2.8. In vivo alkaline comet assay

This study was performed as a follow‐up study to confirm the positive result of the in vitro chromosomal aberration test. This in vivo comet assay was performed according to OECD T G489. Six male Sprague‐Dawley rats (8 weeks old, 258.13‐290.16 g) were orally administered the test article once a day for three consecutive days at doses of 0, 1250, 2500 and 5000 mg/kg/day. The animals of the positive control group were treated with ethyl methane sulfonate (200 mg/kg/day) once orally on the day of the third administration. All animals were killed approximately 3 hours after the final administration and a cell suspension generated from a portion of the left lobe of the liver was used to make specimens. Electrophoresis was conducted on the following day (Silva, de Freitas, Marinho, Speit, & Erdtmann, 2000). According to the “Atlas of Comet Assay Images,” cells were classified into scorable, non‐scorable and hedgehog; only scorable cells were analyzed (JEMS‐MMS, 2014). A total of 150 cells were counted per animal and the median value of the percentage tail DNA was calculated (Olive, Banáth, & Durand, 1990).