Intercalibration study in the evaluation of toxicity with rainbow trout hepatocytes

An intercalibration exercise was carried out evaluating the transferability and reproducibility of the rainbow trout hepatocyte cytotoxicity test. First, rainbow trout hepatocytes were plated in 96-well microplates, packed at 4°C, and sent to eight different laboratories. Each laboratory was instructed to initiate 24-h exposure on two blind samples, one of which was toxic. Second, five laboratories were trained to perform the hepatocyte cytotoxicity test independently with their own fish source. Afterward, they were invited to evaluate toxicity of three samples: reference toxicant (KCl), toxic, and nontoxic industrial effluent. The labs were instructed to evaluate cell viability with either propidium iodide exclusion test or with neutral red uptake assay. These two assays were proposed to accommodate laboratories' constraints (i.e., available spectrophotometers or fluorometers, microplate vs tube centrifuges). Results showed all groups were able to identify toxic sample (i.e., KCl) from nontoxic one. Cytotoxic concentration at which 50% of hepatocytes are killed (CC50) varied between 70–131 mM, when cell viability was determined with PI test, while CC50 values varied between 82–118 mM with NRU assay. These values were in the same range as data obtained in our laboratory during an in-house intercalibration exercise. Moreover, 4/5 of the independent laboratories could correctly classify toxicity of the (3) samples. The rainbow trout hepatocyte cytotoxicity assessment test does, therefore, appear to be reproducible and transferable to other laboratories, producing statistically similar classifications and results for measurement endpoints such as identification of nontoxic samples, lowest observable effect concentration, and CC50 values. ©1999 John Wiley & Sons, Inc. Environ Toxicol 14: 429–437, 1999