The oxidative potential of nanomaterials: an optimized high-throughput protocol and interlaboratory comparison for the ferric reducing ability of serum (FRAS) assay

• Published in: Nanotoxicology 2024-12, Vol.18 (8), p.724-738
• Main Authors: Ruijter, Nienke, Boyles, Matthew, Braakhuis, Hedwig, Ayerbe Algaba, Rafael, Lofty, Morgan, di Battista, Veronica, Wohlleben, Wendel, Cassee, Flemming R, Candalija, Ana
• Format: Article
• Language: English
• Subjects: Assaying; Ferric Compounds - chemistry; High-Throughput Screening Assays - methods; Humans; Nanomaterials; Nanostructures - chemistry; Nanostructures - toxicity; Nanotechnology; Oxidation-Reduction; Oxidative stress; Oxidative Stress - drug effects; Product development; Protocol; Reactive oxygen species; Reactive Oxygen Species - metabolism; Screening; Serum - chemistry
• ISSN: 1743-5390; 1743-5404; 1743-5404
• DOI: 10.1080/17435390.2024.2438116

Successful implementation of Safe and Sustainable by Design (SSbD) and grouping approaches requires simple, reliable, and cost-effective assays to facilitate hazard screening at early stages of product development. Especially for nanomaterials (NMs), which exist in many different forms, efficient hazard screening is of utmost importance. Oxidative potential (OP), which is the ability of a substance to induce reactive oxygen species (ROS), is an important indicator of the potential to induce oxidative damage and oxidative stress. A frequently used assay to measure OP of NMs is the ferric reducing ability of serum (FRAS) assay. Although the widely used cuvette-based FRAS protocol is considered a robust assay, its low throughput makes the screening of multiple materials challenging. Here, we adapt the original cuvette-based FRAS assay protocol, into a 96-well format and thereby improve its user-friendliness, simplicity, and screening capacity. The adapted protocol allows for the screening of multiple NMs per plate, and multiple plates per day, where the original protocol allows for the screening of one NM dose-range per day. When comparing the two protocols, the adapted protocol showed slightly decreased assay precision as compared to the original protocol. The results obtained with the adapted protocol were compared using eight reference NMs in an interlaboratory study and showed acceptably low intra- and interlaboratory variation. We conclude that the adapted FRAS assay protocol is suitable to be used for hazard screening to facilitate SSbD and grouping approaches.