucleate cells for scoring. Preparation of cultures 14. Established cell lines and strains: cells are propagated from stock cultures, seeded in culture medium at a density such that the cultures will not reach confluency in monolayers, and suspension cultures will not reach excessive density before the time of harvest, and incubated at 37 °C. 15. Lymphocytes: whole blood treated with an anti-coagulant (e.g. heparin), or separated lymphocytes, are cultured in the presence of a mitogen e.g. phytohaemagglutinin (PHA) prior to exposure to the test substance and cytoB. Metabolic activation 16. Exogenous metabolising systems should be used when using cells with inadequate endogenous metabolic capacity. The most commonly used system is a co-factor-supplemented post-mitochondrial fraction (S9) prepared from the livers of rodents treated with enzyme-inducing agents such as Aroclor 1254 (45) (46) or a combination of phenobarbitone and β-naphthoflavone (46) (47) (48) (49). The latter combination does not conflict with the Stockholm Convention on Persistent Organic Pollutants (50) and Regulation (EC) No 850/2004 on Persistent Organic Pollutants (66) and has been shown to be as effective as Aroclor 1254 for inducing mixed-function oxidases (46) (47) (48) (49). The S9 fraction typically is used at concentrations ranging from 1-10 % (v/v) in the final test medium. The condition of a metabolic activation system may depend upon the class of chemical being tested and in some cases it may be appropriate to utilise more than one S9 concentration. 17. Genetically engineered cell lines expressing specific human or rodent activating enzymes may eliminate the need for an exogenous metabolic activation system, and may be used as the test cells. In such cases the choice of the cell lines used should be scientifically justified, e.g. by relevance of the mixed function oxidases for the metabolism of the test substance (51), and their responsiveness to known clastogens and aneugens (see separate section on Acceptability Criteria). It should be recognised that the substance being tested may not be metabolised by the expressed mixed function oxidase(s); in this case, the negative results would not indicate that the test substance cannot induce micronuclei. Test substance preparation 18. Solid chemicals should be dissolved in appropriate solvents or vehicles and diluted, if appropriate, prior to treatment of the cells. Liquid chemicals may be added directly to the test systems and/or diluted prior to treatment. Gases or volatile chemicals should be tested by appropriate modifications to the standard protocols, such as treatment in sealed vessels (52) (53). Fresh preparations of the test substance should be used unless stability data demonstrate the acceptability of storage. Test Conditions Solvents/vehicles 19. The solvent/vehicle should not react with the test substance, or be incompatible with the survival of the cells or with the maintenance of S9 activity at the concentration used. If other than well established solvent/vehicles (e.g. water, cell culture medium, dimethyl sulfoxide) are used, their use should be supported by data indicating their compatibility with the test substance and their lack of genetic toxicity. It is recommended that, wherever possible, the use of an aqueous solvent/vehicle should be considered first. Use of cytoB as a cytokinesis blocker 20. One of the most important considerations in the performance of the MNvit assay is ensuring that the cells being scored have completed mitosis during the treatment or the post-treatment incubation period, if one is used. CytoB is the agent that has been most widely used to block cytokinesis because it inhibits actin assembly, and thus prevents separation of daughter cells after mitosis, leading to the formation of binucleated cells (5) (54) (55). Micronucleus scoring, therefore, can be limited to cells that have gone through mitosis during or after treatment. The effect of the test substance on cell proliferation kinetics can be measured simultaneously. CytoB should be used as a cytokinesis blocker when human lymphocytes are used because cell cycle times will be variable within cultures and among donors and because not all lymphocytes will respond to PHA. Other methods have been used when testing cell lines to determine if the cells being scored have divided; these are addressed below (see Paragraph 26). 21. The appropriate concentration of cytoB should be determined by the laboratory for each cell type to achieve the optimal frequency of binucleated cells in the solvent/vehicle control cultures. The appropriate concentration of cytoB is usually between 3 and 6 μg/ml. Measuring cell proliferation and cytotoxicity and choosing exposure concentrations 22. When determining the highest test substance concentration to be tested, concentrations that have the capability of producing artefactual positive responses, such as those producing excessive cytotoxicity, precipitation in the culture medium, and marked changes in pH or osmolality (39) (40) (41), should be avoided. 23. Measurements of cell proliferation are made to ensure that the treated cells have undergone mitosis during the assay and that the treatments are conducted at appropriate levels of cytotoxicity (see Paragraph 29). Cytotoxicity should be determined with and without metabolic activation in cells that require metabolic activation using the relative increase in cell counts (RICC) or relative population doubling (RPD) (see Appendix 2 for formulas) unless cytoB is used. When cytoB is used, cytotoxicity can be determined using the replication index (RI) (see Appendix 2 for formula). 24. Treatment of cultures with cytoB, and measurement of the relative frequencies of mononucleate, binucleate, and multi-nucleate cells in the culture, provides an accurate method of quantifying the effect on cell proliferation and the cytotoxic or cytostatic activity of a treatment (5), and ensures that only cells that divided during or after treatment are scored. 25. In studies with cytoB, cytostasis/cytotoxicity can be quantified from the cytokinesis-block proliferation index (CBPI) (5) (26) (56) or may be derived from the RI from at least 500 cells per culture (see Appendix 2 for formulas). When cytoB is used to assess cell proliferation, a CBPI or RI should be determined from at least 500 cells per culture. These measurements among others can be used to estimate cytotoxicity by comparing values in the treated and control cultures. Assessment of other markers of cytotoxicity (e.g. confluency, cell number, apoptosis, necrosis, metaphase counting) can provide useful information. 26. In studies without cytoB, it is necessary to demonstrate that the cells scored in the culture have undergone division during or following treatment with the test substance, otherwise false negative responses may be produced. Methods that have been used for ensuring that divided cells are being scored include incorporation and subsequent detection of bromodeoxyuridine (BrdU) to identify cells that have replicated (57), the formation of clones when cells from permanent cell lines are treated and scored in situ on a microscope slide (Proliferation Index (PI)) (25) (26) (27) (28), or the measurement of Relative Population Doubling (RPD) or Relative Increase in Cell Count (RICC) or other proven methods (16) (56) (58) (59) (see Appendix 2 for formulas). Assessment of other markers for cytotoxicity or cytostasis (e.g. confluency, cell number, apoptosis, necrosis, metaphase counting) can provide useful information. 