Abstract: Objective To evaluate the mutagenicity risk and hepatocyte DNA damage risk of bumetanide impurity-N-nitrosobumetanil in vivo. Methods SD rats were randomly divided into six groups: control group (0.5% CMC-Na), 100 mg·kg^-1 dose group, 300 mg·kg^-1 dose group, 1 000 mg·kg^-1 dose group, positive control group 1 (N-ethyln-nitrosourea, ENU, 40 mg·kg^-1) and positive control group 2 (ethyl mesylate, EMS, 200 mg·kg^-1). Both positive control groups consisted of six animals and the other groups had twelve animals in each. Rats were administered with N-nitrosobumetanil by oral intragastric administration for fourteen days. Peripheral blood was collected at about 14 days and 28 days after the initial administration for the detection of Pig-a gene mutation rates. Liver cells were collected about 3 hours after the last administration for comet test. Results During the study, N-nitrosobumetanil did not result in abnormal clinical symptoms and there was no change in animal body mass or food intake. The averaged tail% DNA values (mean/median) of animal hepatocytes in 100 mg·kg^-1, 300 mg·kg^-1 and 1 000 mg·kg^-1 dosage groups were 2.90±0.38/2.34±0.46, 3.58±0.27/2.87±0.51 and 3.45±0.59/2.21±1.44, respectively, which showed no difference compared with the solvent control group, and no significant dose-effect correlation was observed. Fourteen days after the initial administration, the mean incidence of RBC^CD59- and RET^CD59- per million cells (RBC^CD59-/RET^CD59-) in 100 mg·kg^-1 dose group, 300 mg·kg^-1dose group and 1 000 mg·kg^-1 dose group were 2.9±1.6/1.2±0.8, 2.8±2.4/1.3±1.5 and 2.4±1.0/1.3±1, respectively. Twenty-eight days after the initial administration, the mean incidence of RBC^CD59- and RET^CD59- per million cell (RBC^CD59-/RET^CD59-) in the 100 mg·kg^-1 dose group, 300 mg·kg^-1 dose group and 1 000 mg·kg^-1 dose group was 5.1±1.5/2.2±0.6, 4.3±1.5/3.5±3.6, and 4.8±2.4/2.5±2.7, respectively. The above values were not different from those of the solvent control group at the same time point, and no correlation of dose effect was observed by statistical analysis. Conclusion After fourteen days of oral administration of N-nitrosobumetanilis, the maximum tolerance of SD rats exceeds 1 000 mg·kg^-1, and the risk of peripheral blood gene mutation and hepatocyte DNA damage is not detected. The data can be used to back up the regulation of genotoxic impurities in drugs and the safe use of drugs containing N-nitroso impurities.

Key words: N-nitrosobumetanil, mutagenicity, genotoxicity, SD rat, Pig-a gene mutation assay, comet assay, intragastric administration