The RTgill-W1 cell line assay describes a 24-well plate format fish cell line acute toxicity test using the permanent cell line from rainbow trout (Oncorhynchus mykiss) gill, RTgill-W1. After 24 h of exposure to the test chemical, cell viability is assessed based on three fluorescent cell viability indicator dyes, measured on the same set of cells. Resazurin enters the cells in its non-fluorescent form and is converted to the fluorescent product, resorufin, by mitochondrial, microsomal or cytoplasmic oxidoreductases. A reduction in the fluorescence of resorufin indicates a decline in cellular metabolic activity, including disruption of mitochondrial membranes. The data are expressed as the percent cell viability of unexposed control values versus the test chemical concentration. The resulting concentration-response curves serve to determine the effective concentrations causing 50% loss in cell viability, i.e. the EC50 value. The test is designed to (i) predict fish acute toxicity in product testing; (ii) range-finding and pre-screening before conducting a full fish acute or other fish-based toxicity test; (iii) generation of toxicity information to be used for hazard assessment in combination with other lines of evidences (e.g., Quantitative Structure Activity Relationships (QSAR), weight of evidence (WoE)) within Integrated Testing Strategy (ITS)/Integrated Approach to Testing and Assessment (IATA).