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Published on: September 25, 2017
Optimizing the detection of N-nitrosamine mutagenicity in the Ames test
Robert H Heflich1, Michelle E Bishop1, Roberta A Mittelstaedt1
1U.S. Food and Drug Administration, National Center for Toxicological Research, USA.
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.
Insights
Determining the mutagenicity of N-nitrosamine drug impurities is crucial for safety. Optimized Ames test conditions, including specific bacterial strains and 30-minute preincubation with 30% hamster liver S9, effectively detect these mutagenic hazards.
Area of Science:
- Pharmaceutical Chemistry
- Toxicology
- Genetics
Background:
- N-nitrosamines are potent mutagens and carcinogens found as impurities in pharmaceuticals.
- Accurate assessment of mutagenicity is vital for drug safety and regulatory compliance.
- The bacterial reverse mutagenicity (Ames) test is a standard method for detecting mutagenic potential.
Purpose of the Study:
- To evaluate and optimize assay sensitivity for detecting mutagenicity of small-molecule N-nitrosamines and nitrosamine drug substance-related impurities (NDSRIs).
- To identify the most effective conditions within the Ames test for identifying mutagenic N-nitrosamine impurities.
Main Methods:
- Tested 12 small-molecule N-nitrosamines and 17 NDSRIs using the Ames test.
- Employed five activation conditions: no exogenous metabolic activation, and metabolic activation with 10% and 30% liver S9 from induced rats and hamsters.
- Varied preincubation times (30 and 60 minutes) and utilized Salmonella typhimurium strains (TA98, TA100, TA1535, TA1537) and Escherichia coli strain WP2 uvrA (pKM101).
Main Results:
- Eighteen out of 29 N-nitrosamine test substances showed mutagenicity under at least one condition.
- Optimal detection of all 18 positive N-nitrosamines was achieved using tester strains TA1535 and WP2 uvrA (pKM101).
- A 30-minute preincubation period with 30% hamster liver S9 mix proved most effective for detecting mutagenicity.
- Mutagenic conditions for NDSRIs generally mirrored those for small-molecule N-nitrosamines.
Conclusions:
- The study identified specific Ames test conditions that enhance sensitivity for detecting mutagenic N-nitrosamine impurities.
- A combination of tester strains TA1535 and WP2 uvrA (pKM101), 30-minute preincubation, and 30% hamster liver S9 is recommended for improved N-nitrosamine mutagenicity assessment.
- These optimized conditions are critical for identifying potential cancer hazards associated with N-nitrosamine impurities in pharmaceuticals.