27. At least three analysable test concentrations should be evaluated. In order to achieve this, it may be necessary to perform the experiment using a larger number of closely spaced concentrations and analyse micronucleus formation in those concentrations providing the appropriate range of cytotoxicities. An alternative strategy is to perform a preliminary cytotoxicity test to narrow the range for the definitive test. 28. The highest concentration should aim to produce 55 ± 5 % cytotoxicity. Higher levels may induce chromosome damage as a secondary effect of cytotoxicity (60). Where cytotoxicity occurs, the test concentrations selected should cover a range from that producing 55 ± 5 % cytotoxicity, to little or no cytotoxicity. 29. If no cytotoxicity or precipitate is observed, the highest test concentration should correspond to 0,01 M, 5 mg/mL or 5 μl/mL, whichever is the lowest. The concentrations selected for analysis should, in general, be separated by a spacing of no more than 10. For test substances that exhibit a steep concentration-response curve, it may be necessary to more closely space the test substance concentrations so that cultures in the moderate and low toxicity ranges also will be scored. 30. When solubility is a limiting factor, the maximum concentration, if not limited by cytotoxicity, should be the lowest concentration at which minimal precipitate is visible in cultures, provided there is no interference with scoring. Evaluation of precipitation should be done by methods such as light microscopy, noting precipitate that persists, or appears during culture (by the end of treatment). Controls 31. Concurrent positive and solvent/vehicle controls both with and without metabolic activation should be included in each experiment. 32. PC are needed to demonstrate the ability of the cells used, and the test protocol, to identify clastogens and aneugens, and to affirm the metabolic capability of the S9 preparation. The PC should employ known inducers of micronucleus formation at concentrations expected to give small, but reproducible increases over background, and demonstrate the sensitivity of the test system. PC concentrations should be chosen so that the effects are clear but do not immediately reveal the identity of the coded slides to the reader. 33. A clastogen that requires metabolic activation (e.g. cyclophosphamide; benzo(a)pyrene) should be used to demonstrate both the metabolic competence and the ability of the test system to detect clastogens. Other PC may be used if justified. Because some PC that need metabolic activation may be active without exogenous metabolic activation under certain treatment conditions or in certain cell lines, the need for metabolic activation, and the activity of the S9 preparation, should be tested in the selected cell line and at the selected concentrations. 34. At the present time, no aneugens are known that require metabolic activation for their genotoxic activity (16). Currently accepted PC for aneugenic activity are, for example, colchicine and vinblastine. Other chemicals may be used if they induce micronuclei solely, or primarily, through aneugenic activity. To avoid the need for two PC (for clastogenicity and aneugenicity) without metabolic activation, the aneugenicity control can serve as the PC without S9, and the clastogenicity control can be used to test the adequacy of the metabolic activation system used. PC for both clastogenicity and aneugenicity should be used in cells that do not require S9. Suggested PC are included in Appendix 3. 35. The use of chemical class-related PC may be considered, when suitable chemicals are available. All PC used should be appropriate for the cell type and activation conditions. 36. Solvent/vehicle controls should be included for every harvest time. In addition, untreated NC (lacking solvent/vehicle) should also be used unless there are published or laboratory historical control data demonstrating that no genotoxic or other deleterious effects are induced by the chosen solvent at the concentrations used. TEST PROCEDURE Treatment Schedule 37. In order to maximise the probability of detecting an aneugen or clastogen acting at a specific stage in the cell cycle, it is important that sufficient numbers of cells are treated with the test substance during all stages of their cell cycles. The treatment schedule for cell lines and primary cell cultures may, therefore, differ somewhat from that for lymphocytes which require mitogenic stimulation to begin their cell cycle and these are considered in Paragraphs 41-43 (16). 38. Theoretical considerations, together with published data (18) indicate that most aneugens and clastogens will be detected by a short term treatment period of 3 to 6 hrs in the presence and absence of S9, followed by removal of the test substance and a growth period of 1,5-2,0 cell cycles (6). Cells are sampled at a time equivalent to about 1,5-2,0 times the normal (i.e. untreated) cell cycle length either after the beginning or at the end of treatment (See Table 1). Sampling or recovery times may be extended if it is known or suspected that the test substance affects the cell cycling time (e.g. when testing nucleoside analogues). 39. Because of the potential cytotoxicity of S9 preparations for cultured mammalian cells, an extended exposure treatment of 1,5-2,0 normal cell cycles is used only in the absence of S9. In the extended treatment, options are offered to allow treatment of the cells with the test chemical in the absence or presence of cytoB. These options address situations where there may be concern regarding possible interactions between the test substance and cytoB. 40. The suggested cell treatment schedules are presented in Table 1. These general treatment schedules may be modified depending on the stability or reactivity of the test substance or the particular growth characteristics of the cells being used. All treatments should commence and end while the cells are growing exponentially. These schedules are presented in more details in paragraphs 41-47 following. 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) Lymphocytes, primary cells, and cell lines with cytoB 41. For lymphocytes, the most efficient approach is to start the exposure to the test substance at 44-48 hrs after PHA stimulation, when cycle synchronisation will have disappeared (5). In the initial assay, cells are treated for 3 to 6 hrs with the test substance in the absence and presence of S9. The treatment medium is removed and replaced with fresh medium containing cytoB, and the cells are harvested 1,5-2,0 normal cell cycles later. 42. If both initial tests of the short (3-6 hrs) treatment are negative or equivocal, a subsequent, extended exposure treatment without S9 is used. Two treatment options are available and are equally acceptable. However, it might be more appropriate to follow Option A for stimulated lymphocytes where exponential growth may be declining at 96 hrs following stimulation. Also, cultures of cells should not have reached confluence by the final sampling time in Option B. — 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. 