Abstract
Considering the increasing production of engineered nanomaterials (ENMs), new approach methodologies (NAMs) are essential for safe-by-design approaches and risk assessment. Our aim was to enhance screening strategies with a focus on reactivity-triggered toxicities. We applied in vitro tests to 10 selected benchmark ENMs in two cell models, lung epithelial A549 and differentiated THP-1 macrophage-like cells. Previously, we categorized ENMs based on surface reactivity. Here we elucidated their reactivity-triggered cytotoxicity and mode of action using the WST-1 assay (metabolic activity), LDH assay (cell membrane integrity), autophagosome detection, and proteomics. Nonreactive SiO(2) NM-200 showed no significant impact on cell viability. Conversely, highly reactive CuO and ZnO (NM-110 and NM-111) disrupted cell homeostasis. Interestingly, moderately reactive TiO(2) (NM-101 and NM-105) and CeO(2) (NM-211 and NM-212), apparently without an adverse effect, induced autophagosome formation, evidencing autophagy as a defensive mechanism. Our improved in vitro testing strategy, combined with state-of-the-art reactivity information, screens ENMs for potential reactivity-triggered toxicity.