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

Abstract

In order to evaluate the utility of the mouse lymphoma assay (MLA) for detecting in vitro clastogens and spindle poisons and to compare it with the in vitro chromosomal aberration test (CA), we conducted an international collaborative 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 negative in the bacterial reverse mutation assay, six were negative in both assays and one was positive in both. We assayed mutations of the thymidine kinase (TK) locus (tk) of L5178Y tk ϩ/-mouse lymphoma cells using the microwell method. According to our standard protocol, cells were exposed to the chemical for 3 h, cultured for 2 days and TK-deficient mutants were expressed in 96-well plates under trifluorothymidine. Each chemical was coded and tested by two or three laboratories. Among the 34 CA-positive chemicals, positive MLA results were obtained for 20 and negative results were obtained for nine. The remaining five chemicals were inconclusive or equivocal because of discrepant inter-laboratory results or reproduced discrepant results, respectively. Among the six CA-negative chemicals, one was negative in the MLA, two were positive and three were inconclusive. Thus, the MLA could detect 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 responses in the CA only after long continuous treatment. These might also be genotoxic in the MLA with long continuous treatment. Improvement of the MLA protocol, including alteration of the duration of the treatment, might render the MLA as sensitive as the CA. IntroductionThe genotoxicity tests that have been developed and validated over the years differ in the biological system used (prokaryotic,

Mono-Ubiquitination of Nuclear Annexin A1 and Mutagenesis

“…Under these conditions, the mutation rate of the thymidine kinase gene increased from 23 x 10 -6 (vehicle control) to 67 x 10 -6 and 104 x 10 -6 with AsCl3 or MMS, respectively, as measured by the number of colonies with trifluorothymidine resistance according to the method described by Honma et al (1999). These observations suggest that DNA damage was induced under these conditions.…”

Models for Detection of Genotoxicity in vivo: Present and Future

“…Inactivating the WT allele induces trifluorothymidine (TFT) resistance, and tk−/− mutants can be selected for in a background of tk+/− non-mutant cells (45)(46)(47). Colony size is an indicator of the type of mutation involved: large colonies typically correspond to small TK-inactivating mutations, while small colonies often indicate clastogenic damage.…”

Scientific Opinion on Flavouring Group Evaluation 20, Revision 4 (FGE.20Rev4): Benzyl alcohols, benzaldehydes, a related acetal, benzoic acids, and related esters from chemical groups 23 and 30

“…Cytotoxicity was reported above 500 µg/ml with and without S9. (Honma et al, 1999a) Published collaborative study on 40 chemicals. Protocol was in accordance with OECD Guideline 476, except that no colony sizing was performed.…”