43. Primary cells and cell lines should be treated in a similar manner to lymphocytes except that it is not necessary to stimulate them with PHA for 44-48 hrs. Cells other than lymphocytes should be exposed such that at the time of study termination, the cells are still in log-phase growth. Cell lines without cytoB 44. Cells should be treated for 3-6 hrs in the presence and absence of S9. The treatment medium is removed and replaced with fresh medium, and the cells are harvested 1,5-2,0 normal cell cycles later. 45. If both initial tests of the short (3-6 hrs) treatment are negative or equivocal, a subsequent, extended exposure treatment (without S9) is used. Two treatment options are available, both of which are equally acceptable: — 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. 46. In monolayers, mitotic cells (identifiable as being round and detaching from the surface) may be present at the end of the 3-6 hr treatment. Because these mitotic cells are easily detached, they can be lost when the medium containing the test substance is removed. Care should be taken to collect these when cultures are washed, and to return them to the cultures, to avoid losing cells that are in mitosis, and at risk for micronuclei, at the time of harvest. Number of cultures 47. Duplicate cultures should be used for each test substance concentration and for the vehicle/solvent and NC cultures. Where minimal variation between duplicate cultures can be demonstrated from historical laboratory data, it may be acceptable for single cultures to be used. If single cultures are used, it is recommended that an increased number of concentrations be analysed. Cell harvest and slide preparation 48. Each culture is harvested and processed separately. Cell preparation may involve hypotonic treatment, but this step is not necessary if adequate cell spreading is otherwise achieved. Different techniques can be used in slide preparation provided that high-quality cell preparations for scoring are obtained. Cell cytoplasm should be retained to allow the detection of micronuclei and (in the cytokinesis-block method) reliable identification of binucleate cells. 49. The slides can be stained using various methods, such as Giemsa or fluorescent DNA specific dyes (59). The use of a DNA specific stain (e.g. acridine orange (61) or Hoechst 33258 plus pyronin-Y (62)) can eliminate some of the artefacts associated with using a non-DNA specific stain. Anti-kinetochore antibodies, FISH with pancentromeric DNA probes, or primed in situ labelling with pancentromere-specific primers, together with appropriate DNA counterstaining, can be used to identify the contents (chromosome/chromosomal fragment) of micronuclei if mechanistic information of their formation is of interest (15) (16). Other methods for differentiation between clastogens and aneugens may be used if they have been shown to be effective. Analysis 50. All slides, including those of the solvent/vehicle and the controls, should be independently coded before the microscopic analysis. Alternatively, coded samples can be analysed using a validated, automated flow cytometric or image analysis system. 51. In cytoB-treated cultures, micronucleus frequencies should be analysed in at least 2 000 binucleated cells per concentration (at least 1 000 binucleated cells per culture; two cultures per concentration). If single cultures are used, at least 2 000 binucleated cells per concentration should be scored from that culture. If substantially fewer than 1 000 binucleate cells per culture, or 2 000 if a single culture is used, are available for scoring at each concentration, and if a significant increase in micronuclei is not detected, the test should be repeated using more cells, or at less toxic concentrations, whichever is appropriate. Care should be taken not to score binucleate cells with irregular shapes or where the two nuclei differ greatly in size; neither should binucleate cells be confused with poorly spread multi-nucleate cells. Cells containing more than two main nuclei should not be analysed for micronuclei, as the baseline micronucleus frequency may be higher in these cells (63) (64) Scoring of mononucleate cells is acceptable if the test substance is shown to interfere with cytoB activity. 52. In cell lines assayed without cytoB treatment, micronuclei should be scored in at least 2 000 cells per concentration (at least 1 000 cells per culture; two cultures per concentration). Where only one culture per concentration is used, at least 2 000 cells should be scored from that culture. 53. When cytoB is used, a CBPI or an RI should be determined to assess cell proliferation (see Appendix 2) using at least 500 cells per culture. When treatments are performed in the absence of cytoB, it is essential to provide evidence that the cells being scored have proliferated, as discussed in Paragraphs 24-27. Acceptability criteria 54. A laboratory proposing to use the MNvit assay described in this TM should demonstrate its ability to reliably and accurately detect chemicals of known aneugenic and clastogenic activity, with and without metabolic activation, as well as known negative chemicals, using the reference chemicals in Appendix 3. As evidence of its ability to perform this TM correctly, the laboratory should provide evidence that the cells being scored for micronucleus formation have completed one nuclear division if the test is performed without the use of cytoB. 55. The chemicals in Appendix 3 are recommended for use as reference chemicals. Substitute or additional chemicals can be included 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. Justification could 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. 56. Solvent/vehicle control and untreated cultures should give reproducibly low and consistent micronuclei frequencies (typically 5-25 micronuclei/1 000 cells for the cell types identified in paragraph 11). Other cell types may have different ranges of responses which should be determined when validating them for use in the MNvit assay. Data from negative, solvent, and PC should be used to establish historical control ranges. These values should be used in deciding the adequacy of the concurrent NC/PC for an experiment. 57. If minor changes to the protocol (e.g. use of automated instead of manual scoring techniques; use of a new cell type) are proposed for the assay, then the effectiveness of the change should be demonstrated before the modified protocol can be considered acceptable for use. Demonstration of effectiveness includes demonstration that the major mechanisms of chromosome breakage and gain or loss can be detected, and that appropriate positive and negative results can be achieved for the class of the individual substance, or the broad range of substances, to be tested. DATA AND REPORTING Treatment of results 58. If the cytokinesis-block technique is used, only the frequencies of binucleate cells with micronuclei (independent of the number of micronuclei per cell) are used in the evaluation of micronucleus induction. Scoring of the numbers of cells with one, two, or more micronuclei could provide useful information, but is not mandatory. 59. Concurrent measures of cytotoxicity and/or cytostasis for all treated and solvent/vehicle control cultures should be determined (58). The CBPI or the RI should be calculated for all treated and control cultures as measurements of cell cycle delay when the cytokinesis-block method is used. In the absence of cytoB, the RPD or the RICC or PI should be used (see Appendix 2). 