Reliability and relevance of the ES®-RHE model for in vitro skin irritation test application.

INTRODUCTION: In vitro methods have been widely used to assess adverse effects. Reconstructed Human Epidermis (RHE) poses as a fascinating test system employed to assess the dermal irritation hazard potential of chemicals. Although several RHE models are reported in the OECD Test Guideline No. 439, the OECD Document No. 220 encourages the scientific community to develop and validate new RHE test systems due to its relevance for socio-economic advancement. METHODS: Following the criteria documented in the OECD No. 220, a blind study for skin irritation (OECD 439) was conducted employing the Minimum List of Reference Chemicals for Determination of Reproducibility and Predictive Capacity using ES®-RHE. Structural and functional characteristics were assessed alongside the prediction model. RESULTS: The model has shown reproducibility of optical density and barrier function, similarly to internationally validated methods. Furthermore, it shows the cell layers' development and differentiation ability due to Cytokeratin14, Cytokeratin10, and filaggrin expression. The prediction model resulted in sensitivity, specificity and accuracy rates of 100, 70, and 77 %, respectively. CONCLUSIONS: The ES®-RHE demonstrated reliability and relevance, with similar structural and functional characteristics comparable to internationally validated models, in addition to the accepted predictive capacity according to OECD required minimum criteria, thus confirming the suitability of the national ES®-RHE in the hazard prediction of dermal irritation based on OECD Test Guideline No. 439.

Immortalized keratinocytes cells generates an effective model of Epidermal Human Equivalent for irritation and corrosion tests.

Three-dimensional skin models, also named 3D skin models, human skin equivalents (HSEs), or Human Epidermal Equivalents (HEEs), have been increasingly used for chemical assessments in terms of efficacy and safety. Considering this, we developed an HEE model using immortalized HaCaT cells, aiming to overcome the limitation of primary tissue source. Our 3D model (HaCaT-HEE) exhibited important markers of cell differentiation (CK10, CK14, involucrin, and filaggrin), although the stratum corneum was shown to be modest. Besides, the model showed a good prediction potential considering membrane permeability, sensitivity, specificity, and accuracy in distinguishing irritant and corrosive effects after exposure to selected chemicals recommended by the OECD protocols. We also validated the formazan determination for the MTT method using High-Performance Liquid Chromatography (HPLC). For that, we considered carry over, linearity, reproducibility/robustness, accuracy, precision, selectivity, and matrix effect, according to the Food and Drug Administration (FDA) guideline. Based on our results, we can conclude that our model has an acceptable predictive value for the safety evaluation of compounds after skin exposure, with the great advantage of being constructed using immortalized cells.

Immortalized equivalent human epidermis as a platform to evaluation hair dyes toxicity: Efficiency comparison between 3D and monolayer culture.

The use of 3D models in various scientific applications is becoming more popular to replace traditional monolayers models. In this work, we used a three-dimensional in-house model of epidermis using HaCaT immortalized cells to evaluate the dermal toxicity induced by Basic Blue 99 and Basic Red 51, both present in commercial hair dye formulations. Our data show that cells cultured in the 3D model respond differently to those cultured in monolayer. Basic Red 51 dye induces apoptosis an DNA breaks in both models, however, these effects is more pronounced in cells cultured in monolayer. The toxic mode of action of Basic Blue 99 seems to be the induction of cell death, without genotoxic effects, but while the necrotic pathway is observed in HaCaT monolayer cell culture, was apoptosis seen in the Equivalent Human Epidermis (EHE) model. We could also confirm that cells in EHE model, an environment that could better mimic human effects, react differently to chemical stressors than the cells cultivated in 2D.