OBJECTIVE  To evaluate the risk of gene mutation of nitrosamine drug substance-related impurities(NDSRI) V6-NO in valsartan by combining genotoxicity software prediction and bacterial reverse mutation test(Ames test).

METHODS Classification of V6-NO was firstly predicted by the CASE Ultra software, followed by the evaluation of the mutagenicity of V6-NO by the Ames test using the standard plate incorporation method(±S₉: 10% rat liver S₉ mixture), as well as by the Mini-Ames test using the pre-incubation method(±S₉: 30% rat liver S₉ mixture, pre-incubated at 37 ℃ for 1 h).

RESULTS  Because of the N-nitroso alert structure of V6-NO, the prediction results were positive based on statistical-based model and expert rule-based model, which fell into category 3 of ICH M7 classification, though the prediction results of statistical-based model indicated a total of six deactivating features of impurity structure. Ames test showed that in presence and absence of S₉ conditions, the number of revertant colonies of V6-NO at a dose range of 62 μg per plate to 5 000 μg per plate did not exceed 2 times of that vehicle control group using standard plate incorporation method. Similarly, Mini-Ames test showed that the number of revertant colonies of V6-NO at a dose range of 62.5 μg per well to 1 000 μg per well did not exceed 2 times of that vehicle control group using pre-incubation method. Both the Ames test and the Mini-Ames test results of V6-NO were non-mutagenic.

CONCLUSION  When the prediction result of NDSRI software is positive and inconsistent with the Ames test results, it is necessary to further verify and analyze the results comprehensively, the analysis should incorporate the activating and deactivating feature of the structure of the test compound, followed by improving the Ames test conditions, in order to analyze and understand the mutagenicity risk of NDSRI comprehensively, and provide valuable reference information for drug development.