The FDA published a new literature that utilizes HepaRG spheroids as an alternative in vitro model for detecting NDSRI-induced genotoxicity.
Highlights
•All five EAT-positive NDSRIs significantly increased % tail DNA in both 2D and 3D HepaRG cell models.
•These compounds also increased micronucleus (MN) frequency and γH2A.X formation in 3D spheroids.
•In contrast, the five EAT-negative NDSRIs did not induce DNA damage or MN formation in either cell model.
•Under the tested conditions, N-nitroso-nortriptyline was the most potent genotoxicant in HepaRG cells.
•3D HepaRG spheroids were more sensitive than 2D HepaRG cells in detecting NDSRI-induced genotoxicity.
Discussion
Regulatory agencies recommend the EAT to evaluate the mutagenic risks of N-nitrosamines (FDA, 2023; Health-Canada, 2023). Besides a negative finding in the EAT, the FDA currently recommends additional data to support increasing a CPCA-derived AI limit to 1500 ng/day including a second in vitro mammalian cell mutation assay and in vitro metabolism data (including using human hepatocytes or microsomes) (FDA, 2025).
HepaRG cells are human-derived and possess an endogenous metabolic activation.