From TD₅₀ to benchmark dose in nitrosamine risk assessment: evidence from N-Nitrosotrimetazidine carcinogenicity and...

The presence of N-nitrosamine drug substance-related impurities (NDSRIs) in pharmaceuticals represents a significant regulatory and safety challenge due to their classification as a "cohort of concern." 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 and the later published value of 400 ng/day, established using the Carcinogenic Potency Categorization Approach (CPCA). The evaluation followed a tiered approach: NTMZ was first confirmed as mutagenic in vitro in a standard Ames test. To further investigate its genotoxic potential, two in vivo studies were conducted in Wistar and transgenic rats. DNA strand breaks in the liver and duodenum (comet assay) and positive results in the Transgenic Rodent (TGR) confirmed an in vivo mutagenic mode of action. Benchmark Dose (BMD) analysis of the TGR data yielded a BMDL₅₀ of 7 mg/kg/day in male rat livers. To characterize long-term carcinogenic risk, a GLP-compliant 2-year carcinogenicity study was conducted in Wistar rats. Chronic exposure induced liver (hemangiosarcomas, hepatocellular carcinomas and adenomas) and intestinal tumors (adenomas and adenocarcinomas), leading to a Tumor Dose 50 (TD₅₀) of 23 mg/kg/day in male rats. BMD analysis of tumor incidence identified the lowest BMDL₁₀ of 2.6 mg/kg/day in females, used to derive an AI of 13 µg/day. Strong concordance between the transgenic rat BMD, carcinogenicity-study BMDL₁₀ and TD₅₀ supported derivation of a conservative AI of 13 µg/person/day based on the BMDL₁₀ and further support regulatory acceptance and use of BMD-derived approaches for nitrosamine evaluation.