Mechanistic hazard differentiation of cerium oxide and barium sulfate nanoparticles using a human-relevant lung model.

Increasing regulatory pressure to reduce animal testing has accelerated the development of New Approach Methodologies (NAMs) for hazard assessment. This study employed a Calu-3/monocyte-derived macrophages (MDMs) co-culture at the air-liquid interface to assess the biological effects of cerium oxide (CeO₂) and barium sulfate (BaSO₄) nanoparticles, two occupationally relevant materials. Doses ranged from 6 to 324 µg/cm² for BaSO4 and 9 to 324 ug/cm2 for CeoO2, and samples were analyzed on days 1, 3, and 7. Epithelial barrier integrity, cytotoxicity, inflammatory cytokine secretion, and targeted transcriptional responses were mapped onto the adverse outcome pathway for inflammation-induced pulmonary fibrosis (AOP173) and compared with published rodent inhalation and human data. CeO₂ induced sustained basolateral interleukin-8 secretion with NF-κB activation and upregulation of oxidative stress, DNA damage, and proteotoxic stress markers peaking at day 3 at the middle dose (108 µg/cm²). Epithelial barrier integrity remained intact, while molecular endpoints detected early AOP-relevant stress responses. BaSO₄ elicited minimal activity across all endpoints, consistent with in vivo and human data, and was correctly identified as a low-hazard material. By integrating multi-endpoint responses within an AOP framework, this study demonstrates that human cell-based co-culture models can mechanistically discriminate nanoparticles with distinct hazard profiles.