Quantum chemical calculations of nitrosamine activation and deactivation pathways for carcinogenicity risk assessment.

N-nitrosamines and nitrosamine drug substance related impurities (NDSRIs) became a critical topic for the development and safety of small molecule medicines following the withdrawal of various pharmaceutical products from the market. To assess the mutagenic and carcinogenic potential of different N-nitrosamines lacking robust carcinogenicity data, several approaches are in use including the published carcinogenic potency categorization approach (CPCA), the Enhanced Ames Test (EAT), mutagenicity studies as well as read-across to analogue molecules with robust carcinogenicity data. We employ quantum chemical calculations as a pivotal tool providing insights into the likelihood of reactive ion formation and subsequent DNA alkylation for a selection of molecules including e.g., carcinogenic N-nitrosopiperazine (), N-nitrosopiperidine (), together with N-nitrosodimethylamine () as well as non-carcinogenic N-nitrosomethyl-tert-butylamine () and bis (butan-2-yl) (nitros)amine (). In addition, a series of nitroso-methylaminopyridines is compared side-by-side. We draw comparisons between calculated reaction profiles for structures representing motifs common to NDSRIs and those of confirmed carcinogenic and non-carcinogenic molecules with data from cancer bioassays. Furthermore, our approach enables insights into reactivity and relative stability of intermediate species that can be formed upon activation of several nitrosamines. Most notably, we reveal consistent differences between the free energy profiles of carcinogenic and non-carcinogenic molecules. For the former, the intermediate diazonium ions mostly react, kinetically controlled, to the more stable DNA adducts and less to the water adducts via transition-states of similar heights. Non-carcinogenic molecules yield stable carbocations as intermediates that, thermodynamically controlled, more likely form the statistically preferred water adducts. In conclusion, our data confirm that quantum chemical calculations can contribute to a weight of evidence approach for the risk assessment of nitrosamines.