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.