Abstract: Objective To evaluate the mutagenicity risk of N-nitroso bumetanide in vitro and provide data for its safety assessment. Methods Structure-activity relationships were analyzed using Derek Nexus and Sarah Nexus software to predict mutagenicity and structural alerts. An enhanced bacterial reverse mutation test was conducted according to EMA guidelines using Salmonella typhimurium TA98, TA100, TA1535, TA1537, and Escherichia coli WP2 uvrA(pKM101) strains in the absence of post-mitochondrial fraction (S9), and also in the presence of 30% rat liver s9, as well as 30% hamster liverS9,followed by 30 min of pre-incubation. Additionally, a mouse lymphoma assay (MLA) was performed to detect cytotoxicity and mutation frequency under different treatment. Results In terms of structure-activity, Derek Nexus predicted the bacterial mutagenicity risk (rated as “plausible”) due to the N-nitroso structure while Sarah Nexus rated it as “ambiguous”. In the enhanced bacterial reverse mutation test, no obvious increase in revertant colonies of any strain was observed at doses of 125-5 000 μg per plate. In the MLA, the relative total growth (RTG) pointed to no obvious toxicity at concentrations of 312.5-2 500 μg·mL^-1, and the mutation frequencies did not differ significantly from those of the negative control, with all the results being negative. Conclusion N-nitroso bumetanide does not pose any mutagenicity risk when tested in vitro.

Key words: N-nitroso Bumetanide, Mutagenicity, Bacterial Reverse Mutation Test, Mouse Lymphoma Assay, Structure-Activity Analysis, Drug Safety Evaluation