60. Individual culture data should be provided. Additionally, all data should be summarised in tabular form. 61. Chemicals that induce micronuclei in the MNvit assay may do so because they induce chromosome breakage, chromosome loss, or a combination of the two. Further analysis using anti-kinetochore antibodies, centromere specific in situ probes, or other methods may be used to determine whether the mechanism of micronucleus induction is due to clastogenic and/or aneugenic activity. Evaluation and interpretation of results 62. There is no requirement for verification by additional testing of a clear positive or negative response. Equivocal results may be clarified by analysis of another 1 000 cells from all the cultures to avoid loss of blinding. If this approach does not resolve the result, further testing should be performed. Modification of study parameters over an extended or narrowed range of conditions, as appropriate, should be considered in follow-up experiments. Study parameters that might be modified include the test concentration spacing, the timing of treatment and cell harvest, and/or the metabolic activation conditions. 63. There are several criteria for determining a positive result, such as a concentration-related increase or a statistically significant increase in the number of cells containing micronuclei. The biological relevance of the results should be considered first. Consideration of whether the observed values are within or outside of the historical control range can provide guidance when evaluating the biological significance of the response. Appropriate statistical methods may be used as an aid in evaluating the test results (65). However, the results of statistical testing should be assessed with respect to dose-response relationship. Reproducibility and historical data should also be taken into consideration. 64. Although most experiments will give clearly positive or negative results, in some cases the data set will preclude making a definite judgement about the activity of the test substance. These equivocal or questionable responses may occur regardless of the number of times the experiment is repeated. 65. Positive results from the MNvit assay indicate that the test substance induces chromosome breakage or chromosome loss, in cultured mammalian cells. Negative results indicate that, under the test conditions used, the test substance does not induce chromosome breaks and/or gain or loss in cultured mammalian cells. Test Report 66. The test report should include at least the following information, if relevant to the conduct of the study: 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 LITERATURE (1) Kirsch-Volders, M. 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Appendix 1 Definitions Aneugen: 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. Aneuploidy: 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). Apoptosis: 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. Cell proliferation: increase in cell number as a result of mitotic cell division. Centromere: DNA region of a chromosome where both chromatids are held together and on which both kinetochores are attached side-to-side. Clastogen: any substance or process which causes structural chromosomal aberrations in populations of cells or organisms. Cytokinesis: the process of cell division immediately following mitosis to form two daughter cells, each containing a single nucleus. Cytokinesis-Block Proliferation index (CBPI): the proportion of second-division cells in the treated population relative to the untreated control (see Appendix 2 for formula). Cytostasis: inhibition of cell growth (see Appendix 2 for formula). Cytotoxicity: harmful effects to cell structure or function ultimately causing cell death. Genotoxic: 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. Interphase cells: cells not in the mitotic stage. Kinetochore: 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. Micronuclei: 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. Mitosis: division of the cell nucleus usually divided into prophase, prometaphase, metaphase, anaphase and telophase. Mitotic index: the ratio of cells in metaphase divided by the total number of cells observed in a population of cells; an indication of the degree of cell proliferation of that population. Mutagenic: produces a heritable change of DNA base-pair sequences(s) in genes or of the structure of chromosomes (chromosome aberrations). Non-disjunction: failure of paired chromatids to disjoin and properly segregate to the developing daughter cells, resulting in daughter cells with abnormal numbers of chromosomes. Polyploidy: numerical chromosome aberrations in cells or organisms involving entire set(s) of chromosomes, as opposed to an individual chromosome or chromosomes (aneuploidy). Proliferation Index (PI): method for cytotoxicity measurement when cytoB is not used (see Appendix 2 for formula). Relative Increase in Cell Count (RICC): method for cytotoxicity measurement when cytoB is not used (see Appendix 2 for formula). Relative Population Doubling (RPD): method for cytotoxicity measurement when cytoB is not used (see Appendix 2 for formula). Replication Index (RI): 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). Test chemical (also referred to as test substance): Any substance or mixture tested using this TM. Appendix 2 Formulas for Cytotoxicity Assessment 1. When cytoB is used, evaluation of cytotoxicity should be based on the Cytokinesis-Block Proliferation Index (CBPI) or Replicative Index (RI) (16) (58). The CBPI indicates the average number of cell cycles per cell during the period of exposure to cytoB, and may be used to calculate cell proliferation. The RI indicates the relative number of nuclei in treated cultures compared to control cultures and can be used to calculate the % cytostasis: % Cytostasis = 100 – 100{(CBPIT – 1) ÷ (CBPIC – 1)} And: T = test chemical treatment culture C = vehicle control culture Where: Thus, a CBPI of 1 (all cells are mononucleate) is equivalent to 100 % cytostasis. Cytostasis = 100 – RI T = treated cultures C = control cultures 2. Thus, an RI of 53 % means that, compared to the numbers of cells that have divided to form binucleate and multinucleate cells in the control culture, only 53 % of this number divided in the treated culture, i.e. 47 % cytostasis. 3. When cytoB is not used, evaluation of cytotoxicity based on Relative Increase in Cell Counts (RICC) or on Relative Population Doubling (RPD) is recommended (58), as both take into account the proportion of the cell population which has divided. where: Population Doubling = [log (Post-treatment cell number ÷ Initial cell number)] ÷ log 2 4. Thus, a RICC, or a RPD of 53 % indicates 47 % cytotoxicity/cytostasis. 