Integrating Adverse Outcome Pathways with In Vitro-to-In Vivo Extrapolation for Risk Assessment: A Case Study of Cadmium-Induced Bone Toxicity.

Recent advances in regulatory science have promoted the development of next-generation risk assessment, with an increasing emphasis on chemical safety evaluation via new approach methodologies. Herein, an integrated approach combining adverse outcome pathways (AOPs) with in vitro-to-in vivo extrapolation (IVIVE) was established, enabling the systematic organization of mechanistic evidence and the quantitative translation of early molecular events into human health risk metrics. Using cadmium (Cd) as a case study, this framework incorporated toxicogenomic data, bioinformatics, and human-relevant in vitro cellular assays to elucidate the AOPs underlying Cd-induced bone toxicity. Two AOP frameworks were proposed, highlighting key events (KEs) involving the RANKL/OPG, Wnt, BMP, and MAPK signaling pathways as well as osteogenic differentiation and cell apoptosis. Benchmark dose and physiologically based toxicokinetic modeling were further applied within IVIVE to derive human-equivalent doses (HEDs) for KEs in the proposed AOPs. Of these, the HED derived from Wnt and MAPK signaling pathways (both 0.03 μg/kg bw/day) was considered an appropriate metric for risk characterization, with its scientific validity further supported by existing epidemiological evidence. This study presents an AOP-IVIVE integration strategy that advances mechanism-informed and human-relevant risk assessment, providing scientific evidence to support regulatory decision-making.