Mutagenicity of EAT-positive NDSRIs in HepaRG spheroids

The Enhanced Ames Test (EAT) is currently recommended by regulatory authorities for evaluating the mutagenicity of N-nitrosamine drug impurities, including nitrosamine drug substance-related impurities (NDSRIs). In previous studies, we developed protocols to evaluate the genotoxicity and mutagenicity of NDSRIs in both two-dimensional (2D) and three-dimensional (3D) HepaRG cell cultures as a follow-up to verify EAT findings. In this study, we investigated the genotoxicity and mutagenicity of six EAT-positive NDSRIs, N-nitroso-desmethyl-diphenhydramine, N-nitroso-duloxetine, N-nitroso-lorcaserin, N-nitroso-nortriptyline, N-nitroso-propranolol, and N-nitroso-sertraline, in 3D HepaRG spheroids using a 14-day treatment protocol. Genotoxicity was evaluated using the CometChip DNA damage assay and the micronucleus (MN) assay, while mutagenesis was measured with an error-corrected sequencing (ECS) method, High-Fidelity sequencing (HiFi-seq). The six EAT-positive NDSRIs induced time- and concentration-dependent cytotoxicity in HepaRG spheroids. All except N-nitroso-lorcaserin caused DNA damage, while only N-nitroso-propranolol induced MN formation at acceptable levels of cytotoxicity. Notably, all six EAT-positive NDSRIs significantly increased mutation frequency in HepaRG spheroids, each exhibiting distinct mutational spectra. Quantitative analysis revealed varying levels of DNA damage and mutagenic potency among the six NDSRIs and two previously reported NDSRIs (N-nitroso-varenicline and N-nitroso-fluoxetine). These findings represent an important first step towards supporting the use of 3D HepaRG spheroids as a human-relevant in vitro new approach methodology (NAM) for mutagenicity assessment as a follow-up to the EAT. The results also provide evidence for genotoxic potency ranking of NDSRIs and offer mechanistic insights through mutational spectrum analysis.