The increasing production of engineered nanomaterials (ENMs) raises significant concerns about human and environmental exposure, making it essential to understand the mechanisms of their interaction with biological systems to manage the associated risks. To address this, we propose categorizing ENM reactivity using in chemico methodologies. Surface analysis through methanol chemisorption and temperature-programmed surface reaction allows for the determination of reactive surface sites, providing accurate estimates of effective ENM doses in toxicity studies. Additionally, antioxidant consumption assays (dithiothreitol, cysteine, and glutathione) and reactive oxygen species (ROS) generation assays (RNO and DCFH2-DA) are employed to rank the oxidative potential of ENM surface sites in a cell-free environment. Our study confirms the classification of ZnO NM-110, ZnO NM-111, CuO, and carbon black as highly oxidant ENMs, while TiO2 NM-101 and NM-105 exhibit low oxidative potential due to their acidic surface sites. In contrast, CeO2 NM-211 and NM-212 demonstrate redox surface sites. SiO2 nanomaterials (NM-200 and NM-201) are shown to be inert, with low oxidation rates and minimal reactive surface density, despite their high surface area. Quantifying reactive surface sites offers a refined dose metric for assessing ENM toxicity, advancing safe-by-design nanomaterial development.