5. By using a Proliferation Index (PI), cytotoxicity may be assessed via counting the number of clones consisting of 1 cell (cl1), 2 cells (cl2), 3 to 4 cells (cl4) and 5 to 8 cells (cl8) 6. The PI has been used as a valuable and reliable cytotoxicity parameter also for cell lines cultured in situ in the absence of cytoB (25) (26) (27) (28). Appendix 3 Reference chemicals recommended for assessing performance (16) 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 B.50. SKIN SENSITISATION: LOCAL LYMPH NODE ASSAY: DA INTRODUCTION 1. OECD Guidelines for the Testing of Chemicals and EU Test Methods are periodically reviewed in light of scientific progress, changing regulatory needs, and animal welfare considerations. The first Test Method (TM) (B.42) for the determination of skin sensitisation in the mouse, the Local Lymph Node Assay (LLNA; OECD Test Guideline 429) has been revised (1) The details of the validation of the LLNA and a review of the associated work have been published (2) (3) (4) (5) (6) (7) (8) (9). In the LLNA, radioisotopic thymidine or iodine is used to measure lymphocyte proliferation and therefore the assay has limited use where the acquisition, use, or disposal of radioactivity is problematic. The LLNA: DA (developed by Daicel Chemical Industries, Ltd) is a non-radioactive modification to the LLNA, which quantifies adenosine triphosphate (ATP) content via bio-luminescence as an indicator of lymphocyte proliferation. The LLNA: DA test method has been validated and reviewed and recommended by an international peer review panel as considered useful for identifying skin sensitising and non-sensitising chemicals, with certain limitations (10) (11) (12) (13). This TM is designed for assessing skin sensitisation potential of chemicals (substances and mixtures) in animals. Chapter B.6 of this Annex and OECD Test Guideline 406 utilise guinea pig tests, notably the guinea pig maximisation test and the Buehler test (14) The LLNA (Chapter B.42 of this Annex; OECD Test Guideline 429) and the two non-radioactive modifications, LLNA: DA (Chapter B.50 of this Annex; OECD Test Guideline 442 A) and LLNA: BrdU-ELISA (Chapter B.51 of this Annex; OECD Test Guideline 442 B), all provide an advantage over the guinea pig tests in B.6 and OECD Test Guideline 406 (14) in terms of reduction and refinement of animal use. 2. Similar to the LLNA, the LLNA: DA studies the induction phase of skin sensitisation and provides quantitative data suitable for dose-response assessment. Furthermore, an ability to detect skin sensitisers without the necessity for using a radiolabel for DNA eliminates the potential for occupational exposure to radioactivity and waste disposal issues. This in turn may allow for the increased use of mice to detect skin sensitisers, which could further reduce the use of guinea pigs to test for skin sensitisation potential (i.e. B.6; OECD Test Guideline 406) (14). DEFINITIONS 3. Definitions used are provided in Appendix 1. INITIAL CONSIDERATIONS AND LIMITATIONS 4. The LLNA: DA is a modified LLNA method for identifying potential skin sensitising chemicals, with specific limitations. This does not necessarily imply that in all instances the LLNA: DA should be used in place of the LLNA or guinea pig tests (i.e. B.6; OECD Test Guideline 406) (14), but rather that the assay is of equal merit and may be employed as an alternative in which positive and negative results generally no longer require further confirmation (10) (11). The testing laboratory should consider all available information on the test substance prior to conducting the study. Such information will include the identity and chemical structure of the test substance; its physicochemical properties; the results of any other in vitro or in vivo toxicity tests on the test substance; and toxicological data on structurally related chemicals. This information should be considered in order to determine whether the LLNA: DA is appropriate for the test substance (given the incompatibility of limited types of chemicals with the LLNA: DA (see paragraph 5) and to aid in dose selection. 5. The LLNA: DA is an in vivo method and, as a consequence, will not eliminate the use of animals in the assessment of allergic contact sensitising activity. It has, however, the potential to reduce animal use for this purpose when compared to the guinea pig tests (B.6; OECD Test Guideline 406) (14). Moreover, the LLNA: DA offers a substantial refinement (less pain and distress) of the way in which animals are used for allergic contact sensitisation testing, since unlike the B.6 and OECD Test Guideline 406, the LLNA: DA does not require that challenge-induced dermal hypersensitivity reactions be elicited. Despite the advantages of the LLNA: DA over B.6 and OECD Test Guideline 406 (14), there are certain limitations that may necessitate the use of B.6 or OECD Test Guideline 406 (e.g. the testing of certain metals, false positive findings with certain skin irritants (such as some surfactant-type substances) (6) (1 and Chapter B.42 in this Annex), solubility of the test substance). In addition, chemical classes or substances containing functional groups shown to act as potential confounders (16) may necessitate the use of guinea pig tests (i.e. B.6; OECD Test Guideline 406 (14)). Limitations that have been identified for the LLNA (1 and Chapter B.42 in this Annex) have been recommended to apply also to the LLNA: DA (10). Additionally, the use of the LLNA: DA might not be appropriate for testing substances that affect ATP levels (e.g. substances that function as ATP inhibitors) or those that affect the accurate measurement of intracellular ATP (e.g. presence of ATP degrading enzymes, presence of extracellular ATP in the lymph node). Other than such identified limitations, the LLNA: DA should be applicable for testing any substances unless there are properties associated with these substances that may interfere with the accuracy of the LLNA: DA. In addition, consideration should be given to the possibility of borderline positive results when Stimulation Index (SI) values between 1,8 and 2,5 are obtained (see paragraphs 31-32). This is based on the validation database of 44 substances using an SI ≥ 1,8 (see paragraph 6) for which the LLNA: DA correctly identified all 32 LLNA sensitisers, but incorrectly identified three of 12 LLNA non-sensitisers with SI values between 1,8 and 2,5 (i.e. borderline positive) (10). However, as the same dataset was used for setting the SI-values and calculating the predictive properties of the test, the stated results may be an over-estimation of the real predictive properties. PRINCIPLE OF THE TEST METHOD 6. The basic principle underlying the LLNA: DA is that sensitisers induce proliferation of lymphocytes in the lymph nodes draining the site of test substance application. This proliferation is proportional to the dose and to the potency of the applied allergen and provides a simple means of obtaining a quantitative measurement of sensitisation. Proliferation is measured by comparing the mean proliferation in each test group to the mean proliferation in the vehicle treated control (VC) group. The ratio of the mean proliferation in each treated group to that in the concurrent VC group, termed the SI, is determined, and should be ≥ 1,8 before further evaluation of the test substance as a potential skin sensitiser is warranted. The procedures described here are based on the use of measuring ATP content by bioluminescence (known to correlate with living cell number) (17) to indicate an increased number of proliferating cells in the draining auricular lymph nodes (18) (19). The bioluminescent method utilises the luciferase enzyme to catalyse the formation of light from ATP and luciferin according to the following reaction: 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). DESCRIPTION OF THE ASSAY Selection of animal species 7. The mouse is the species of choice for this test. Validation studies for the LLNA: DA were conducted exclusively with the CBA/J strain, which is therefore considered the preferred strain (12) (13). Young adult female mice, which are nulliparous and non-pregnant, are used. At the start of the study, animals should be between 8-12 weeks old, and the weight variation of the animals