Bacterial Reverse Mutation Test

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.