Serious concerns over potential toxicity of new engineered nanomaterials (ENMs) require the development of reliable and high throughput screening assays that impart critical information regarding potential toxicity, guide in-depth toxicity evaluations, justify exposure controls, and aid in the development of sustainable nanomanufacturing. Reactive oxygen species (ROS) generation or biological oxidative damage (BOD) is an important mechanism of ENM toxicity. Several acellular assays have been used to screen for ENMs toxicity based on ROS generation. The dichlorofluorescin (DCFH) assay has gained popularity to determine the degree of ROS generation due to its low cost and automation. However, a rigorous evaluation of this method is lacking. Our study; (i) evaluates the performance of the acellular DCFH assay; (ii) compares the performance of the DCFH and the newly developed Ferric Reducing Ability of Serum (FRAS) assay; (iii) explores the variability in physicochemical characterizations (PCs) of ENMs and their relationship to ROS generation and FRAS-measured BOD; (iv) validates BOD as a metric to determine the effects of individual PCs and their interplays, and further exams the possible utility of ENM-induced BOD for hazard identification of these materials. This study demonstrates that FRAS-measured BOD is a highly informative biologically relevant metric for ENM hazard screening, reflecting the combined effects of multiple PCs. Further, FRAS shows significant potential for hazard identification, as a direct exposure metric, and as a useful tool in responsible nanomanufacturing efforts, providing an opportunity for engineering redesign with the goal of producing less toxic materials while maintaining targeted functional properties
