Detection of in vitro clastogens and spindle poisons by the mouse lymphoma assay using the microwell method : interim report of an international collaborative study

Under the auspices of the Ministry of Health and Welfare of Japan and the Japanese Pharmaceutical Manufacturer Association, a collaborative study of the mouse lymphoma assay (MLA) was conducted by 42 Japanese laboratories and seven overseas laboratories to clarify the performance of the MLA for the detection of in vitro clastogens and spindle poisons. Twenty-one chemicals that were positive in in vitro chromosomal aberration assays (CA) but negative in bacterial reverse mutation assays (BRM) were examined by the MLA using the microwell method. All chemicals were coded, and each chemical was tested by two or three laboratories. Positive responses were obtained with 14 chemicals: mitomycin C (an internal positive control), arsenic trioxide, cadmium sulphate, chlorendic acid, cytosine arabinoside, diethylstilbestrol, eugenol, 5-fluorouracil, griseofulvin, hexamethyl phosphoramide, hydroxyurea, methotrexate, monocrotaline and pentachloroethane. Two chemicals (benzene and chlorodibromomethane) showed positive responses in one of two laboratories and were judged probably positive chemicals. Three chemicals (bromodichloromethane, isophorone and tetrachloroethane) were inconclusive because of a marginal response in one laboratory and a negative response in the other. Urethane was judged probably negative because two laboratories out of three showed clear negative responses. Dideoxycytidine (DDC) was a clear negative chemical in this study. The present results showed that 75.0% of the test chemicals (15/20, excluding mitomycin C) were positive, 15.0% (3/20) were inconclusive, and 10.0% (2/20) were negative. This suggests that the MLA may detect a majority of CA-positive chemicals. The inconclusive chemicals, however, are critical for the judgement of the MLA potential to detect clastogens. The findings that DDC was clearly negative suggests that the MLA may not be able to detect some clastogens. To clarify these issues, we began the second phase of the collaborative study with other BRM-negative and CA-positive chemicals.