1. An Evaluation of the Episkin and the Skinethic RHE Test Methods for Predicting Dermal Toxicity Using the OECD TG404 N. Alepée¹, J.Cotovio¹, N. Seyler², C. Tornier², A. de Brugerolle de Fraissinette, M.H. Grandidier¹, M. Roguet², F. Soler¹, and J.R. Meunier¹
¹ SkinEthic Laboratories, Lyon, France
² L’Oréal Research & Innovation


The Globally Harmonised System for the classification and labelling of chemical substances and mixtures (GHS) defined skin corrosion as the production of irreversible tissue damage in the skin. In vitro human reconstructed epidermal models have been used to develop protocols able to discriminate corrosives from non corrosives. The SkinEthic RHE test method, using 0.63 cm2 inserts, was validated by ESAC (ECVAM advisory board) in 2006. Due to manufacturing constraints, the SkinEthic RHE model is now produced on 0.5 cm2 instead of 0.63 cm2.


The present study first aim to demonstrate that the RHE skin corrosion protocol could be adapted from 0.63 cm2 to 0.5 cm2 RHE tissues samples. For such purposes, the protocol size adaptation was performed using the 25 chemicals including the 12 OECD TG431 reference test substances. To test the robustness and relevance of the test method, particular attention was given to choose chemicals correctly classified (non corrosive, corrosive) but also misclassified chemicals (false positives / negatives).


In addition, the objective was to show that the quality of RHE tissues was not only maintained but also improved. The quality control, performed on 136 (0.63 cm2) and 262 (0.5 cm2) RHE batches, showed a mean of 0.991 and 1.169, respectively. This similarity over the 9 years demonstrates the high quality production of the tissues using both viability and morphology parameters.


Results obtained with the 0.5 cm2 skin corrosion test method showed that all corrosives (12) were correctly classified, and 11 out of 13 chemicals were identified as non corrosives. The 2 misclassified chemicals were also identified as false positives in the reference validated EpiSkin test method. The overall accuracy over the 25 chemicals was 92%. The specificity and the sensitivity of the 12 OECD TG431 reference chemicals were 100%.


In conclusion, the quality of 0.5 cm2 SkinEthic RHE tissues was thoroughly maintained over 9 years and the performance of the skin corrosion test method fully meet the OECD and ECVAM requirements.

2. Evaluation of the in vitro EpiSkin and SkinEthic RHE Skin Irritation Test Methods for Hazard Identification of Chemicals D. Lelièvre¹, N. Alépée¹, J. Cotovio¹, M. H. Grandidier¹, A. de Brugerolle de Fraissinette², C. Tornier², and J.R. Meunier¹
¹ L’Oréal Research & Innovation
² SkinEthic Laboratories, Nice, France


On July 22 nd, 2010, the in vitro skin irritation OECD Test Guideline TG439 and the supportive Background Document were adopted. As reflected in the OECD TG439, the EpiSkinTM, designated as the Validated Reference Method and the SkinEthicTM RHE method can be considered to be applied to a wide range of physico-chemical substances and mixtures. The evaluation of both the reproducibility and the reliability of these methods was performed to confirm their ability to predict the classification of chemicals in the former and the recent implemented Global Harmonization System (GHS) classifications applied in Europe.


The present study purpose was to investigate the performances of both test methods with an expanded set of 62 chemicals in both classifications. Chemicals with in vivo scores between 2 to 2.3 are now considered as non-irritants. Consequently to the new Classification Labelling Packaging (CLP) system, the number of No category substances were increased from 37 to 45 whereas the irritant category number decreased to 17.


When comparing the obtained results in the old versus the new European classification systems, the sensitivities of both, the in vitro Validated Reference EpiSkinTM method and the SkinEthicTM RHE test method were improved to 88.2 and 94.1%, respectively. Accuracies of both test methods were 83.9 and 77.4 respectively. Overall performances were in accordance with criteria defined in the Annex 2 of OECD TG439.


These results confirm that these two test methods can be considered as reliable in vitro screening tools to assess the skin irritation potential of chemicals for the REACh registration and the European Cosmetic Directive legislation.

3. Cutaneous Metabolism J. Eilstein, G. Léreaux, J.R. Meunier, J. Leclaire and D. Duché
L’Oréal Research & Innovation


According to the literature, the metabolism of other organs than the liver such the skin seems much less studied. Indeed, skin represents the major protective barrier of the body to the environment and chemicals exposure but is not really yet considered as an organ involved in xenobiotic metabolism. It appears to be a tissue of weak catalytic activity generating less diverse metabolites and less funny reaction mechanisms. However, this assertion could be due to the lack of specific tools to study skin metabolism such as particular sample preparation protocols or analytical methods which are accurate and sensitive enough.

Thus, as skin is the largest organ of the human body, even if weak enzymatic activities are observed, they can become consequent when considering its total surface area. Consequently, research on skin metabolism would require a real scientific effort and dynamism to characterize skin metabolizing enzymes and their activities. In addition, the 7th European amendment to the cosmetic directive forbids the use of animal testing to assess the effectiveness and safety of new cosmetics. This policy has forced the cosmetic industry to develop in vitro tools as alternative methods to animal experiments. Reconstructed human skin models are a part of them. For these reasons, these models have to be characterized and compared with normal human skin in terms of metabolic capabilities. This work presents a review of the L’Oreal research strategy and main results in the characterization of skin metabolic equipment and its catalytic capabilities. Thus, characterization for the expression of several enzymes (CYP450, Esterase, NAT, GST, UGT, SULT…) and their catalytic activities (Apparent Km, Vmax and clearance) in various reconstructed skins were compared to normal human skin samples.

