Chinese Journal of Pharmacovigilance ›› 2026, Vol. 23 ›› Issue (5): 487-493.
DOI: 10.19803/j.1672-8629.20260219

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Mutagenic Risk Prediction and Experimental Validation of Heterocyclic Nitroso Impurities

WEN Hairuo1,2, JIN Longlong3△, KOU Xiaoxuan1,2, JIANG Chenchen1,2, YE Xiao1,2, ZHANG Ruiqiu1,2, TIAN Ye1,2#, WU Xianfu3,*   

  1. 1Institute of Safety Evaluation, National Institutes for Food and Drug Control, Beijing 100176, China;
    2State Key Laboratory of Drug Regulatory Science, Beijing 102629, China;
    3National Institutes for Food and Drug Control, Center of Reference Standards and Standardization, Beijing 102629, China
  • Received:2026-03-17 Published:2026-05-20

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Abstract: Objective To evaluate the mutagenic risk of three drug-related heterocyclic nitroso impurities, and to provide data for setting regulatory limits. Methods Three (quantitative) structure-activity relationship [(Q)SAR] prediction software packages, namely Lhasa, Leadscope, and CASE Ultra, were used to predict the mutagenic risk of N-nitroso-paroxetine, N-nitroso-reboxetine, and N-nitroso-vortioxetine hydrobromide. Enhanced bacterial reverse mutation tests were conducted, involving Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and Escherichia coli strain WP2 uvrA (pKM101), both in the absence and presence of a 30% hamster S9 metabolic activation kit to evaluate their mutagenic potential. The benchmark dose (BMD) levels of these heterocyclic nitroso impurities were compared using the PROAST software (an R-based package) to study the correlations between the strength of mutagenic risk and structural modifications. Results As was predicted by (Q)SAR, Lhasa and Leadscope, based on structural characteristics, suggested a mutagenic risk for all the three heterocyclic nitroso impurities. In contrast, CASE Ultra, which relied on internal research data alone, predicted that N-nitroso-paroxetine posed no risk of bacterial reverse mutation. In the enhanced bacterial reverse mutation tests, all the test substances yielded negative results under non-metabolic activation. However, under metabolic activation with hamster S9, all the heterocyclic N-nitroso impurities, except N-nitroso-paroxetine, induced an increase in the number of revertant colonies, indicating a mutagenic risk. Based on BMDL values, N-nitroso-reboxetine was believed to be the most mutagenically potent for strain TA98, and N-nitroso-vortioxetine hydrobromide for strain TA100. Conclusion The three common heterocyclic nitroso impurities, except N-nitroso-paroxetine, demonstrate bacterial mutagenicity. Nitrosamine impurities containing a nitroso group within a six-membered ring are likely to induce frameshift mutations. These findings are expected to provide data for regulatory control of impurities in heterocyclic drugs and for risk prediction of nitrosamine impurities based on chemical structure-activity relationships.

Key words: Heterocyclic Nitroso Impurities, Mutagenicity, Structure-Activity Relationship Prediction, Enhanced Bacterial Reverse Mutation Test, Benchmark Dose Level

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