Two novel prediction models improve predictions of skin corrosive sub-categories by test methods of OECD Test Guideline No. 431

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Implementation, availability and regulatory status of an OECD accepted Reconstructed Human Epidermis model in Brazil

Introduction: In 2014, Brazil has joined the growing list of countries to ban cosmetic products from being tested on animal models. The new legislation comes into force in 2019. As a result, the interest for validated alternative testing methods for safety assessment has been increasing in academia, industry and associations. However, the lack of specific legislation on the use of biological material of human origin for toxicological tests makes the access to alternative in vitro models difficult. Furthermore, importation to Brazil is not possible on timely manner. Method: In this article, we report the implementation process of a Reconstructed Human Epidermis (SkinEthic™ RHE), an alternative model internationally accepted by OECD, through a technology transfer from EPISKIN® Lyon to Brazil. Regulatory evolution has been motivating the implementation and wide use of alternative methods to animal testing in several industry segments including cosmetic and pharmaceutical. Results: Protocol has been shown to be robust and highly reproducible. Quality control parameters (histological analysis, barrier function test and tissue viability) were performed on 24 batches assembled in Brazil. SkinEthic™ RHE model use allows the full replacement of animal test methods for skin hazards identification. It has regulatory acceptance for several toxicological endpoints, such as the Draize test for skin irritation and corrosion. It allows the reduction and refining of pre-clinical protocols through tiered strategies. Implementation of SkinEthic™ RHE protocol is just a first and important step towards a new approach of toxicological safety testing in Brazil. Conclusion: The implementation was successfully done and reported here. However, in order to follow completely the new legislation up to 2019, the availability of validated models is essential. Quality control tests done on RHE batches produced in Brazil demonstrate that the model met OECD acceptance criteria and therefore can be used for reliable prediction of irritation and corrosion classification.TÍTULO PT: Implementação, disponibilidade e contexto regulatório de um modelo de Epiderme Humana Reconstruída no Brasil aceito pela OECDIntrodução: Em 2014, o Brasil aderiu à crescente lista de países a banir testes de produtos cosméticos em modelos animais. A nova legislação entra em vigor em 2019. Como resultado, o interesse em métodos de testes alternativos validados para avaliação de segurança tem aumentado na academia, indústria e associações. No entanto, a falta de legislação específica sobre o uso de material biológico de origem humana para testes toxicológicos dificulta o acesso aos modelos alternativos in vitro. Além disso, a importação no Brasil não é possível em tempo hábil. Método: Neste artigo, relatamos o processo de implementação de um modelo de Epiderme Humana Reconstruída (SkinEthic™ RHE) internacionalmente aceito pela OECD, através de uma transferência tecnológica da Episkin Lion para o Brasil, bem como discutimos a evolução regulatória que tem motivado a implementação e a ampla utilização de métodos alternativos à experimentação animal em diversos segmentos além do cosmético e farmacêutico. Resultados: O protocolo de fabricação dos tecidos mostrou-se robusto e altamente reprodutível, considerando os parâmetros de controle de qualidade (análise histológica, função barreira e viabilidade tecidual) analisados em 24 lotes fabricados no Brasil. Conclusões: A implementação do modelo SkinEthic™ RHE é apenas um primeiro e importante passo em direção a uma nova abordagem para testes de segurança toxicológica no Brasil, realizada com êxito e aqui relatada. No entanto, para seguir plenamente a nova legislação até 2019, a disponibilidade de modelos validados é essencial. Os testes de controle de qualidade realizados nos lotes RHE produzidos no Brasil demonstram que o modelo atende aos critérios de aceitação da OCDE e, portanto, pode ser usado para uma previsão confiável de irritação e classificação de compostos corrosivos

Retrospective analysis of the Draize test for serious eye damage/eye irritation: importance of understanding the in vivo endpoints under UN GHS/EU CLP for the development and evaluation of in vitro test methods

