Development of an integrated testing strategy using in vitro models to predict lung carcinogenesis

Carcinogenicity testing has traditionally been conducted using long-term animal studies, as specified in OECD TG 451 and 453 guidelines. These studies typically use rats and last for two years, requiring significant time and resources. Consequently, there is a pressing need to develop alternative toxicity testing methods that can efficiently predict lung cancer risks caused by chronic chemical exposure. In this study, we designed integrated testing strategies (ITS) to assess carcinogenesis by focusing on cell survival, clonal growth, and metastasis using the BEAS-2B cell model. Non-tumorigenic BEAS-2B cells were exposed to Benzo(a)pyrene (B(a)P), Ethyl carbamate (EC), Epichlorohydrin (ECH), and chloromethyl methyl ether (CMME) for 4 months (#40 passages). After treatment, the BEAS-2B cells showed enhanced anchorage-dependent and anchorage-independent colony formation. Furthermore, cell migration and invasion assays using transwell chambers revealed a significant increase in these malignant characteristics in treated BEAS-2B cells. Collectively, our findings demonstrate that prolonged exposure of non-tumorigenic BEAS-2B cells to B(a)P, EC, ECH, and CMME can lead to the acquisition of metastatic potential and multiple malignant characteristics. These integrated testing strategies for assessing carcinogenic potential could serve as a valuable tool for identifying unknown carcinogens.