Nitrosamine drug substance-related impurities cause DNA methylation adducts in vitro and in primary hepatocytes upon Cytochrome P450-dependend metabolic activation

N -nitrosamines are DNA alkylating agents found in food, cosmetics, tobacco products and, more recently, drugs. Following Cytochrome P450 (CYP)-mediated metabolic activation, these compounds cause DNA damage and mutations. Unlike well-characterized compounds like N -nitrosodimethylamine (NDMA), data on the genotoxicity of nitrosamine drug substance-related impurities (NDSRIs) remain limited. Given their regulatory relevance, this study assessed the genotoxic potential of three NDSRIs — N -nitrosobetahistine (NBH), N -nitrosofluoxetine (NFluo), and N -nitrosonortriptyline (NNT) —compared to NDMA. The NDSRIs demonstrated distinct DNA methylating potential, confirmed by elevated levels of N 7-methyl-deoxyguanosine ( N7 -MedG) and O 6 -methyl-deoxyguanosine ( O 6 -MedG) in a DNA alkylation assay with metabolic activation. Recombinant CYP isoforms contributed differentially to the bioactivation of each NDSRI, highlighting enzyme-specific pathways of toxification. Subsequently, we demonstrated that all NDSRIs cause DNA methylation adducts ( N 7-MedG > O 6 -MedG) in primary rat hepatocytes, with generally higher levels than those caused by NDMA. Consistently, the NDSRIs generated more DNA strand breaks than NDMA, which followed the DNA adduct kinetics. Furthermore, all NDSRIs showed cytotoxicity after 24 h, whereas no cytotoxic effect was observed for NDMA. Taken together, our study provided evidence that the three NDSRIs are genotoxic in primary rat hepatocytes, which warrants further investigation with regard to their mutagenic potential.