Complex versus simple N-nitrosamines: Comprehensive genotoxicity and in silico carcinogenicity assessment toward...

The detection of N-nitrosamines in pharmaceuticals has been a focus for industry and regulatory authorities since 2018. This has prompted extensive research into their mutagenic potential and mitigation strategies. We present a comprehensive evaluation of 25 structurally diverse N-nitrosamines using in vitro (Ames test), in vivo (liver comet, Big Blue transgenic mutation, and duplex sequencing), and in silico methods [quantum mechanical modeling and cytochrome P450 (CYP)-binding analysis]. Results show strong concordance across assays and reveal that complex active pharmaceutical ingredient (API)-derived or process-related impurity (PRI)-derived N-nitrosamines are often not mutagenic or have low mutagenic potency. Quantitative in vivo data enabled the derivation of compound-specific acceptable intakes (AIs), frequently exceeding regulatory limits based on an assessment of structural features. Duplex sequencing demonstrated superior sensitivity over traditional assays, and quantum mechanical models proved effective for potency prediction. A weight-of-evidence decision tree to derive AIs that integrates experimental and computational data is proposed, which reduces animal testing and supports drug product remediation efforts that are commensurate with risk.