Evaluation of Mutagenic Potential of Nitrosamines Using the Enhanced Ames Test with Hamster Liver S9 Activation

Nitrosamines (NAs) and Nitrosamine Drug Substance-Related Impurities (NDSRIs) have recently drawn significant attention because of their strong links to cancer and genetic damage. The standard Ames test, described in Organisation for Economic Co-operation and Development (OECD) Guideline 471, is widely used to assess mutagenicity, but it often struggles with certain nitrosamines, especially those requiring complex metabolic activation or producing weak signals at low doses. To address these limitations, regulators now recommend the Enhanced Ames Test (EAT) for improved sensitivity. In this study, we assessed three nitrosamines, N-nitrosodimethylamine (NDMA), 1?cyclopentyl?4?nitrosopiperazine (CPNP), and N-nitrosodiethylamine (NDEA), using the EAT. We began with cytotoxicity evaluations in Salmonella typhimurium ( S. typhi .) strains TA100 and TA1535 to determine the highest non-toxic doses. Each compound was then preincubated with 30% liver S9 enzymes from rats and hamsters for 30 minutes to better replicate metabolic activation. Hamster S9 produced stronger mutagenic responses than rat S9. Among the tested strains, TA1535 showed the highest sensitivity. NDMA produced 2.3-2.7?fold higher mutagenicity in TA1535 compared with other strains. CPNP showed an even stronger effect, with a five?fold increase. NDEA was the most potent, generating nearly a five?fold stronger response with hamster S9 than with rat S9 (88.7?fold vs. 17.2?fold per dose). Overall, these findings highlight that the EAT, combined with optimized metabolic activation, is a robust approach for detecting mutagenic nitrosamines that may otherwise remain undetected.