IN-VIVO RODENT ERYTHROCYTE MICRONUCLEUS ASSAY

The following summary represents a consensus of the working group except where noted. The items discussed are listed in the order in which they appear in the OECD guideline (474) for easy reference. Selection of species. Any commonly used laboratory rodent species is acceptable. There is no strain preference. Number and sex. The size of experiment (i.e., number of cells per animal, number of animals per group) should be finalized based on statistical considerations. Although a consensus was not achieved, operationally it was agreed that 2000 cells per animal and four animals per group was a minimum requirement. In general, the available database suggests that the use of one gender is adequate for screening. However, if there is evidence indicating a significant difference in the toxicity between male and female, then both sexes should be used. Treatment schedule. No unique treatment schedule can be recommended. Results from extended dose regimens are acceptable as long as positive. For negative studies, toxicity should be demonstrated or the limit dose should be used, and dosing continued until sampling. Dose levels. At least three dose levels separated by a factor between 2 and root 10 should be used. The highest dose tested should be the maximum tolerated dose based on mortality, bone marrow cell toxicity, or clinical symptoms of toxicity. The limit dose is 2 g/kg/day for treatment periods of 14 days or less and 1 g/kg/day for treatment periods greater than 14 days. A single dose level (the limit dose) is acceptable if there is no evidence of toxicity. Controls. Concurrent solvent (vehicle) controls should be included at all sampling times. A pretreatment sample, however, may also be acceptable only in the short treatment period peripheral blood studies. A concurrent positive control group should be included for each experiment. It is acceptable that the positive control may be administered by a route different from the test agent and sampled at only a single time. Performance of the test. For the bone marrow assay, two sample times (24 and 48 h) should be used after a single administration, or one sample time between 18 and 24 h after the last of two or more repeated administrations. For the peripheral blood assay, two sample times (48 and 72 h) are used after a single administration, or one sample time between 36 and 48 h is used after the last of two or more repeated administrations. For a longer treatment study, one sample time any time up to 48 h after the last dose is acceptable. Analysis. All slides should be coded. The frequency of micronucleated immature erythrocytes is the principal endpoint. The frequencies of micronucleated mature erythrocytes in the mouse can also be the endpoint for the longer repeated treatment study (4 weeks or more). When analyzing slides, the percentage of immature erythrocytes among total erythrocytes should not be less than 20% of the control value (i.e., 10% for 50% in the control). To assess cytotoxicity, at least 200 bone marrow erythrocytes per animal or at least 1000 peripheral blood erythrocytes per animal should be observed. Treatment of results. Although a consensus was not achieved, it was generally agreed that both a trend test and also pairwise tests were appropriate. Historical negative control data should be used to determine if the concurrent control response is acceptable.