Quantitative genotoxicity assessment of N-nitrosodimethylamine in mice by error-corrected next-generation sequencing and DNA methylation quantification for toxicity threshold determination

The contamination of N-nitrosamines (N-NAs) in pharmaceuticals has raised global concerns, due to potential carcinogenic risks. N-nitrosodimethylamine (NDMA) is a rodent carcinogen and the most prevalent N-NAs impurity in drug products. Current acceptable intake of NDMA (96 ng/day) is a simple linear extrapolation from rodent carcinogenicity data, which may not comprehensively characterize its genotoxic potential in humans. Most N-NAs react with DNA to form DNA adducts after metabolic activation, leading to mutations and carcinogenesis. Here, we combined high-accuracy LC–MS/MS with error-corrected next-generation sequencing (ecNGS) to assess NDMA-induced DNA adduction and somatic mutations in mice using the quantitative benchmark dose (BMD) approach. NDMA induced dose-dependent increases in both DNA adduction and somatic mutations in liver tissues, with significant increases in mutation frequencies observed at ≥ 1 mg/kg/day. NDMA induced mutations mainly with CG > TA and TA > CG transitions, exhibiting a signature resembling COSMIC signature 11, the mutational profile of alkylating agents. BMD₅₀ (90% CI) was estimated as 0.08–0.32 mg/kg for N7-methylguanine and 1.64–3.83 mg/kg for mutations. Permitted daily exposure (PDE) for NDMA was calculated from the lower confidence limit of BMD₅₀ (BMDL₅₀) of genotoxic endpoints using appropriate uncertainty factors, revealing a PDE value of 245 ng/day for NDMA-induced mutation. This PDE value was higher than the limit of 96 ng/day recommended by regulatory agencies. Based on the quantitative genotoxicity assessment, our findings may improve the risk assessment of NDMA-contaminated pharmaceuticals and demonstrate the potential implementation of ecNGS in regulatory decision-making.