Evaluation of the division arrest method of the CHO/HGPRT mutation assay

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Abstract
We have evaluated the division arrest method of the CHO/HGPRT mutation assay following the procedure described by O'Neill et al. Environ. Mutagen. 4, 421–434 (1982). This method simplifies the culture manipulations required during the expression period and can be readily adopted for screening mutagenic compounds. The mutation frequencies derived from the subculture method and the division arrest (no subculture) method after treatment of CHO‐K1BH4 cells with ethyl methanesulfonate were similar at all concentrations. The background mutation frequencies observed in the medium and solvent (0.5% DMSO) controls ranged from 0 to 9 colonies per 1 × 106 cells. Twenty to 24 h treatments, using the direct‐acting compounds ethyl methanesulfonate, N‐methyl‐N′‐nitro‐N‐nitrosoguanidine, ICR‐191 acridine, 5‐Bromo‐2′‐deoxyuridine, mitomycin C and high concentrations of thymidine produced positive mutagenic responses, while 2′‐deoxyuridine and trifluorothymidine gave negative responses. Four‐h treatments with Benzo[a]pyrene, diethylnitrosamine, 2−acetylaminofluorene, 3‐methylcholanthrene and 2‐aminoanthracene in the presence of S9 also produced positive results. Anthracene, fluorene and pyrene with S9 were negative. However we found that a toxic, non‐mutagenic compound (anthracene) can yield sporadic increases in mutation frequencies (1 out of 10 replicates). Overall, our results indicate that the division arrest method of the CHO/HGPRT mutation assay can be reliably used for routine screening.