Studies on the activities of benzo[ <italic>a</italic> ]pyrene, benzidine and ethyl methanesulphonate in the L5178Y TK <sup>+/-</sup> mouse lymphoma mutagenicity assay using standardized and non-standardized protocols

Abstract

As part of the third UKEMS collaborative trial, ethylmethane sulphonate (EMS), benzo[a]pyrene (B[a]P) and benzidine (BZD) were assayed for mutagenicity using the L5178Y mouse lymphoma TK +/- forward mutation system. Exogenous metabolic activation was achieved with two different sources of rat liver S9 which were used under optimized conditions for B[a]P and BZD, the latter being tested also without S9. EMS was assayed in the absence of S9 only. Mutants were selected for trifluorothymidine (TFT) resistance after 48 and 72 h expression time. Mutant frequency (MF) data were subjected to ANOVA analysis and t-tests for differences between replicate cultures, linear trend and differences from solvent controls. Large TFT resistant colonies are believed to be due to induction of small amounts of genetic damage (point mutations, small deletions) at the tk locus, whilst small colonies are thought to be a result of larger effects. The relative proportions of the two colony types were determined in some experiments. All three compounds induced dose-related increases in MF under all conditions. B[a]P induced equal proportions of large and small colonies at all doses. EMS induced predominantly large colonies at all doses. The effect of BZD on colony size was variable. The source of S9 did not exert any consistent effect on toxicity, mutagenicity or mutant colony size. Maximum MF values occurred at 72 h for B[a]P and BZD but the results with EMS were variable. No consistent differences were apparent between estimates of toxicities made either by cloning immediately after treatment (day 0 cloning) or by estimating the relative total growth (RTG) at expression time.