The mutagenic potential of BCP was evaluated by a bacterial reverse mutation assay, according to OECD TG471 (OECD, 2020 [22]). The four histidine auxotroph strains of Salmonella typhimurium TA100, TA1535, TA98, and TA1537, along with a tryptophan auxotroph strain of Escherichia coli, WP2 uvrA (pKM101), were used for bacterial reverse mutation testing (Green and Muriel, 1976; Maron and Ames, 1983 [23, 24]). To induce a metabolic activation system, S9 fraction (Molecular toxicology Inc., Lot No. 4230), a mitochondrial fraction of liver homogenated in SD rats, was added with Cofactor-1 (Genogen Co. Ltd, Lot No. 2009608 Ⅰ). The test strains were exposed to the test article using the pre-incubation method. Based on the results of a range-finding test conducted on the test article, dose ranges were determined using the five test strains in both the presence and absence of the metabolic activation system with two plates per dose. In this study, the highest dose was set at 5,000 μg/plate for all test strains, and six-serial diluted concentrations (5,000, 1,250, 313, 78.1, 19.5, and 4.88 μg/plate) were tested in the main study. The colonies were counted using an automated colony counter.

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.

Male 6-week-old CrljOri: CD1(ICR) mice were purchased from Samtako Laboratory Animal, Inc. (Gyeonggi-do, Korea) and were used for experiments at 8 weeks of age after acclimatization for 1 week. The animals were housed in polycarbonate cages. An ambient temperature of 22 ± 3°C, a relative humidity of 50 ± 10%, and a photoperiod of 12 h were maintained throughout the study. All animals used in this study were cared for in accordance with the principles outlined in the “Guide for the Care and Use of Laboratory Animals,” a NIH publication. ICR mice were divided into five groups (n = 5) on the basis of their body weights. BCP and negative control was orally administered twice every 24 h to mice at doses of 0, 62.5, 125, and 250 mg/kg. Mitomycin C (MMC) (Lot no. SLBX4310, Sigma-Aldrich), positive control, was administered once intraperitoneally at 2 mg/kg 24 h before sacrifice. The bone marrow cells were then centrifuged at 4°C and 1,000 rpm for 5 min and smeared on a clean slide glass. Smeared slides were air-dried, fixed in methanol, and then stained with 3% Giemsa solution for 30 min. Additionally, the stained slides were observed under the fluorescence microscope at 600x ∼ ×1,000 magnification (B × 51, Olympus, Japan). The number of polychromatic erythrocytes (PCEs), micronucleated polychromatic erythrocytes (MNPCEs), and normochromic erythrocytes (NCEs) among the red blood cells (RBCs, PGE + NCE) were counted. A total of 4,000 PCEs were scored per animal by the same observer for determining the frequencies of micronucleated polychromatic erythrocytes (MNPCEs). PCE/(PCE + NCE) ratio was calculated by counting 500 cells. This animal study was conducted in accordance with OECD guideline for testing chemicals No. 474 (OECD, 2016b) and approval of the Institute Animal Care and Use Committees of Hoseo University [Approval No.: HSIACUC-20-061(1)].

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