Adjusting the Preincubation Conditions to Enhance the Ames Test for Detecting the Mutagenicity of N-Nitrosamines.

Conducting S9-mediated preincubations at slightly acid pHs (e.g., pH 5.0 to pH 6.5) has been reported to enhance the mutagenicity of small-molecule nitrosamines in the Ames test. In this study, we have evaluated the effect of adjusting the preincubation mix used in the Enhanced Ames Test (EAT) from pH 7.4 to pH 6.0 and supplying the activation mix with preformed reducing equivalents (NADPH and NADH). Abbreviated EAT assays were conducted on five small-molecule N-nitrosamines and 10 nitrosamine drug substance-related impurities (NDSRIs) with tester strains TA1535 and WP2 uvrA (pKM101) and using S9 mixes containing 30% hamster liver S9. Testing on small-molecule nitrosamines found that the pH 6.0 preincubation mix enhanced the mutagenicity of N-nitroso-dimethylamine and N-nitroso-diethylamine but not N-nitroso-diphenylamine, N-nitroso-methyl-4-aminobutyric acid or N-(2,2-diethoxyethyl)-2,2-diethoxy-N-nitrosoethanamine. Of the 10 NDSRIs that were tested, N-nitroso-phenylephrine was consistently positive with the pH 6.0 preincubation mix, while it was generally negative with pH 7.4 preincubation mixes. The mutagenicity of the nine other NDSRIs that were tested was not changed by the slightly acid preincubation mix. The results indicate that performing preincubation reactions under slightly acid conditions increases the mutagenicity of some small-molecule nitrosamines, and in one case, produced a positive mutagenic response with an NDSRI that was otherwise negative in the EAT.