Oroxcell Cellular Toxicity

Transcription

1 Oroxcell 2009 Cellular Toxicity

2 Table of contents Cellular Toxicity 1. Episkin Episkin model by SkinEthic Skin corrosion Skin irritation 2. Reconstructed Human Epidermis (RHE) RHE model by SkinEthic Validation of skin irritation assay 3. Human Corneal Epithelium (HCE) HCE model by SkinEthic Ocular irritation assay 4. Advantages of alternative methods Advantages of cellular assays Oroxcell

3 Episkin Episkin model by SkinEthic Skin Culture and Source Human keratinocytes, from mammary/abdominal samples from healthy consenting donors, are cultured on a collagen base. After terminal differentiation an epidermis is reconstituted with a functional horny layer. Applications Skin corrosion of chemicals Skin irritation of chemicals and cosmetics Skin permeability and metabolism Validation status Skin corrosion test - OECD TG 431 guidelines Validated by ECVAM for skin irritation as an alternative method to animal testing In vivo In vitro Oroxcell

4 Episkin Skin corrosion of chemicals The classification obtained by Oroxcell using in vitro Episkin model was in accordance with in vivo classification for all the tested compounds N Product In vivo classification (C/NC) 3 min 1 hour Oroxcell Cell viablity Cell viablity classification ± SD ± SD (%) (%) (C/NC) 1 Acetic acid (99.8%) (C+) C C 2 Acrylic acid (99%) C C 3 Octanoic acid (99%) C C 4 Nitric acid (65%) C C 5 Sodium chloride (0.9%) (C-) NC NC 6 4-amino-1,2,4-triazole (99%) NC NC 7 Tetrachloroethylene (99%) NC NC 8 Eugenol (99%) NC NC 9 Sodium hydroxyde (10% aq) C na C corrosive NC non corrosive na not applicable, according to the guidelines: dramatically damaged tissues C+ positive control C- negative control Oroxcell

5 Episkin Skin irritation of chemicals Classification of tested compounds Test compounds were classified according to the following scheme Cell Viability (MTT) Cell viability and IL1α release were assessed after 15 min contact with the test substances followed by 42h incubation Results were in accordance with bibliography (Tornier et al. 2005) Internal validation of the assay was performed 50% Irritant > 50% IL-1α release (42 h) 60 pg/ml > 60 pg/ml See results Non-irritant Irritant Oroxcell

6 Episkin Skin irritation of chemicals N Compound 1 SDS (5% aq.) I 2 Heptanal (92%) I 3 Tri-isobutyl phosphate (97%) I 4 2,4 xylidine (98%) I 5 Alpha terpineol (90%) I 6 10-Undecenoic Acid (99%) I 7 1-Bromopentane (>98%) I 8 d-limonene (98%) I 9 cis-cyclooctene (95%) NI 10 di-n propyl disulfide (97%) I 11 Hydroxycitronellal (95%) I 12 PBS NI 13 Heptyl butyrate (98%) NI 14 Isopropyl myristate (90%) NI 15 Isopropanol (99,8%) NI 16 3,3' dipropionique Acid (99%) NI 17 4-Amino-1,2,4-triazole (99%) NI Oroxcell

7 Reconstructed Human Epidermis RHE model by SkinEthic In vivo Skin Culture and source Normal human keratinocytes are cultured on an inert polycarbonate filter at the air-liquid interface, in a chemically defined medium. Histologically similar to the in vivo human epidermis. Applications Skin corrosion of chemicals Skin Irritation of chemical and cosmetics Permeability and metabolism prediction Active ingredients Rank ordering of formulations In vitro Validation status Skin corrosion test - OECD TG 431 guidelines Validated by ECVAM as an alternative method to the animal use for skin irritation Oroxcell

8 Reconstructed Human Epidermis Validation of skin irritation assay Oroxcell has contributed to the multicenter study for ECVAM validation Predictive values for the MTT endpoint of the validated in vitro test for skin irritation using RHE in comparison to the fully validated reference method of the ECVAM skin irritation validation study using EPISKIN EPISKIN Considering the MTT endpoint, the validated method has predictive values for the 20 reference chemicals tested The test method meets the values of predictivity indicated in the performance standards (specificity = 80% and sensitivity = 70%) RHE Specificity 80% 80% Sensitivity 70% 90% False positive rate 20% 20% False negative rate 30% 10% Accuracy 75% 85% Oroxcell

9 Human Corneal Epithelium HCE model by SkinEthic Corneal Culture and Source Cultivated immortalized Human Corneal Epithelial cells from the cell line HCE reconstruct a corneal epithelial tissue. Devoid of stratum corneum, the model is ultra-structurally (tissue morphology and thickness) similar to the corneal mucosa of the human eye. Applications Ocular irritation assay Ocular permeability and metabolism In vitro Validation status ECVAM pre-validation phase as alternative method to animal testing for ocular irritation Oroxcell

10 Human Corneal Epithelium Ocular irritation assay Comparison of Oroxcell results vs. those obtained in the published ECVAM prevalidation multicenter study (Van Goethem et al. 2006) Viability of HCE was assessed following 3h incubation with 20 test substances Oroxcell s results are online with those obtained in the prevalidation study Oroxcell Multicenter study Oroxcell

11 Advantages of alternative methods Advantages of cellular assays Quality Excellent predictive capacity (higher than rabbit skin Draize test) Reproducibility within and between laboratories Adapted to large screening studies Accordance with international directives Chemicals : REACH Cosmetics : 7th Amendment to the EU Cosmetics Directive Ethics Impact on 3R Principles: replacement of animal models by alternative methods Price Lower costs than in vivo Draize test Oroxcell

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