N-NITROSO-BETAHISTINE IMPURITY INDUCED MUTAGENICITY METABOLICALLY ACTIVATED BY S9-MIX FROM RAT LIVER: A CASE STUDY

Resumo

INTRODUCTION: N-nitrosamines, or nitrosamines, are organic compounds containing a nitrous group, which are impurities found in human medicines with potential genotoxic and carcinogenic properties. Regarding nitrosamine impurity qualification, the European Medicines Agency (EMA) has published adaptations for the Ames test based on literature data (2023). OBJECTIVE: This study aimed to test the N-Nitroso-Betahistine impurity for its ability to induce reverse mutations at the his locus in four Salmonella typhimurium. MATERIALS AND METHODS: The N-Nitroso-Betahistine was provided by sponsor (confidential) and dissolved in sterile deionized water. The tests were conducted with strains TA98, TA100, TA1537 and TA1535 using the pre-incubation method with and without metabolic activation (20% and 30% S9-mix from rat liver) following OECD 471 and the EMA’s recommendations  with modifications. Although EMA also recommended hamster S9 fraction, this study was conducted with rat liver S9 only. Reference chemicals like NMor, NDEA, and NDMA, known mutagenic nitrosamine impurities, were also included in the experiments. This study is case study from laboratory of toxicology at NSF International conducted in November 2023.  RESULTS AND CONCLUSION: In strains TA100 and TA1535, in presence of metabolization, N-Nitroso-Betahistine showed positive results evaluated by mutagenicity index. For strains TA98 and TA1537, the N-Nitroso-Betahistine did not induce statistically significant reverse mutations, either with or without metabolic activation. When metabolically activated, the nitrosamine NMor significantly increased the frequency of mutations in the TA1535 and TA100 strains, while no response was observed in TA98 and TA1537. However, NDEA and NDMA did not increase mutation frequency when activated metabolically. The tests with the nitrosamine NMor using the pre-incubation protocol confirm that the genetic toxicity of nitrosamines occurs through activation by the P-450 microsomal system (CYP 450) via enzyme action. In conclusion, the nitrosamine impurity tested, induced point mutations by frame shifts mutation in the genome of Salmonella typhimurium, using the pre-incubation method with rat liver metabolic activation. Although our results indicate that the rat S9 fraction did not activate the controls NDEA and NDMA, but activated NMor, the N-Nitroso-Betahistine impurity tested was clearly mutagenic. Both concentrations of rat S9-mix, 20% or 30%, in the mutagenicity assessment, showed efficient metabolic activation.