should be minimal and not exceed 20 % of the mean weight. Alternatively, other strains and males may be used when sufficient data are generated to demonstrate that significant strain and/or gender-specific differences in the LLNA: DA response do not exist. Housing and feeding conditions 8. Mice should be group-housed (21), unless adequate scientific rationale for housing mice individually is provided. The temperature of the experimental animal room should be 22 ± 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. For feeding, conventional laboratory diets may be used with an unlimited supply of drinking water. Preparation of animals 9. The animals are randomly selected, marked to permit individual identification (but not by any form of ear marking), and kept in their cages for at least five days prior to the start of dosing to allow for acclimatisation to the laboratory conditions. Prior to the start of treatment all animals are examined to ensure that they have no observable skin lesions. Preparation of dosing solutions 10. Solid chemicals should be dissolved or suspended in solvents/vehicles and diluted, if appropriate, prior to application to an ear of the mice. Liquid chemicals may be applied neat or diluted prior to dosing. Insoluble chemicals, such as those generally seen in medical devices, should be subjected to an exaggerated extraction in an appropriate solvent to reveal all extractable constituents for testing prior to application to an ear of the mice. Test substances should be prepared daily unless stability data demonstrate the acceptability of storage. Reliability check 11. Positive control chemicals (PC) are used to demonstrate appropriate performance of the assay by responding with adequate and reproducible sensitivity to a sensitising test substance for which the magnitude of the response is well characterised. Inclusion of a concurrent PC is recommended because it demonstrates competency of the laboratory to successfully conduct each assay and allows for an assessment of intra- and inter-laboratory reproducibility and comparability. Some regulatory authorities also require a PC for each study and therefore users are encouraged to consult the relevant authorities prior to conducting the LLNA: DA. Accordingly, the routine use of a concurrent PC is encouraged to avoid the need for additional animal testing to meet such requirements that might arise from the use of a periodic PC (see paragraph 12). The PC should produce a positive LLNA: DA response at an exposure level expected to give an increase in the SI ≥ 1,8 over the negative control (NC) group. The PC dose should be chosen such that it does not cause excessive skin irritation or systemic toxicity and the induction is reproducible but not excessive (e.g. SI > 10 would be considered excessive). Preferred PC are 25 % hexyl cinnamic aldehyde (Chemical Abstracts Service (CAS) number 101-86-0) and 25 % eugenol (CAS number 97-53-0) in acetone: olive oil (4:1, v/v). There may be circumstances in which, given adequate justification, other PC, meeting the above criteria, may be used. 12. While inclusion of a concurrent PC group is recommended, there may be situations in which periodic testing (i.e. at intervals ≤ 6 months) of the PC may be adequate for laboratories that conduct the LLNA: DA regularly (i.e. conduct the LLNA: DA at a frequency of no less than once per month) and have an established historical PC database that demonstrates the laboratory’s ability to obtain reproducible and accurate results with PCs. Adequate proficiency with the LLNA: DA can be successfully demonstrated by generating consistent positive results with the PC in at least 10 independent tests conducted within a reasonable period of time (i.e. less than one year). 13. A concurrent PC group should always be included when there is a procedural change to the LLNA: DA (e.g. change in trained personnel, change in test method materials and/or reagents, change in test method equipment, change in source of test animals), and such changes should be documented in laboratory reports. Consideration should be given to the impact of these changes on the adequacy of the previously established historical database in determining the necessity for establishing a new historical database to document consistency in the PC results. 14. Investigators should be aware that the decision to conduct a PC study on a periodic basis instead of concurrently has ramifications on the adequacy and acceptability of negative study results generated without a concurrent PC during the interval between each periodic PC study. For example, if a false negative result is obtained in the periodic PC study, negative test substance results obtained in the interval between the last acceptable periodic PC study and the unacceptable periodic PC study may be questioned. Implications of these outcomes should be carefully considered when determining whether to include concurrent PCs or to only conduct periodic PCs. Consideration should also be given to using fewer animals in the concurrent PC group when this is scientifically justified and if the laboratory demonstrates, based on laboratory-specific historical data, that fewer mice can be used (22). 15. Although the PC should be tested in the vehicle that is known to elicit a consistent response (e.g. acetone: olive oil; 4:1, v/v), there may be certain regulatory situations in which testing in a non-standard vehicle (clinically/chemically relevant formulation) will also be necessary (23). If the concurrent PC is tested in a different vehicle than the test substance, then a separate VC for the concurrent PC should be included. 16. In instances where substances of a specific chemical class or range of responses are being evaluated, benchmark substances may also be useful to demonstrate that the test method is functioning properly for detecting the skin sensitisation potential of these types of substances. Appropriate benchmark substances should have the following properties: — 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. TEST PROCEDURE Number of animals and dose levels 17. A minimum of four animals is used per dose group, with a minimum of three concentrations of the test substance, plus a concurrent NC group treated only with the vehicle for the test substance, and a PC (concurrent or recent, based on laboratory policy in considering paragraphs 11-15). Testing multiple doses of the PC should be considered, especially when testing the PC on an intermittent basis. Except for absence of treatment with the test substance, animals in the control groups should be handled and treated in a manner identical to that of animals in the treatment groups. 18. Dose and vehicle selection should be based on the recommendations given in references (2) and (24). Consecutive doses are normally selected from an appropriate concentration series such as 100 %, 50 %, 25 %, 10 %, 5 %, 2,5 %, 1 %, 0,5 %, etc. Adequate scientific rationale should accompany the selection of the concentration series used. All existing toxicological information (e.g. acute toxicity and dermal irritation) and structural and physicochemical information on the test substance of interest (and/or structurally related substances) should be considered, where available, in selecting the three consecutive concentrations so that the highest concentration maximises exposure while avoiding systemic toxicity and/or excessive local skin irritation (24) (25). In the absence of such information, an initial pre-screen test may be necessary (see paragraphs 21-24). 