AUTOMATION OF AN IN VITRO CITOTOXICITY ASSAY USED TO ESTIMATE STARTING DOSES IN ACUTE ORAL SYSTEMIC TOXICITY TESTS

The development, optimisation and validation of alternative methods that can be used for regulatory safety assessment is a challenging endeavour. Moreover, the demand for reliable and relevant tests that can be widely deployed has never been greater. New technologies offer significant opportunities for improving the performance of in vitro assays both in terms of the quality of the data produced and the value of the information derived. Automation of in vitro methods has had a major impact in the pharmaceutical sector where High Throughput Screening (HTS) of large molecular libraries has become common place. Traditionally however the HTS approaches employed have focused on therapeutic targets and only in recent years has attention shifted to include toxicological profiling, in an effort to reduce safety-related attrition rates. Application of assay automation and HTS to the regulatory safety assessment and prioritisation of nonpharmaceutical chemicals is still in its infancy but shows great promise in terms of facilitating better understanding of toxicological modes-of-action, reducing the reliance on animal testing, and allowing more data-poor chemicals to be assessed at a reasonable cost. To promote the uptake and acceptance of HTS approaches for supporting regulatory decision making, we describe in a stepwise manner how a well known cytotoxicity assay (uptake of neutral red by 3T3 fibroblasts) can be automated so that the essential features and reliability of the assay are retained while the throughput is increased. To demonstrate the performance of the automated assay, results generated with selected reference chemicals were directly compared with data generated during a previous international validation study, where the aim was to evaluate if the assay could be used to predict acute systemic toxicity in rodents. The automated assay was then included in a formal ECVAM validation study to further evaluate the relevance of the assay which involved the blind-testing of 56 reference chemicals on the HTS platform. Finally, the assay was adapted to a format more suited to higher throughput testing without compromising the quality of the concentration-response data obtained.

BOUHIFD Mounir;  BORIES Gilles;  CASADO POBLADOR Juan;  COECKE Sandra;  NORLEN Hedvig;  PARISSIS Nicholaos;  RODRIGUES Robim;  WHELAN Maurice; 

2012-12-31

PERGAMON-ELSEVIER SCIENCE LTD

JRC65946

0278-6915,   

https://publications.jrc.ec.europa.eu/repository/handle/JRC65946,   

10.1016/j.fct.2012.03.046,