TA-100

The Ames test is a critical assay for assessing the mutagenic potential of chemical compounds; however, its conventional version (OECD TG 471) falls short in detecting nitrosamine mutagenicity due to insufficient metabolic activation. Given the ubiquity of nitrosamines as environmental contaminants and drug impurities, an enhanced protocol incorporating 30 % v/v hamster and rat liver S9 fractions with a 30-minute pre-incubation is warranted. This study streamlined the enhanced Ames test by evaluating the sensitivity of S. typhimurium strains TA1535 and TA100, and E. coli WP2 uvrA (pKM101), against N‑nitrosodimethylamine (NDMA) and 1‑cyclopentyl‑4‑nitrosopiperazine (CPNP). Dose‑normalized fold‑induction metrics revealed that TA1535 offers superior sensitivity across varied S9 conditions. Hamster liver S9 generally enhanced mutagenic responses compared to rat liver S9. Notably, CPNP was more potent in TA1535 and TA100, while NDMA was more active in WP2 uvrA (pKM101) with rat S9. This optimized screening strategy minimizes resources and is recommended for initial nitrosamine impurity mutagenicity detection.

The Ames test is required by regulatory agencies worldwide for assessing the mutagenic and carcinogenic potential of chemical compounds. This test uses several strains of bacteria to evaluate mutation induction: positive results in the assay are predictive of rodent carcinogenicity. As an initial step to understanding how well the assay may detect mutagens present as constituents of complex mixtures such as botanical extracts, a cross‐sector working group examined the within‐laboratory reproducibility of the Ames test using the extensive, publicly available National Toxicology Program (NTP) Ames test database comprising more than 3000 distinct test articles, most of which are individual chemicals. This study focused primarily on NTP tests conducted using the standard Organization for Economic Co‐operation and Development Test Guideline 471 preincubation test protocol with 10% rat liver S9 for metabolic activation, although 30% rat S9 and 10 and 30% hamster liver S9 were also evaluated. The reproducibility of initial negative responses in all strains with and without 10% S9, was quite high, ranging from 95% to 99% with few exceptions. The within‐laboratory reproducibility of initial positive responses for strains TA98 and TA100 with and without 10% rat liver S9 was ≥90%. Similar results were seen with hamster S9. As expected, the reproducibility of initial equivocal responses was lower, <50%. These results will provide context for determining the optimal design of recommended test protocols for use in screening both individual chemicals and complex mixtures, including botanicals.