The toxicologic and carcinogenic potential of chemicals is usually determined through a sequence of acute, sub-acute (14-day), sub-chronic (90-day) and chronic (two-year) studies in rats and mice of both sexes. The US National Toxicology Program (NTP) does not conduct acute toxicity studies. Dose levels for 14-day toxicity studies are typically estimated from information in the literature, if available. The toxicology information obtained from 14-day studies is used in the selection of doses for 90-day studies. The protocol for 14-day studies consists of five doses and control groups and five animals per group of each sex and species, resulting in the use of 120 animals per study. At present, in addition to refining the current testing protocols, the NTP is evaluating the potential for in vitro test methods to partially or completely avoid the need for 14-day toxicity studies, especially for chemicals where the dermal route of exposure is used. The in vitro assays used were the EpiDerm bioassay to estimate dermal irritation, the neutral red uptake (NRU) bioassay to estimate systemic toxicity and the primary rat hepatocyte cytotoxicity (PRHC) assay to estimate hepatotoxicity. The purpose of using these assays was to assess their potential for predicting relative in vivo toxicity and to support dose selection decisions for 90-day studies. In general, based on these limited number of studies, the EpiDerm and NRU tests were predictive of the responses observed in in vivo studies. However, a larger comparative database is needed to derive definitive conclusions regarding the value of in vitro tests in the prediction of in vivo effects.