The Report of the International Workshop on In Vitro Methods for Assessing Acute Systemic Toxicity concludes that none of the available in vitro methods for assessing acute systemic toxicity have been evaluated adequately to replace the use of animals. In addition, the document recommends the development of a simple predictive system for gut absorption, which would optimize the ability of in vitro assays to predict in vivo LD50 values. Consequently, this proposal outlines a series of studies whose aim is to develop a cell culture technique with the potential to screen representative chemicals for their effect on gastrointestinal absorption (GIA) concomitantly with acute cytotoxicity. Effect of chemicals on GIA in vitro is determined using markers for paracellular permeability, including Lucifer yellow, FITC-dextran, [3H]-mannitol permeability, and transepithelial electrical resistance (TEER) measurements. Acute cytotoxicity is monitored with the MTT and NRU assays, recommended cytotoxic indicators for cell viability. Twenty chemical agents in the Registry of Cytotoxicity, suggested by the ICCVAM Guidance Document will be evaluated. Acute 3-hour and 24-hour exposures are performed with continuous Caco-2 monolayers. Dose-response curves are generated; 50% effective concentrations (EC50s) for GIA and 50% inhibitory concentrations (IC50s) for acute toxicity are extrapolated. Regression curves for both sets of data are calculated according to the Guidance Document. EC50s and IC50s are then compared to each other and to animal LD50s, human toxic and lethal concentrations, and human oral bioavailability data available for these chemicals. It is anticipated that a cell culture model that can simultaneously and systematically screen for acute toxicity and distinguish from the effect on GIA, may be used to predict starting doses for in vivo lethality studies and to improve the ability of in vitro cytotoxicity data to predict in vivo LD50 values.