For more than two decades, scientists have been trying to replace the regulatory in vivo Draize eye test by in vitro methods, but so far only partial replacement has been achieved. In order to better understand the reasons for this, historical in vivo rabbit data were analysed in detail and resampled with the purpose of (1) revealing which of the in vivo endpoints are most important in driving United Nations Globally Harmonized System/European Union Regulation on Classification, Labelling and Packaging (UN GHS/EU CLP) classification for serious eye damage/eye irritation and (2) evaluating the method’s within-test variability for proposing acceptable and justifiable target values of sensitivity and specificity for alternative methods and their combinations in testing strategies. Among the Cat 1 chemicals evaluated, 36–65 % (depending on the database) were classified based only on persistence of effects, with the remaining being classified mostly based on severe corneal effects. Iritis was found to rarely drive the classification (<4 % of both Cat 1 and Cat 2 chemicals). The two most important endpoints driving Cat 2 classification are conjunctiva redness (75–81 %) and corneal opacity (54–75 %). The resampling analyses demonstrated an overall probability of at least 11 % that chemicals classified as Cat 1 by the Draize eye test could be equally identified as Cat 2 and of about 12 % for Cat 2 chemicals to be equally identified as No Cat. On the other hand, the over-classification error for No Cat and Cat 2 was negligible (<1 %), which strongly suggests a high over-predictive power of the Draize eye test. Moreover, our analyses of the classification drivers suggest a critical revision of the UN GHS/EU CLP decision criteria for the classification of chemicals based on Draize eye test data, in particular Cat 1 based only on persistence of conjunctiva effects or corneal opacity scores of 4. In order to successfully replace the regulatory in vivo Draize eye test, it will be important to recognise these uncertainties and to have in vitro tools to address the most important in vivo endpoints identified in this paper

State-of-the-art and new options to assess T cell activation by skin sensitizers: Cosmetics Europe Workshop

Significant progress has been made in the development and validation of non-animal test methods for skin sensitization assessment. At present, three of the four key events of the Adverse Outcome Pathway (AOP) are assessable by OECD-accepted in vitro methods. The fourth key event describes the immunological response in the draining lymph node where activated dendritic cells present major histocompatibility complex-bound chemically modified peptides to naive T cells, thereby priming the proliferation of antigen-specific T cells. Despite substantial efforts, modelling and assessing this adaptive immune response to sensitizers with in vitro T cell assays still represents a challenge. The Cosmetics Europe Skin Tolerance Task Force organized a workshop, bringing together academic researchers, method developers, industry representatives and regulatory stakeholders to review the scientific status of T cell-based assays, foster a mutual scientific understanding and conceive new options to assess T cell activation. Participants agreed that current T cell assays have come a long way in predicting immunogenicity, but that further investment and collaboration is required to simplify assays, optimize their sensitivity, better define human donor-to-donor variability and evaluate their value to predict sensitizer potency. Furthermore, the potential role of T cell assays in AOP-based testing strategies and subsequent safety assessment concepts for cosmetic ingredients was discussed. It was agreed that it is currently difficult to anticipate uses of T cell assay data for safety assessment and concluded that experience from case studies on real-life risk assessment scenarios is needed to further consider the usefulness of assessing the fourth AOP key event.JRC.F.3-Chemicals Safety and Alternative Method

Systematic evaluation of non-animal test methods for skin sensitisation safety assessment.

The need for non-animal data to assess skin sensitisation properties of substances, especially cosmetics ingredients, has spawned the development of many in vitro methods. As it is widely believed that no single method can provide a solution, the Cosmetics Europe Skin Tolerance Task Force has defined a three-phase framework for the development of a non-animal testing strategy for skin sensitisation potency prediction. The results of the first phase - systematic evaluation of 16 test methods - are presented here. This evaluation involved generation of data on a common set of ten substances in all methods and systematic collation of information including the level of standardisation, existing test data, potential for throughput, transferability and accessibility in cooperation with the test method developers. A workshop was held with the test method developers to review the outcome of this evaluation and to discuss the results. The evaluation informed the prioritisation of test methods for the next phase of the non-animal testing strategy development framework. Ultimately, the testing strategy - combined with bioavailability and skin metabolism data and exposure consideration - is envisaged to allow establishment of a data integration approach for skin sensitisation safety assessment of cosmetic ingredients