Establishment of a tested 3D epidermis model of an immortalized cells as a platform for assessing dermal toxicity induced by hair dyes

Author(s)

Camila Alessandra Mini

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Ciências Farmacêuticas de Ribeirão Preto (PCARP/BC)
• Defense date: 2020-08-12

Examining board members

Danielle Palma de Oliveira; Lusania Maria Greggi Antunes; Lorena Rigo Gaspar Cordeiro; Daniela Morais Leme

• Advisor: Danielle Palma de Oliveira; Silvya Stuchi Maria Engler

Abstract

With the adoption of alternative methods to the use of animals in the scientific community, several groups worked towards the development of new models capable of being applied in the evaluation of the toxicity of substances / products. In the case of dermal exposure, the reconstructed skin / epidermis models were developed worldwide, but the acquisition of these models in Brazil is quite difficult. Because of this, the objective proposed here was the development of a three-dimensional model of epidermis, using immortalized keratinocytes (HaCaT-EHE) for application in the evaluation of toxicity of dyes present in hair dyes, in addition to comparing the effects with cell culture in monolayer . After building the three-dimensional model, cell differentiation was evaluated by histological / immunohistochemical methods, validation of the analytical method for determining formazan salt used in corrosion and irritation tests. The permeability of the membrane and the proficiency of the model in differentiating irritating, non-irritating, corrosive and non-corrosive substances were verified. For evaluation of toxicity endpoints, genotoxicity assay (Comet assay), viability / cytotoxicity (MTT, cell viability test by annexin V / PI and Tunel test) and irritation and corrosion were performed. The model showed cell differentiation and expression of the biomarkers of development of the layers, in addition it presented membrane permeability similar to the models already validated. The method for determining formazan salt has proven to be selective, accurate, precise and robust. In addition, it had a recovery rate greater than 85% and variation less than 15%, and the presence of salt residues was not observed after an injection with high concentration. In addition, the model demonstrated good prediction for irritation (100% selectivity, 75% specificity and 90% accuracy) and also for corrosion (100% selectivity, 100% specificity and 100% accuracy) for the tested substances. As for the dyes, Basic Blue 99 (BB99) did not demonstrate genotoxic effects in both models, however, it showed a decrease in cell viability, in addition to intense staining with PI and double staining (annexin and PI) indicating cell death by necrosis in the 2D model. In the 3D model, intense fluorescein staining was observed at all concentrations tested, indicating apoptosis. As expected, BB99 was irritating after exposure for 30 minutes, although it did not induce a corrosive effect. The Basic Red 51 (BR51) dye induced genotoxic effects in both models, in addition it caused a drop in cell viability in the 2D model and in the 3D model it induced fluorescein staining. The dye had no irritating or corrosive effects. In view of the above, we can conclude that the model presented a good prediction and can be used as a platform for evaluating the safety / toxicity of products after dermal exposure. The dyes tested demonstrated toxic effects at concentrations below the limit recommended by the European Scientific Committee and, therefore, the use in commercial hair dye products should be reviewed. (AU)

• FAPESP's process: 16/15660-4 - Local toxic effects evaluation in epidermis due dyes exposure used in hair dyes
• Grantee: Camila Alessandra Mini
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)