Genotoxicity study of 2-methoxyethanol and benzalkonium chloride through Comet assay using 3D cultured HepG2 cells

Though the key data in identifying carcinogenicity is experience in human, long-term carcinogenicity tests using experimental animals are more realistic. Because carcinogenicity tests require much time and cost, performing the test is minimized through pre-screening. Recently, as bioethics has been strengthened, it is required to minimize animal testing in screening tests as well as carcinogenicity tests. The replacement of the micronucleus assay in experimental animal is the beginning, and the ultimate goal is to replace the carcinogenicity test using experimental animals. The micronucleus assay and the comet assay in 3D culture system of human-derived cells is considered as the most applicable practical measures at this stage. This study was conducted to provide more diverse information in the evaluation of carcinogenicity by establishing the comet test method in a three-dimensional cell culture system. In this study, HepG2 cells were cultured for 4 days in hang-in drop method, and then cultured for 7 days on a low adhesion plate to prepare spheroids. The methods were confirmed by d-mannitol (negative control), ethylmethane sulfonate (positive control), and cyclophosphamide (positive control for metabolite). 2-methoxyethanol and benzalkonium chloride were selected as test substances. Though 2-methoxyethanol is positive in in vivo comet assay and in vitro mammalian chromosome aberration test, it is considered negative in the comprehensive genotoxicity evaluation based on negative in bacterial reverse mutation assay, in vitro mammalian cell gene mutation test and mammalian chromosome aberration test. Benzalkonium chloride has been questioned on carcinogenicity because it is a disinfectant ingredient that has become a social issue in Korea. As a result of the Comet assay for 2-methoxyethanol and benzalkonium chloride in the cultured HepG2 cell line, 2-methoxyethanol was evaluated as positive in the metabolic activation system, but benzalkonium chloride was evaluated as negative in both the presence and absence of the metabolic activation system. Therefore, in order to clarify the carcinogenic potential of 2-methoxyethanol, it is judged that additional studies based on mechanistic studies are needed.