Evaluation of the mouse lymphoma tk assay (microwell method) as an alternative to the in vitro chromosomal aberration test

In order to evaluate the utility of the mouse lymphoma assay (MLA) for dete cting in vitro clastogens and spindle poisons and to compare it with the in vitro chromosomal aberration test (CA), we conducted an international coll aborative study of the MLA that included 45 Japanese laboratories and seven overseas laboratories under the cooperation of the Ministry of Health and Welfare of Japan and the Japanese Pharmaceutical Manufacturer's Association . We examined 40 chemicals; 33 were reportedly positive in the CA but negat ive in the bacterial reverse mutation assay, six were negative in both assa ys and one was positive in both. We assayed mutations of the thymidine kina se (TK) locus (tk) of L5178Y tk(+/-) mouse lymphoma cells using the microwe ll method. According to our standard protocol, cells were exposed to the ch emical for 3 h, cultured for 2 days and TK-deficient mutants were expressed in 96-well plates under trifluorothymidine. Each chemical was coded and te sted by two or three laboratories. Among the 34 CA-positive chemicals, posi tive MLA results were obtained for 20 and negative results were obtained fo r nine. The remaining five chemicals were inconclusive or equivocal because of discrepant inter-laboratory results or reproduced discrepant results, r espectively. Among the six CA-negative chemicals, one was negative in the M LA, two were positive and three were inconclusive, Thus, the MLA could dete ct only 59% (20/34) of CA-positive chemicals. We concluded that the MLA was not as sensitive as the CA. Some MLA-negative chemicals evoked positive re sponses in the CA only after long continuous treatment. These might also be genotoxic in the MLA with long continuous treatment. Improvement of the ML A protocol, including alteration of the duration of the treatment, might re nder the MLA as sensitive as the CA.