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Ekwall et al. (ATLA 17:83-100, 1989) have proposed that ˜80% of chemical-induced systemic toxicity is the result of disruption of basic cellular processes common to most cell types in the body, and that systemic toxicity for many chemicals could be estimated in in vitro cultures. Strickland et al (The Toxicologist, Abs#761, 2003) reported on a joint European/USA validation to evaluate two cytotoxicity assays in NHEK and BALB/c 3T3 cells to predict acute systemic toxicity using a neutral red uptake (NRU) viability endpoint. We report on a two-lab validation study to evaluate the ATP viability endpoint using the optimized protocol from the aforementioned validation. Cytotoxicity was measured as a dose-dependent reduction in ATP from which an IC50 was determined. Initially, 20 chemicals from the Multicenter Evaluation of In Vitro Cytotoxicity (MEIC) program were tested to determine the relationship between the ATP IC50 values in vitro and the human LC50 values [log LC50 µM = 0.794(log IC50 µM)+0.176; r2 = 0.887]. Subsequently, 50 chemicals from Strickland et al were tested to determine the relationship between the ATP IC50 values and the rodent oral LD50 values [log LD50 mmol/kg = 0.495 (log IC50 mM)+0.413; r2 = 0.399] and is similar to the prediction model published in the Registry of Cytotoxicity (Halle, 1998). A high correlation (r2 = 0.930) was demonstrated between ATP IC50 values and NRU50 values obtained in the cytotoxicity validation study. The results demonstrate that the NHEK assay with the ATP endpoint may be used to predict systemic toxicity, or rodent oral LD50 doses, and that the ATP endpoint is an acceptable alternative to the NRU endpoint.