N-nitroso-hydrochlorothiazide: in vivo mutagenicity evaluation in a transgenic rodent model.

N-nitrosamine drug substance related impurities (NDSRIs) are considered to be within the "cohort of concern" (CoC) due to their potential mutagenic carcinogenicity. However, increasing evidence indicates variability in mutagenic potency across this class. N-nitroso-hydrochlorothiazide (N-nitroso-HCTZ), an impurity associated with hydrochlorothiazide, has shown positive results in bacterial mutagenicity (Ames) assays, although mechanistic studies suggest these findings are likely driven by reactive degradation products rather than the parent compound. To further characterize mutagenic potential in vivo, a transgenic rodent (TGR) gene mutation assay was conducted in the Muta™Mouse test system. Male mice were administered N-nitroso-HCTZ by oral gavage at 0, 18.5, 55.7, 167, 500, or 750 mg/kg/day for 28 consecutive days. Mutant frequency (lacZ) was evaluated in liver and glandular stomach at 167, 500, and 750 mg/kg/day. Systemic exposure was confirmed in a toxicokinetic study following a single 750 mg/kg dose. N-nitroso-HCTZ was well tolerated, and no statistically significant increases in mutant frequency were observed in either tissue at any evaluated dose. All values were within historical control ranges, while the positive control demonstrated the anticipated response. Under the conditions of this study, N-nitroso-HCTZ did not induce gene mutations in vivo.