4. L'Oréal Commitment in the Development, Evaluation and Validation of Screening and Testing Approaches Contributing to the 3Rs J. R. Meunier, N. Alépée, J. Cotovio, D. Duché, G. Ouédraogo, L. Marrot,
S. Teissier, J.M. Ovigne and J Leclaire
L’Oréal Research & Innovation


Dissemination of advances on alternative methods represents a step to promote alternatives to animal testing in line with the EU Cosmetics Directive. Over the past decade there have been great efforts in the industry or academic laboratories to develop alternative methods either in silico or in vitro that would comply with regulatory constraints. The implementation is still limited given that it is a great challenge to replace historical approaches by good and well predictive in vitro tests.


L’Oréal has, based on these principles, developed test methods to screen and test potential effects on chemicals.


We have focused initially on approaches for skin irritation, since this is an important endpoint for chemicals used in Cosmetics. A peer review on various aspects of alternative techniques was performed at all stages of the research and development with a focus on in vitro methods improvement of chemicals selection (screening) as well as quality testing. Following the development of the screening test, we have implemented some protocols (i.e. EpiSkinTM and SkinEthicTM RHE) for pre-validation and validation processes according to ECVAM’s recommendations. To ensure quality and objectivity, experts from international committees oversee the content. Details of the approach will be presented for both skin corrosion and irritation with a set of 20 reference chemicals.


Moreover, the poster will also describe the practical approaches developed by L’Oréal in the areas of eye irritation (SkinEthicTM HCE defined with 90 chemicals), skin sensitization (MUSST assay optimized with 50 chemicals), phototoxicity and genotoxicity.


By participating actively in relevant forums, L’Oréal and other industries will continue to promote the development of new tools and methodologies as well as favor the acceptance of in vitro alternatives by authorities.

5. Evaluation of an Oral Care Product Safety Screening Program Utilizing the In Vitro Skinethic Human Gingival Epithelium (Rhg) and Oral Buccal (Rho) Models L. Wurzburger¹, P. Kazmi¹, T. Re¹, A. Alonso², B. Bertino², N. Barnes3, A. de Brugerolle de Fraissinette², A. Hilberer3, H. Raabe3, N. Wilt3, V. Srinivasan¹
¹ L'Oreal USA Products, Clark, NJ, United States.
² SkinEthic Laboratories, Nice, France.
3 Institute for In Vitro Sciences, Inc., Gaithersburg, MD, United States.


Assuring the safety of personal care products without testing in animals is a goal common to many personal care products manufacturers, due to both ethical concerns for animal welfare, as well as the limited relevancy of animal models to predict human responses. Towards this goal, the cosmetics and personal care industry has increasingly relied upon human cell-based 3–dimensional reconstructed tissues to evaluate the safety of their product candidates in various target tissues. Accordingly, we have developed a program for screening the potential irritancy of teeth whitening products in oral mucosal tissues using commercially-available oral buccal (SkinEthic RHO) and gingival (SkinEthic RHG) models. Four formulations containing H2O2 at various concentrations and one sodium bicarbonate were tested at four exposure times to determine ET50 values. Three irritancy endpoints were measured after each exposure: viability using the MTT conversion assay, the amount of IL-1a released from the tissues, and histological changes. Both models presented the same rank order of the five materials, with increases in the ET50 values correlating with decreases in H2O2 concentration. The formula containing sodium bicarbonate, however, was non-toxic in both models. Histological analysis confirmed the MTT results and provided evidence of the chemical impact upon cellular and tissue morphology. IL-1a release did not appear to be as sensitive as the MTT assay at shorter exposure times, although it may be useful to differentiate among formulations predicted by the MTT assay to be of low irritation potential. Our results suggest that the MTT viability and histology endpoints in 3–D human oral reconstructed tissues can provide useful predictive information to support an oral care products safety program.

6. In vitro Assessment of Eye Tolerance using the SkinEthic HCE Test Method Applied to Ingredients Used in Cosmetic M. H. Grandidier, N. Alépée, S. Blond, J. Eilstein, D. Duché, J. Cotovio and J. R.Meunier
L’Oréal Research & Innovation


To comply with the EU Cosmetic Directive, accurate sensitive and predictive alternative methods are required in order to evaluate eye damage potential of chemicals. Linked to the complexity of both the eye irritation mechanism and the diversity of the intrinsic molecular behavior of chemicals, assessment of the potential eye irritation of ingredients is a complicated issue.


The role of corneal epithelium is crucial because it represents the first line of defense against many types of injury. Thus we have used the standardized SkinEthic reconstructed human corneal epithelial (HCE) model to evaluate in vitro eye irritancy (Cotovio et al, 2010).


We have determined the chemical reactivity parameter by measuring the depletion of synthetic peptide containing Cysteine or Lysine residues in contact with chemicals. The reactivity peptide binding assay (RPBA-HCE) defining the reactivity status of chemicals was optimized from the DPRA assay (skin sensitization evaluation) for assessing the eye irritancy using the SkinEthic HCE test method.


The Prediction Model (PM), applied for a set of more than 180 chemicals, is based on a 5.95 % depletion cut off, with 2 categorizations (reactive and non reactive).


In parallel, SkinEthic HCE protocol (10 min and 1 hr + 16 hrs post-exposure incubation) used with the same set of chemicals confirmed that the PM based on a 50 % viability cut-off allowed the drawing up of irritants and non irritants classes. Results (~1/3 reactive versus ~2/3 non reactive chemicals) showed that the measure of molecular reactivity of these chemicals used to orientate the tested chemicals towards the suitable time exposure allowed to discriminate irritants from non irritants. Consequently, the resulting performances of the testing battery combining SkinEthic HCE assays and chemical reactivity measurement greatly enhanced the applicability domain of the SkinEthic HCE assay.