Optimization of the RTgill-W1 assay for commercial testing applications

The RTgill-W1 in vitro assay is a new approach method designed as an alternative to one of the most widely used toxicity tests globally, the fish acute lethality test. The RTgill-W1 assay is standardized (Organisation for Economic Co-operation and Development; International Organisation of Standardization) but the test conditions could be optimized to allow for higher throughput, better replication, and lower costs. This study explores potential optimizations regarding the culturing conditions, plate format, and reference toxicant testing to make the RTgill-W1 assay more practical for widespread implementation. We demonstrate that the RTgill-W1 culture can be routinely split 1:3 without impacting test sensitivity (p = 0.207 to 0.612), which allows all work to be contained to a standard 5-day work week and 1.3x more tests over the current test methods. The test can adopt a 96-well plate format without impacting sensitivity (p = 0.672 to 0.889), dramatically improving the replication of the exposure wells and test controls and generating test data from a single plate. The fluorescent signal does not bleed across the smaller wells in the 96-well format to impact test endpoints (p = 0.465 to > 0.999). The reference toxicity test concentrations can be modified to increase confidence in the point estimate (EC50), allowing for more effective monitoring of assay performance. These optimizations improve the practicality and decrease the costs of the RTgill-W1 assay, which is particularly desirable for implementation in commercial and government laboratories that conduct regulatory toxicity testing.