Abstract
ABSTRACT
Nitrosamine drug impurities, including both small‐molecule nitrosamines and nitrosamine drug substance‐related impurities (NDSRIs), are members of a cohort of concern for pharmaceutical safety assessment. As the toxicity of most nitrosamine impurities requires metabolic activation by cytochrome P450 enzymes, this study used human HepaRG cells, which have metabolic activity similar to that of human hepatic cells, to evaluate methods for nitrosamine safety assessment. The genotoxicity of four small‐molecule nitrosamines,
N‐
bis(2,2‐diethoxyethyl) nitrous amide (BDEA),
N
‐nitrosodiphenylamine,
N,N
‐nitroso‐
N
‐ethylaniline (NEPA), and 1‐methyl‐4‐nitrosopiperazine (MNP), and five NDSRIs,
N
‐nitroso‐bumetanide,
N
‐nitroso‐ciprofloxacin,
N
‐nitroso‐dabigatran etexilate,
N
‐nitroso‐desmethyl‐diphenhydramine, and
N
‐nitroso‐sertraline, was evaluated in two‐dimensional (2D)/attached and three‐dimensional (3D)/spheroid HepaRG cell cultures using two high‐throughput genetic toxicity assays, the CometChip DNA damage assay and the flow‐cytometric micronucleus (MN) assay. These test substances exhibited a range of responses in other genotoxicity assays, including strong positive, negative, and inconclusive results. Following a 24‐h treatment, two NDSRIs,
N
‐nitroso‐desmethyl‐diphenhydramine and
N
‐nitroso‐sertraline, and two small‐molecule nitrosamines, BDEA and MNP, induced DNA damage, with the small‐molecule nitrosamines positive only in 3D HepaRG spheroids. MN formation was detected only in spheroids exposed to
N
‐nitroso‐desmethyl‐diphenhydramine. Benchmark concentration (BMC) values derived from the DNA damage data were lower for the genotoxic NDSRIs compared to the genotoxic small‐molecule nitrosamines, which correlated with higher cytotoxicity for the NDSRIs. These results show that, while HepaRG spheroids were a more sensitive model for the hazard identification of small‐molecule nitrosamines, the responses produced by NDSRIs were more complex, with NDSRIs tested producing similar levels of DNA damage in 2D and 3D cultures and inducing MN formation only in 3D spheroids.