Results of the final phase of a validation study to evaluate in vitro cytotoxicity assays for estimating rodent acute systemic toxicity

WS Stokes¹, MW Paris², JA Strickland², S Casati³, RR Tice², H Raabe⁴, C Cao⁵, R Clothier⁶, JHarbell⁴, G Mun⁴, A Sizemore⁴, G Moyer⁴, J Madren-Whalley⁵, C Krishna⁵, M Owen⁶, N Bourne⁶, J Haseman², P Crockett⁷, E Harvey⁷, R Lee⁷, M Wenk⁸, M Vallant⁹

1. NIEHS, NICEATM, RTP, NC, USA
2. ILS. Inc., NIEHS/NICEATM, RTP, NC, USA
3. ECVAM, JRC, Ispra, Italy
4. IIVS, Gaithersburg, MD, USA
5. US Army ECBC, Aberdeen Proving Ground, MD, USA
6. Univ. of Nottingham, Nottingham, United Kingdom
7. Constella Group, Durham, NC, USA
8. BioReliance Corp., Rockville, MD, USA
9. NIEHS, RTP, NC, USA

Recent studies have identified a correlation between in vitro basal cytotoxicity and in vivo acute oral toxicity. NICEATM and ECVAM subsequently initiated a three-phase multi-laboratory validation study to evaluate the usefulness of two standardized neutral red uptake (NRU) assays for estimating acute rodent toxicity and to determine the extent that they may reduce animal use. Seventy-two coded chemicals (12 from each of five acute oral hazard categories and 12 unclassified/nontoxic chemicals) were tested using the NRU endpoint with mouse 3T3 fibroblasts and normal human epidermal keratinocytes (NHK). Three chemicals were tested in Phase Ib, nine chemicals in Phase II, and 60 chemicals in the final Phase III. Based upon preliminary analyses, the results for the positive control, sodium laurel sulfate, were reproducible over the entire study. IC50 results from all phases were used with rodent oral LD50 values to calculate linear regressions for each lab and assay. Although the NHK data were more reproducible than the 3T3 data, the 3T3 data yielded a better regression fit. Comparison of the regressions for both assays to the Registry of Cytotoxicity (RC) regression indicated that the new regressions were statistically different from the RC, but that their predictions of toxicity category were generally similar. The new regressions will be used in conjunction with computer simulations to determine animal savings that may result by using in vitro data as the basis for starting doses for acute toxicity studies. Supported by: N01-ES-35504, N01-ES-75408; EPA IAG DW-75-93893601-0; European Commission 19416-2002-04 F2ED ISP GB.

P187

Date: Tuesday, August 23, 2005, 13.00-14.00 h

5.6 Session: In Vitro Aproaches for Determining Acute Systemic Toxicity.