Optimizing the detection of N-nitrosamine mutagenicity in the Ames test

Abstract

Accurately determining the mutagenicity of small-molecule N-nitrosamine drug impurities and nitrosamine drug substance-related impurities (NDSRIs) is critical to identifying mutagenic and cancer hazards. In the current study we have evaluated several approaches for enhancing assay sensitivity for evaluating the mutagenicity of N-nitrosamines in the bacterial reverse mutagenicity (Ames) test. Preincubation assays were conducted using five activation conditions: no exogenous metabolic activation and metabolic activation mixes employing both 10% and 30% liver S9 from hamsters and rats pretreated with inducers of enzymatic activity. In addition, preincubations were conducted for both 60 min and 30 min. These test variables were evaluated by testing 12 small-molecule N-nitrosamines and 17 NDSRIs for mutagenicity in Salmonella typhimurium tester strains TA98, TA100, TA1535, and TA1537, and Escherichia coli strain WP2 uvrA (pKM101). Eighteen of the 29 N-nitrosamine test substances tested positive under one or more of the testing conditions and all 18 positives could be detected by using tester strains TA1535 and WP2 uvrA (pKM101), preincubations of 30 min, and S9 mixes containing 30% hamster liver S9. In general, the conditions under which NDSRIs were mutagenic were similar to those found for small-molecule N-nitrosamines.

Keywords: Acceptable intake limit; Ames test; Carcinogenic potency categorization approach; Drug impurities; Hamster liver S9; Mutagenicity dose-response ranking; Nitrosamine drug substance-related impurities; Preincubation; Rat liver S9; Small-molecule N-Nitrosamines.

Fig. 1.. Performance of Ames tester strains in detecting the mutagenicity of N-nitrosamines.

(A) Heatmap shows the number of test conditions that detected positive responses with each tester strain. Small-molecule nitrosamines and NDSRIs are listed separately in the order of their molecular weight. A total of 10 Ames test conditions (5 S9 conditions 2 preincubation time) were evaluated for each strain. (B) Summary of the total number of positive small-molecule nitrosamines and NDSRIs detected by each tester strain.

Fig. 2.. Performance of different S9 conditions in detecting the mutagenicity of N-nitrosamines.

(A) Heatmap showing the number of test conditions that detected positive response with each S9 condition. Small-molecule nitrosamines and NDSRIs are listed separately in the order of their molecular weight. A total of 10 Ames test conditions (5 tester strains × 2 preincubation time) were evaluated for each S9 activation condition. (B) Summary of the total number of Ames-positive small-molecule nitrosamines and NDSRIs detected by each S9 activation condition.