Session E6: Eye Toxicity

Chairs: Odile de Silva (France) and Dan Bagley (USA)

E6: The Research Program of the Steering Committee on Alternatives to Animal Testing (SCAAT)
Odile de Silva and David Basketter on behalf of the Colipa Steering Committee on Alternatives to Animal Testing (SCAAT). L'Oréal Recherche Centre C, Zviak, 90 rue du Général Roguet, 92583 Clichy Cedex, France. odesilva@recherche.loreal.com.

COLIPA'S Steering Committee on Alternatives to Animal Testing is currently focusing its research activities on two areas: skin tolerance and eye irritation. SCAAT has set up two 3-year projects on skin sensitization, one 3-year project on skin irritation, and a 1-year project on eye irritation. The sensitization projects aim at the standardization and optimization of a method using human dendritic cells and the identification of up-regulated genes following exposure of these cells to chemicals. The skin irritation project aims to identify new markers of irritation with the help of genomic and proteomic technologies. These three projects will be carried out by industry. The scope of the eye irritation project is to identify new in vitro endpoints, predictive of the in vivo response to chemical injury. It involves collaboration with ophthalmologists from the universities of Aachen and Bristol. The team in Aachen will try to identify if the kinetics and patterns of changes in physiological function and signals of injury released from the cornea in vitro can predict a chemical's potential to damage the eye, with a focus on recovery. The Bristol team will work on human corneal cell cultures and 3D constructs and will study chemically induced injury and recovery. All these projects aim at developing and optimizing alternative methods for further pre-validation.

E6: A Reproducible Tissue Model for Ocular Irritancy Testing
J. Sheasgreen¹, M. Klausner¹, J. Kubilus¹, and J. Harbell². ¹MatTek Corporation, 200 Homer Avenue, Ashland, MA 01721, USA; ²Institute for In Vitro Sciences, Gaithersburg, MD 20878, USA. jsheasgreen@mattek.com.

The utility of any toxicological test system or method critically depends on the reproducibility of the target tissue. Over the past six years, quantitative quality control (QC) test methods utilizing the MTT assay have established a high level of intra-lot and inter-lot reproducibility for MatTek's organotypic skin model, EpiOcular^TM. QC testing on each lot of tissue consists of exposing the tissue to 0.3% Triton X-100 (TX). Using the MTT assay, an exposure time which reduced the tissue viability to 50% (ET-50) is determined. For OCL-200 produced in 1996 (47 lots) versus 2001 (77 lots), the ET-50 for TX averaged 24.9 ± 6.3 and 23.8 ± 5.7 minutes, respectively. Interestingly, the MTT assay can distinguish between standard EpiOcular (OCL-200) and hydrocortisone-free EpiOcular (OCL-200-HCF); during 2000-2001, the ET-50 for OCL-200-HCF was 15.3 ± 5.5 minutes (17 lots). Direct comparison between laboratories shows that the shipping effects on the tissue are negligible. During 2000 and 2001, 50 lots of OCL-200 were tested by the Institute of In Vitro Sciences (IIVS). ET-50 values at IIVS for TX averaged 26.7 ± 5.3 minutes; these same lots tested at MatTek gave 23.4 ± 6.2 minutes, which corresponds well, despite small changes in testing methodology. Two prediction models have been developed that allow companies to interpret their in vitro EpiOcular results in terms of in vivo irritancy. Both methods have been shown to be useful and are used in industry. Thus, EpiOcular provides a reproducible, non-animal means to test materials and/or products for ocular irritancy.

E6: Three-dimensional Construct of the Human Corneal Epithelium for In Vitro Toxicology
D.H. Nguyen, R.W. Beuerman, and B. de Wever. Dept. of Ophthalmology, LSU Eye Center, Louisiana State University School of Medicine, New Orleans, Louisiana 70112, USA. dnguye@lsuhsc.edu.

This study characterizes and explores the use of three-dimensional, human corneal epithelial construct for in vitro toxicology testing. Airlifted cultures of immortalized human corneal epithelial cells were examined by light and electron microscopy and Western blot. The biochemical effects of bezalkonium chloride on structural and signal transduction pathway were determined. Gene expression was analyzed by DNA micro-array. Histologically, the construct appeared as a multi-layered, stratified epithelium with an overall thickness of 60 microns, similar to the normal human corneal epithelium. Ultra-structurally, numerous intermediate filaments, desmosomal and hemidesmosomal junctions were seen. Western blot analysis showed the presence of the corneal specific cytokeratin-3. Structural and biochemical changes were correlated in severity to the concentration of benzalkonium chloride (0.1%, 0.01%, and 0.001%) after 24 hours' contact with the construct. Short-term (1-3 hr) effects of benzalkonium chloride (0.01%) showed levels of the p42/p44 MAPK signal transduction pathway returned to control levels after replacement to saline vehicle, suggesting cell recovery. Of the 12,627 genes, 95 and 11 genes showed a 2-fold increase at 1 and 3 hours; 51 and 431 genes showed a 2-fold decrease. In conclusion, the 3-dimensional human corneal epithelial construct is a valuable alternative to previous methods for in vitro toxicology testing and evaluation.

E6: Evaluation of a New In Vitro Human Cornea Model as an Alternative to the In Vivo Rabbit Eye Draize Test

The development of in vitro alternatives to in vivo eye irritation (Draize) testing in animals is stimulated by scientific and ethical considerations. This multi-center study is aimed at evaluating the predictive power of a new commercially available human cornea model (SkinEthic, Nice, France) to assess acute ocular irritation. When cultivated in vitro on a polycarbonate filter in chemically defined medium, the human corneal epithelial cells of the immortalized cell line HCE (provided by Prof. Roger Beuerman, LSU Eye Center, New Orleans, USA) form a corneal epithelial tissue, histologically resembling the outer corneal cell layers of the human eye. Since the reconstituted epithelium grows at the air-liquid interface, solutions, formulations, and water-insoluble compounds can be assayed for their irritation potential. Reference chemicals, ranging from "non-" to very severe eye irritating, were applied topically to the HCE cultures. The rate of cytotoxicity (tissue viability and histological analysis) was compared to published in vivo acute eye irritation data. The in vitro results will be presented together with the prediction model. In addition to reducing the number of animals required for safety testing, this in vitro eye irritation model is rapid and requires less compound than other in vitro assays.