From TD 50 to Benchmark Dose in Nitrosamine Risk Assessment: Evidence from N-Nitrosotrimetazidine Carcinogenicity and TGR Mutation Data
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The presence of N-nitrosamine drug substance-related impurities (NDSRIs) in pharmaceuticals represents a significant regulatory and safety challenge due to their classification as “cohort of concern” compounds. This paper describes the toxicological evaluation of N-Nitrosotrimetazidine (NTMZ), performed to refine the initial default acceptable intake (AI) limits of 18–26.5 ng/day established by regulatory authorities.
The evaluation followed a tiered approach: NTMZ was first confirmed as mutagenic in vitro via the standard Ames test. To further investigate its genotoxic potential, two in vivo studies were conducted in Wistar and transgenic rats. Detection of DNA strand breaks in the liver and duodenum (comet assay) together with positive results in the c II mutation assay confirmed an in vivo mutagenic mode of action. Benchmark Dose (BMD) analysis of the transgenic rat data yielded a BMDL 50 of 7 mg/kg/day in the male liver.
To characterize long-term carcinogenic risk, a GLP-compliant 2-year carcinogenicity study was conducted in Wistar rats. Chronic exposure induced dose-dependent increases in liver tumors (hemangiosarcomas, hepatocellular carcinomas and adenomas) and intestinal tumors (adenomas and adenocarcinomas), leading to a Tumor Dose 50 (TD 50 ) of 23 mg/kg/day in male rats. Benchmark dose analysis of tumor incidence identified a lowest BMDL 10 of 2.6 mg/kg/day in females, which served as the basis for deriving an AI of 13 µg/day.
This assessment demonstrates a strong predictive correlation between the BMD derived from the in vivo transgenic model, the BMDL 10 and the final TD 50 values obtained in the 2-year carcinogenicity study. These findings provided the scientific basis for establishing a conservative AI of 13 µg/person/day based on the BMDL 10 and further support the regulatory acceptance and use of BMD-derived approaches for the evaluation of nitrosamine impurities.
