An AOP-Based Integrated In Vitro and In Vivo Assessment of the Non-Genotoxic Carcinogenic Potential of Multi-Walled...

Multi-walled carbon nanotubes (MWCNTs) are increasingly incorporated into industrial and consumer products, raising concerns about potential carcinogenicity because their physicochemical properties vary widely among materials. Although Mitsui-7 has been classified as possibly carcinogenic to humans (IARC, Group 2B), the carcinogenic potential of domestically manufactured MWCNTs and the determinants underlying material-specific differences remain insufficiently characterized. Here, we applied an adverse outcome pathway (AOP)-oriented integrated testing strategy (ITS) to compare four domestically manufactured MWCNTs with Mitsui-7 using human bronchial epithelial BEAS-2B cells. Acute responses were assessed by measuring cytotoxicity and intracellular reactive oxygen species (ROS). Exposure concentrations for long-term studies were selected using range-finding assays, and cells were then exposed for four weeks at non-cytotoxic concentrations. Following chronic exposure, transformation-related phenotypes were evaluated using anchorage-independent growth, anchorage-dependent clonogenicity, wound healing migration, and Transwell-Matrigel invasion assays, and tumorigenic potential was examined in xenograft models using colony-derived cells. Highly aggregated MWCNTs elicited stronger oxidative stress and were associated with increased proliferation/clonal expansion, enhanced anchorage-independent colony formation, and increased tumor formation in vivo, whereas other materials showed more limited or endpoint-specific responses. Overall, the results indicate that MWCNT-associated carcinogenic potential is material-dependent rather than a uniform class effect and support the utility of an AOP-aligned ITS for nanosafety assessment and hazard differentiation of carbon-based nanomaterials.