Abstract: OBJECTIVE: To establish a method for genotoxicity evaluation of ethyl methanesulfonate (EMS) in rats by using a combination of Comet and flow micronucleus assays. METHODS: EMS (50, 100 and 200 mg/kg) was administered to rats once daily at 24 h and 45 h intervals. Negative controls received 0.9% physiological saline. Each dose group contained 5 rats. Blood was collected at 3 h after the final administration. From each sample, 2 000 reticulocytes (RET) were counted for MN frequency by flow cytometry. Then, all rats were humanely sacrificed with pentobarbital sodium. Liver, stomach, and kidney were removed to make single cell suspensions. These cells were used for the Comet assay. The Comet parameters were measured using the Comet Assay IV. RESULTS: Based on the Comet assay, EMS induced a significant dose-related increase of the Comet tail DNA percentage in the liver, stomach, and kidney. In the flow micronucleus assay, statistically significant increases in MN frequency were noted in the 100 mg/kg EMS dose group compared with those in the negative controls. CONCLUSION: EMS can cause DNA strand breaks and micronuclei and the combined Comet and MN assays will improve the accuracy of investigations.

Key words: ethyl methanesulfonate, in vivo Comet assay, micronucleus test, multiple endpoints