Protocol optimization during a validation study to evaluate in vitro cytotoxicity assays for estimating rodent acute systemic toxicity

MW Paris¹, JA Strickland¹, WS Stokes², S Casati³, RR Tice¹, H Raabe⁴, C Cao⁵, R Clothier⁶, J Harbell⁴, G Mun⁴, A Sizemore⁴, G Moyer⁴, J Madren-Whalley⁵, C Krishna⁵, M Owen⁶, N Bourne⁶, J Haseman¹, P Crockett⁷, M Wenk⁸, M Vallant⁹

1. Integrated Laboratory Systems, Inc./NICEATM, RTP, NC, USA
2. NIEHS, NICEATM, RTP, NC, USA
3. ECVAM, JRC, Ispra, Italy
4. IIVS, Gaithersburg, MD, USA
5. U.S. Army ECBC, Aberdeen Proving Ground, MD, USA
6. University of Nottingham, Nottingham, UK
7. Constella Group, Durham, NC, USA
8. BioReliance Corp., Rockville, MD, USA
9. NIEHS, RTP, NC, USA

Previous studies have identified a correlation between in vitro cytotoxicity and acute oral toxicity. NICEATM and ECVAM subsequently initiated a three-phase multi-laboratory validation study to evaluate the usefulness of two standardized in vitro basal cytotoxicity assays for estimating acute rodent toxicity and 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 in mouse 3T3 fibroblasts and in normal human epidermal keratinocytes (NHK) using neutral red (NR) uptake assays. Phase Ia established the historical databases for sodium laurel sulfate, the positive control, for each of three laboratories. Three chemicals were tested in Phase Ib and nine chemicals were tested in Phase II. Protocols were optimized after each of the first two phases to minimize intra- and inter-laboratory variation prior to testing 60 chemicals in Phase III. Technical challenges arose in Phases Ia/Ib (i.e., formation of NR dye crystals; uneven growth of NHK cells; slow growth of 3T3 cells) that were resolved with Phase II protocols. Significant variation in NHK growth in Phase II attributable to different lots of media and supplements required prequalification of medium. The optimized final protocols were used for Phase III testing. These studies demonstrate the value of using a phased approach during validation to optimize and standardize a final test method protocol that can then be used for the final validation phase. Supported by: N01-ES-35504, N01-ES-75408; EPA IAG DW-75-93893601-0; European Commission 19416-2002-04 F2ED ISP GB.

P182

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

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