Abstract
Nitrosamine drug substance-related impurities (NDSRIs) are a sub-category of N-nitrosamine drug impurities that share structural similarity to the corresponding active pharmaceutical ingredient. The mutagenicity of NDSRIs is poorly understood. We previously tested a series of NDSRIs using the Enhanced Ames Test (EAT). In this follow-up study, we further examined the genotoxicity and mutagenicity of 15 of these NDSRIs in human TK6 cells. Seven EAT-positive NDSRIs, including N-nitroso-nortriptyline, N-nitroso-fluoxetine, N-nitroso-desmethyl-diphenhydramine, N-nitroso-duloxetine, N-nitroso-lorcaserin, N-nitroso-varenicline, and N-nitroso-sertraline, induced concentration-dependent increases in micronuclei after bioactivation with hamster liver S9. These NDSRIs were also mutagenic in the TK and HPRT gene mutation assays, consistent with their positive EAT results. In the presence of hamster liver S9, the eight EAT-negative NDSRIs were negative in the micronucleus assay and negative for mutation induction. Using TK6 cells endogenously expressing a single human cytochrome P450 (CYP), we found that CYP2C19, CYP2B6, CYP2A6, and CYP3A4 are key enzymes activating the genotoxicity and mutagenicity of these NDSRIs. Overall, the hamster S9-mediated TK6 cell mutagenicity results agreed with those observed in the EAT, indicating consistency in the mutagenic responses produced by NDSRIs across different testing systems. These data support the use of EAT for hazard identification and safety assessment of NDSRIs.