SFTG international collaborative study on in vitro micronucleus test I. General conditions and overall conclusions of the study.

This study, coordinated by the SFTG (French branch of European Environmental Mutagen Society), included 38 participants from Europe, Japan and America. Clastogens (bleomycin, urethane), including base and nucleoside analogs (5-fluorouracil and cytosine arabinoside), aneugens and/or polyploidy inducers (colchicine, diethylstilboestrol, griseofulvin and thiabendazole), as well as non-genotoxic compounds (mannitol and clofibrate), were tested. Four cell types were used, i.e. human lymphocytes in the presence of cytochalasin B and CHO, CHL and L5178Y cell lines, in the presence or absence of cytochalasin B, with various treatment-recovery schedules. Mitomycin C was used as a positive control for all cell types. Mannitol and clofibrate were consistently negative in all cell types and with all treatment-recovery conditions. Urethane, known to induce questionable clastogenicity, was not found as positive. Bleomycin and mitomycin C were found positive in all treatment-recovery conditions. The base and nucleoside analogs were less easy to detect, especially 5-fluorouracil due to the interference with cytotoxicity, while cytosine arabinoside was detected in all cell types depending on the treatment-recovery schedule. Aneugens (colchicine, diethylstilboestrol and griseofulvin) were all detected in all cell types. In this study, the optimal detection was ensured when a short treatment followed by a long recovery was associated with a long continuous treatment without recovery. There was no impact of the presence or absence of cytochalasin B on the detection of micronucleated cells on cell lines. Scoring micronucleated cells in both mononucleated and binucleated cells when using cytochalasin B was confirmed to be useful for the detection and the identification of aneugens. In conclusion, these results, together with previously published validation studies, provide a useful contribution to the optimisation of a study protocol for the detection of both clastogens and aneugens in the in vitro micronucleus test.

SFTG international collaborative study on in vitro micronucleus test V. Using L5178Y cells.

In this report, results are presented from an international study of the in vitro micronucleus assay using mouse lymphoma L5178Y cells. This study was coordinated by an organizing committee supported by the SFTG (the French branch of the European Environmental Mutagen Society). Test chemicals included mannitol, bleomycin, 5-fluorouracil, colchicine and griseofulvin. Mitomycin C was used as a positive control. Each chemical was evaluated in at least two laboratories following a variety of different protocols (short and long exposures, varying recovery times, with and without cytochalasin B) in order to help determine a standard protocol for routine testing in mouse lymphoma L5178Y cells. Mannitol was the only exception, being tested in only one laboratory. Mannitol was negative, while bleomycin induced a concentration-dependent increase in micronucleated cells. Equivocal results were obtained for 5-fluorouracil, colchicine and griseofulvin. High levels of cytotoxicity interfered with the assessment of aneuploidy for colchicine and griseofulvin, preventing the ability to obtain clear results in all the treatment schedules. Experiments with 5-fluorouracil, colchicine and griseofulvin showed that both short and long treatment times are required as each compound was detected using one or more treatment protocol. No clear differences were seen in the sensitivity or accuracy of the responses in the presence of absence of cytochalasin B. It was also found that a recovery period may help to detect compounds which induce a genotoxicity associated to a reduction in cell number or cell proliferation. Overall, the results of the present study show that mouse lymphoma L5178Y cells are suitable for the in vitro micronucleus assay.

Mouse lymphoma thymidine kinase gene mutation assay: International Workshop on Genotoxicity Tests Workgroup report--Plymouth, UK 2002.

The Mouse Lymphoma Assay (MLA) Workgroup of the International Workshop on Genotoxicity Tests (IWGT) met on June 28th and 29th, 2002, in Plymouth, England. This meeting of the MLA group was devoted to discussing the criteria for assay acceptance and appropriate approaches to data evaluation. Prior to the meeting, the group conducted an extensive analysis of data from both the microwell and soft agar versions of the assay. For the establishment of criteria for assay acceptance, 10 laboratories (6 using the microwell method and 4 using soft agar) provided data on their background mutant frequencies, plating efficiencies of the negative/vehicle control, cell suspension growth, and positive control mutant frequencies. Using the distribution curves generated from this data, the Workgroup reached consensus on the range of values that should be used to determine whether an individual experiment is acceptable. In order to establish appropriate approaches for data evaluation, the group used a number of statistical methods to evaluate approximately 400 experimental data sets from 10 laboratories entered into a database created for the earlier MLA Workshop held in New Orleans [Environ. Mol. Mutagen. 40 (2002) 292]. While the Workgroup could not, during this meeting, make a final recommendation for the evaluation of data, a general strategy was developed and the Workgroup members agreed to evaluate this new proposed approach using their own laboratory data. This evaluation should lead to a consensus global approach for data evaluation in the near future.

Mouse lymphoma thymidine kinase gene mutation assay: follow-up International Workshop on Genotoxicity Test Procedures, New Orleans, Louisiana, April 2000.

The Mouse Lymphoma Assay (MLA) Workgroup of the International Workshop on Genotoxicity Test Procedures held a second harmonization meeting just prior to the U.S. Environmental Mutagen Society Meeting in New Orleans, LA, in April 2000. The discussion focused on several important aspects of the MLA, including: 1) cytotoxicity measures and their determination, 2) use of a 24-hr treatment, 3) the ability of the assay to detect aneugens, and 4) concentration selection. Prior to the meeting the group developed Microsoft Excel Workbooks for data entry. Ten laboratories entered their data into the workbooks (primarily as coded chemicals). The Excel Workbooks were used to facilitate data analysis by generating an extensive set of graphs that were evaluated by the meeting participants. Based on the Workgroup's previous agreement that a single cytotoxicity measure should be established for both the microwell and soft agar versions of the assay, the Workgroup analyzed the submitted data and unanimously agreed that the relative total growth (RTG) should be used as the cytotoxicity measure for concentration selection and data evaluation. The Workgroup also agreed that the various cytotoxicity measures should be calculated using the same methods regardless of whether the soft agar or microwell version of the assay was used. In the absence of sufficient data to make a definitive determination, the Workgroup continued to endorse the International Committee on Harmonization recommendation for the use of 24-hr treatment and made some specific 24-hr treatment protocol recommendations. The Workgroup recognized the ability of the MLA to detect at least some aneugens and also developed general guidance and requirements for appropriate concentration selection.