The in vitro chromosomal aberration test was performed according to OECD TG473 (OECD, 2016a). Chinese hamster lung cells (CHL/IU) were obtained from the American Type Culture Collection (ATCC, United States). The cells were maintained with Eagle’s minimum essential medium supplied with 10% fetal bovine serum. The cells were cultured at 37 ± 1°C and 5% CO2-95% air using a humidified incubator and cell culture flasks (culture surface 75 cm2). The cytotoxicity dose ranges of this study were determined by calculating the relative population of doubling (RPD) in substance-treated cultures observed in a dose range-finding test. The RPD value was determined using the following formula.

population doubling = [log (post-treatment cell number/Initial cell number)]/log 2

According to the RPD results of the dose range-finding test, the highest dose was chosen to produce <50% in this test. The assay consisted of short-term (6 h) and continuous (24 h) treatments. Approximately 22 h after the start of the treatment, colcemid was added to each culture at a final concentration of 0.2 μg/ml. The slides of CHL cells were prepared following the hypotonic-methanol-glacial acetic acid-flame drying Giemsa schedule for metaphase plate analysis. More than 300 metaphases were selected and analyzed for each treatment group under ×1,000 magnification using a light microscope. When a significant difference was confirmed after statistical analyses to determine chromosomal aberrations, it was positive.

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