19. The vehicle should not interfere with or bias the test result and should be selected on the basis of maximising the solubility in order to obtain the highest concentration achievable while producing a solution/suspension suitable for application of the test substance. Recommended vehicles are acetone: olive oil (4:1 v/v), N,N-dimethylformamide, methyl ethyl ketone, propylene glycol, and dimethyl sulphoxide (6) but others may be used if sufficient scientific rationale is provided. In certain situations it may be necessary to use a clinically relevant solvent or the commercial formulation in which the test substance is marketed as an additional control. Particular care should be taken to ensure that hydrophilic substances are incorporated into a vehicle system, which wets the skin and does not immediately run off, by incorporation of appropriate solubilisers (e.g. 1 % Pluronic® L92). Thus, wholly aqueous vehicles are to be avoided. 20. The processing of lymph nodes from individual mice allows for the assessment of inter-animal variability and a statistical comparison of the difference between test substance and VC group measurements (see paragraph 33). In addition, evaluating the possibility of reducing the number of mice in the PC group is only feasible when individual animal data are collected (22). Further, some regulatory authorities require the collection of individual animal data. Regular collection of individual animal data provides an animal welfare advantage by avoiding duplicate testing that would be necessary if the test substance results originally collected in one manner (e.g. via pooled animal data) were to be considered later by regulatory authorities with other requirements (e.g. individual animal data). Pre-screen test 21. In the absence of information to determine the highest dose to be tested (see paragraph 18), a pre-screen test should be performed in order to define the appropriate dose level to test in the LLNA: DA. The purpose of the pre-screen test is to provide guidance for selecting the maximum dose level to use in the main LLNA: DA study, where information on the concentration that induces systemic toxicity (see paragraph 24) and/or excessive local skin irritation (see paragraph 23) is not available. The maximum dose level tested should be 100 % of the test substance for liquids or the maximum possible concentration for solids or suspensions. 22. The pre-screen test is conducted under conditions identical to the main LLNA: DA study, except there is no assessment of lymph node proliferation and fewer animals per dose group can be used. One or two animals per dose group are suggested. All mice will be observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Body weights are recorded pre-test and prior to termination (Day 8). Both ears of each mouse are observed for erythema and scored using Table 1 (25). Ear thickness measurements are taken using a thickness gauge (e.g. digital micrometer or Peacock Dial thickness gauge) on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose), Day 7 (24 hours prior to termination) and Day 8. Additionally on Day 8, ear thickness could be determined by ear punch weight determinations, which should be performed after the animals are humanely killed. Excessive local irritation is indicated by an erythema score ≥ 3 and/or ear thickness of ≥ 25 % on any day of measurement (26) (27). The highest dose selected for the main LLNA: DA study will be the next lower dose in the pre-screen concentration series (see paragraph 18) that does not induce systemic toxicity and/or excessive local skin irritation Table 1 Erythema Scores Observation Score No erythema 0 Very slight erythema (barely perceptible) 1 Well-defined erythema 2 Moderate to severe erythema 3 Severe erythema (beet redness) to eschar formation preventing grading of erythema 4 23. In addition to a 25 % increase in ear thickness (26) (27), a statistically significant increase in ear thickness in the treated mice compared to control mice has also been used to identify irritants in the LLNA (28) (29) (30) (31) (32) (33) (34). However, while statistically significant increases can occur when ear thickness is less than 25 % they have not been associated specifically with excessive irritation (30) (31) (32) (33) (34). 24. The following clinical observations may indicate systemic toxicity (35) when used as part of an integrated assessment and therefore may indicate the maximum dose level to use in the main LLNA: DA: changes in nervous system function (e.g. pilo-erection, ataxia, tremors, and convulsions); changes in behaviour (e.g. aggressiveness, change in grooming activity, marked change in activity level); changes in respiratory patterns (i.e. changes in frequency and intensity of breathing such as dyspnea, gasping, and rales), and changes in food and water consumption. In addition, signs of lethargy and/or unresponsiveness and any clinical signs of more than slight or momentary pain and distress, or a > 5 % reduction in body weight from Day 1 to Day 8 and mortality, should be considered in the evaluation. Moribund animals or animals showing signs of severe pain and distress should be humanely killed (36). Main study experimental schedule 25. The experimental schedule of the assay is as follows: — Day 1: Individually identify and record the weight of each animal and any clinical observation. Apply 1 % sodium lauryl sulfate (SLS) aqueous solution to the dorsum of each ear by using a brush dipped in the SLS solution to cover the entire dorsum of each ear with four to five strokes. One hour after the SLS treatment, apply 25 μL of the appropriate dilution of the test substance, the vehicle alone, or the PC (concurrent or recent, based on laboratory policy in considering paragraphs 11-15), to the dorsum of each ear. — Days 2, 3 and 7: Repeat the 1 % SLS aqueous solution pre-treatment and test substance application procedure carried out on Day 1. — Days 4, 5, and 6: No treatment. — Day 8: Record the weight of each animal and any clinical observation. Approximately 24 to 30 hours after the start of application on Day 7, humanely kill the animals. Excise the draining auricular lymph nodes from each mouse ear and process separately in phosphate buffered saline (PBS) for each animal. Details and diagrams of the lymph node identification and dissection can be found in reference (22). To further monitor the local skin response in the main study, additional parameters such as scoring of ear erythema or ear thickness measurements (obtained either by using a thickness gauge, or ear punch weight determinations at necropsy) may be included in the study protocol. Preparation of cell suspensions 26. From each mouse, a single-cell suspension of lymph node cells (LNC) excised bilaterally is prepared by sandwiching the lymph nodes between two glass slides and applying light pressure to crush the nodes. After confirming that the tissue has spread out thinly pull the two slides apart. Suspend the tissue on both slides in PBS by holding each slide at an angle over the Petri dish and rinsing with PBS while concurrently scraping the tissue off of the slide with a cell scraper. Further, the lymph nodes in NC animals are small, so careful operation is important to avoid any artificial effects on SI values. A total volume of 1 mL PBS should be used for rinsing both slides. The LNC