Predicting fish acute toxicity of chemicals in vitro is an attractive alternative method to the conventional approach using juvenile and adult fish. The rainbow trout (Oncorhynchus mykiss) cell line assay with RTgill-W1 cells has been designed for this purpose. It quantifies cell viability using fluorescent measurements for metabolic activity, cell- and lysosomal-membrane integrity on the same set of cells. Results from over 70 organic chemicals attest to the high predictive capacity of this test. We here report on the repeatability (intralaboratory variability) and reproducibility (interlaboratory variability) of the RTgill-W1 cell line assay in a round-robin study focusing on 6 test chemicals involving 6 laboratories from the industrial and academic sector. All participating laboratories were able to establish the assay according to preset quality criteria even though, apart from the lead laboratory, none had previously worked with the RTgill-W1 cell line. Concentration-response modeling, based on either nominal or geometric mean-derived measured concentrations, yielded effect concentrations (EC50) that spanned approximately 4 orders of magnitude over the chemical range, covering all fish acute toxicity categories. Coefficients of variation for intralaboratory and interlaboratory variability for the average of the 3 fluorescent cell viability measurements were 15.5% and 30.8%, respectively, which is comparable to other fish-derived, small-scale bioassays. This study therefore underlines the robustness of the RTgill-W1 cell line assay and its accurate performance when carried out by operators in different laboratory settings.

Eawag Swiss Federal Institute of Aquatic Science and Technology Department of Environmental Toxicology 8600 Dübendorf Switzerland

EPF Lausanne School of Architecture Civil and Environmental Engineering 1015 Lausanne Switzerland

ETH Zürich Institute of Biogeochemistry and Pollutant Dynamics 8093 Zürich Switzerland

Givaudan Schweiz AG Department of In vitro Molecular Screening 8310 Kemptthal Switzerland

IRAS The Institute for Risk Assessment Sciences Toxicology Division Utrecht University 3584 CL Utrecht The Netherlands

Norwegian Institute for Water Research Section for ecotoxicology 0349 Oslo Norway

RECETOX Faculty of Science Masaryk University 625 00 Brno Bohunice Czech Republic

The Procter and Gamble Company Global Product Stewardship Mason Ohio 45040 USA

VITO ABS team Department of Environmental Risk and Health Flemish Institute for Technological Research 2400 Mol Belgium

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