2023 FDA Science Forum
Genotoxicity assessment of eight nitrosamine impurities using 2D and 3D in vitro human HepaRG models.
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Contributing OfficeNational Center for Toxicological Research
Abstract
N-nitrosamine impurities have been detected increasingly in human drugs. This is a safety concern as most nitrosamines are mutagenic in bacteria, genotoxic in mammalian cells and carcinogenic in rodent models. There are, however, only limited data on the genotoxic potency of nitrosamines in human cell systems. The mutagenic and carcinogenic activity of most nitrosamines require metabolic activation by CYP450 enzymes. In this study, we used metabolically competent human HepaRG cells, whose metabolic capability is comparable to that of primary human hepatocytes, to evaluate the genotoxicity of eight nitrosamine impurities [N-cyclopentyl-4-nitrosopiperazine (CPNP), N-nitrosodibutylamine (NDBA), N-nitrosodiethylamine (NDEA), N-nitrosodimethylamine (NDMA), N-nitrosodiisopropylamine (NDIPA), N-nitrosoethylisopropylamine (NEIPA), N-nitroso-N-methyl-4-aminobutyric acid (NMBA), and N-nitrosomethylphenylamine (NMPA)]. Given that three-dimensional (3D) spheroids possess higher levels of CYP activity compared to 2D monolayer cells, the genotoxicity of the eight nitrosamines was investigated in both 2D and 3D HepaRG models using the high-throughput CometChip assay and the flow cytometry-based micronucleus (MN) assay. Following a 24-hr treatment, all the nitrosamines induced DNA damage in 3D spheroids, while only three nitrosamines, NDBA, NDEA, and NDMA produced positive responses in 2D HepaRG cells. In addition, these three nitrosamines also caused significant increases in MN frequency in both 2D and 3D HepaRG models, while NMBA and NMPA were positive only in the 3D HepaRG MN assay. Quantitative benchmark dose analysis showed that under the current experimental conditions, NDMA produced the most compelling response in 3D HepaRG models. Overall, benchmark dose analysis indicated that 3D HepaRG spheroids were more sensitive at detecting the genotoxicity of the eight nitrosamines than 2D cultures. These results suggest that 3D HepaRG spheroids may be useful as a New Approach Method (NAM) for detecting the genotoxicity of nitrosamine impurities that result from human metabolic activation.