suspension in the Petri dish should be homogenised lightly with the cell scraper. A 20 μL aliquot of the LNC suspension is then collected with a micropipette, taking care not to take up the membrane that is visible to the eye, and subsequently mixed with 1,98 mL of PBS to yield a 2 mL sample. A second 2 mL sample is then prepared using the same procedure so that two samples are prepared for each animal. Determination of cellular proliferation (measurement of ATP content of lymphocytes) 27. Increases in ATP content in the lymph nodes are measured by the luciferin/luciferase method using an ATP measurement kit, which measures bioluminescence in Relative Luminescence Units (RLU). The assay time from time of animal sacrifice to measurement of ATP content for each individual animal should be kept uniform, within approximately 30 minutes, because the ATP content is considered to gradually decrease with time after animal sacrifice (12) Thus, the series of procedures from excision of auricular lymph nodes to ATP measurement should be completed within 20 minutes by the pre-determined time schedule that is the same for each animal. ATP luminescence should be measured in each 2 mL sample so that a total of two ATP measurements are collected for each animal. The mean ATP luminescence is then determined and used in subsequent calculations (see paragraph 30). OBSERVATIONS Clinical observations 28. Each mouse should be carefully observed at least once daily for any clinical signs, either of local irritation at the application site or of systemic toxicity. All observations are systematically recorded with records being maintained for each mouse. Monitoring plans should include criteria to promptly identify those mice exhibiting systemic toxicity, excessive local skin irritation, or corrosion of skin for euthanasia (36). Body weights 29. As stated in paragraph 25, individual animal body weights should be measured at the start of the test and at the scheduled humane kill. CALCULATION OF RESULTS 30. Results for each treatment group are expressed as the mean SI. The SI is derived by dividing the mean RLU/mouse within each test substance group and the PC group by the mean RLU/mouse for the solvent/VC group. The average SI for the VCs is then one. 31. The decision process regards a result as positive when SI ≥ 1,8 (10) However, the strength of the dose-response relationship, the statistical significance and the consistency of the solvent/vehicle and PC responses may also be used when determining whether a borderline result (i.e. SI value between 1,8 and 2,5) is declared positive (2) (3) (37). 32. For a borderline positive response between an SI of 1,8 and 2,5, users may want to consider additional information such as dose-response relationship, evidence of systemic toxicity or excessive irritation, and where appropriate, statistical significance together with SI values to confirm that such results are positives (10). Consideration should also be given to various properties of the test substance, including whether it has a structural relationship to known skin sensitisers, whether it causes excessive skin irritation in the mouse, and the nature of the dose-response relationship observed. These and other considerations are discussed in detail elsewhere (4) 33. Collecting data at the level of the individual mouse will enable a statistical analysis for presence and degree of dose-response relationship in the data. Any statistical assessment could include an evaluation of the dose-response relationship as well as suitably adjusted comparisons of test groups (e.g. pair-wise dosed group versus concurrent solvent/vehicle control comparisons). Statistical analyses may include, e.g. linear regression or William’s test to assess dose-response trends, and Dunnett’s test for pair-wise comparisons. In choosing an appropriate method of statistical analysis, the investigator should maintain an awareness of possible inequalities of variances and other related problems that may necessitate a data transformation or a non-parametric statistical analysis. In any case, the investigator may need to carry out SI calculations and statistical analyses with and without certain data points (sometimes called “outliers”). DATA AND REPORTING Data 34. Data should be summarised in tabular form showing the individual animal RLU values, the group mean RLU/animal, its associated error term (e.g. SD, SEM), and the mean SI for each dose group compared against the concurrent solvent/vehicle control group. Test report 35. The test report should contain the following information: Test and control chemicals: — identification data (e.g. CAS number and EC number, if available; source; purity; known impurities; lot number); — physical nature and physicochemical properties (e.g. volatility, stability, solubility); — if mixture, composition and relative percentages of components; Solvent/vehicle: — identification data (purity; concentration, where appropriate; volume used); — justification for choice of vehicle; Test animals: — source of CBA mice; — microbiological status of the animals, when known; — number and age of animals; — source of animals, housing conditions, diet, etc.; Test conditions: — the source, lot number and manufacturer’s quality assurance/quality control data for the ATP kit; — details of test substance preparation and application; — justification for dose selection (including results from pre-screen test, if conducted); — vehicle and test substance concentrations used, and total amount of test substance applied; — details of food and water quality (including diet type/source, water source); — details of treatment and sampling schedules; — methods for measurement of toxicity; — criteria for considering studies as positive or negative; — details of any protocol deviations and an explanation on how the deviation affects the study design and results; Reliability check: — a summary of results of latest reliability check, including information on test substance, concentration and vehicle used; — concurrent and/or historical PC and concurrent negative (solvent/vehicle) control data for testing laboratory; — if a concurrent PC was not included, the date and laboratory report for the most recent periodic PC and a report detailing the historical PC data for the laboratory justifying the basis for not conducting a concurrent PC; Results: — individual weights of mice at start of dosing and at scheduled kill; as well as mean and associated error term (e.g. SD, SEM) for each treatment group; — time course of onset and signs of toxicity, including dermal irritation at site of administration, if any, for each animal; — time of animal termination and time of ATP measurement for each animal; — a table of individual mouse RLU values and SI values for each dose treatment group; — mean and associated error term (e.g. SD, SEM) for RLU/mouse for each treatment group and the results of outlier analysis for each treatment group; — calculated SI and an appropriate measure of variability that takes into account the inter-animal variability in both the test substance and control groups; — dose response relationship; — statistical analyses, where appropriate; Discussion of results: — a brief commentary on the results, the dose-response analysis, and statistical analyses, where appropriate, with a conclusion as to whether the test substance should be considered a skin sensitiser. 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