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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Academic Research Enhancement Awards (AREA) (R15)
Project #
1R15ES012170-01
Application #
6595805
Study Section
Alcohol and Toxicology Subcommittee 4 (ALTX)
Program Officer
Shreffler, Carol K
Project Start
2003-06-01
Project End
2006-05-31
Budget Start
2003-06-01
Budget End
2006-05-31
Support Year
1
Fiscal Year
2003
Total Cost
$161,945
Indirect Cost
Name
St. John's University
Department
Other Health Professions
Type
Schools of Pharmacy
DUNS #
073134744
City
Queens
State
NY
Country
United States
Zip Code
11439
Dholakiya, Sanjay L; Barile, Frank A (2013) Alternative methods for ocular toxicology testing: validation, applications and troubleshooting. Expert Opin Drug Metab Toxicol 9:699-712
Calabro, Anthony R; Gazarian, Dmitry I; Barile, Frank A (2011) Effect of metals on ýý-actin and total protein synthesis in cultured human intestinal epithelial cells. J Pharmacol Toxicol Methods 63:47-58
Barile, Frank A (2010) Validating and troubleshooting ocular in vitro toxicology tests. J Pharmacol Toxicol Methods 61:136-45
Calabro, Anthony R; Konsoula, Roula; Barile, Frank A (2008) Evaluation of in vitro cytotoxicity and paracellular permeability of intact monolayers with mouse embryonic stem cells. Toxicol In Vitro 22:1273-84
Konsoula, Roula; Barile, Frank A (2007) Correlation of in vitro cytotoxicity with paracellular permeability in mortal rat intestinal cells. J Pharmacol Toxicol Methods 55:176-83
Konsoula, Roula; Barile, Frank A (2005) Correlation of in vitro cytotoxicity with paracellular permeability in Caco-2 cells. Toxicol In Vitro 19:675-84