Protein precipitation in vitro as a measure of chemical-induced cytotoxicity: an EDIT sub-programme

As a priority area of the Evaluation-Guided Development of In Vitro Toxicit y and Toxicokinetic Tests (EDIT) programme, an in vitro protein precipitati on (PP) assay was used on the 50 reference chemicals of the Multicentre :Ev aluation of In Vitro Cytotoxicity (MEIC) project, to confirm and extend the MEIC results. Dose-response curves were generated for only 30 of the chemi cals, and the concentrations causing 10% (EC10) and 50% (EC50) protein prec ipitation versus the positive control were chosen as endpoints. The number of chemicals with a positive response increased to 46 when a new endpoint, the minimum effect concentration (MEC) that induces protein precipitation w ith respect to the negative control, was used. When the results were correl ated with in vitro cytotoxicity in human cell lines, a similarly good corre lation was found between the various endpoints of the PP assay at 5 hours a nd the 24-hour IC50 average cytotoxicity in human cell lines, even though t he number of chemicals included in the correlation was larger for the MEG. Using the prediction error, the endpoint that gave the best correlation bet ween the PP assay and human cell cytotoxicity was once more found to be the 5-hour MEG, and this was chosen for the PP assay. The sensitivity of the P P assay is lower than that of th in vitro cell-line cytotoxicity assay, pos sibly due to its shorter exposure period and because precipitation is the u ltimate event in the :sequence of a protein disturbance. It is expected tha t earlier denaturation steps would give better sensitivity. However, this s imple, inexpensive and rapid assay could be useful in the early stages of t esting chemicals.