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In alignment with the global movement toward reducing animal testing, several reconstructed human epidermis (RHE) models have been created for conducting skin irritation tests. These models have undergone development, verification, validation, and integration into OECD TG 439. Our team has introduced a novel in-house RHE named GB-RHE, and we adhere to OECD TG 439 to pre-validate the model and test its potential employment for nanoparticle irritation studies. GB-RHE exhibits morphological, biochemical, and physiological attributes equivalent to the human epidermis, featuring well-differentiated multilayered viable keratinocytes with a robust barrier function. The performance of the GB-RHE model was evaluated using ten reference chemicals, following the performance standard of OECD TG 439. The results demonstrated commendable predictive capacity and showed that titanium dioxide nanoparticles (TiO 2 NPs) are 'non-irritant' to the human epidermis following the globally harmonized classification system. However, although the histological analysis did not show morphological changes, transmission electron micrographs demonstrated that TiO 2 NPs can be internalized, reaching the external viable layers of the epidermis. This study demonstrates that in addition to the potential of the GB-RHE model to evaluate skin irritation, this model also has the potential to evaluate the skin toxicity of NPs and carry out cell internalization studies., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.) more...

The TRPV1 receptor, which is known to contribute significantly to pain perception, has recently been identified as a useful tool for predicting eye stinging potential in cosmetics. In this study, HEK-293 cells with high TRPV1 expression were utilized to evaluate calcium influx related to receptor activation triggered by chemicals and cosmetic formulations. The cells were exposed to increasing concentrations of substances to cause or not some aggression to the eye, and TRPV1 activity was assessed by measuring intracellular FURA-2 AM fluorescence signal. To confirm TRPV1 channel activation, capsazepine, a capsaicin antagonist, was employed in addition to using capsaicin as a positive control. The study's results indicate that this novel model can identify compounds known to cause some aggression to the eye, such as stinging, considering a cut-off value of 60% of Ca 2+ influx exposed to the lowest evaluated concentration (0.00032%). When applied to the cosmetic baby formulation, although the presented model exhibited higher sensitivity by classifying as stinging formulations that had previously undergone clinical testing and were deemed non-stinging, the assay could serve as a valuable in vitro tool for predicting human eye stinging sensation and can be used as a tier 1 in an integrated testing strategy., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Marize Campos Valadares reports financial support was provided by Boticario Group. Marize Campos Valadares reports a relationship with Federal University of Goias that includes: employment and non-financial support. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.) more...

The Brazilian National Network of Alternative Methods (RENAMA), which is linked to the Ministry of Science, Technology and Innovation, is currently comprised of 51 laboratories from CROs, academia, industry and government. RENAMA's aim is to develop and validate new approach methodologies (NAMs), as well as train researchers and disseminate information on their use - thus reducing Brazilian, and consequently Latin American, dependence on external technology. Moreover, it promotes the adoption of NAMs by educators and trained researchers, as well as the implementation of good laboratory practice (GLP) and the use of certified products. The RENAMA network started its activities in 2012, and was originally comprised of three central laboratories - the National Institute of Metrology, Quality and Technology (INMETRO); the National Institute of Quality Control in Health (INCQS); and the National Brazilian Biosciences Laboratory (LNBio) - and ten associated laboratories. In 2022, RENAMA celebrated its 10th anniversary, a milestone commemorated by the organisation of a meeting attended by different stakeholders, including the RENAMA-associated laboratories, academia, non-governmental organisations and industry. Ninety-six participants attended the meeting, held on 26 May 2022 in Balneário Camboriú, SC, Brazil, as part of the programme of the XXIII Brazilian Congress of Toxicology 2022. Significant moments of the RENAMA were remembered, and new goals and discussion themes were established. The lectures highlighted recent innovations in the toxicological sciences that have translated into the assessment of consumer product safety through the use of human-relevant NAMs instead of the use of existing animal-based approaches. The challenges and opportunities in accepting such practices for regulatory purposes were also presented and discussed., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. more...

The skin is the body's largest organ, continuously exposed to and affected by natural and anthropogenic nanomaterials (materials with external and internal dimensions in the nanoscale range). This broad spectrum of insults gives rise to irreversible health effects (from skin corrosion to cancer). Organ-on-chip systems can recapitulate skin physiology with high fidelity and potentially revolutionize the safety assessment of nanomaterials. Here, we review current advances in skin-on-chip models and their potential to elucidate biological mechanisms. Further, strategies are discussed to recapitulate skin physiology on-chip, improving control over nanomaterials exposure and transport across cells. Finally, we highlight future opportunities and challenges from design and fabrication to acceptance by regulatory bodies and industry., Competing Interests: Declaration of interests The authors declare none., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.) more...

This chapter presents the protocols for developing of skin equivalents (SE) and reconstructed human epidermis (RHE) models for dermal toxicity evaluation as an alternative method to animal use in research. It provides a detailed protocol for the in vitro reconstruction of human skin from primary keratinocytes, melanocytes, and fibroblasts obtained from foreskin biopsies, including the procedures for reconstruction of a stratified epidermis on a polyester membrane. SE and RHE developed through these methods have been proven suitable not only for dermal toxicity studies, but also for investigating of pathological conditions in the skin, such as diabetes and invasion of melanoma. more...

Currently, there is a strong global trend towards the development of in vitro models to replace the use of animals in safety evaluation tests. Reconstructed Human Epidermis (RHE) models have been employed as an alternative method to animal testing of skin corrosion and irritation potential of chemical compounds. However, the consequences of an absence of the dermal compartment in these models should be considered since the cross-talk between fibroblasts and keratinocytes is fundamental for promoting proper epidermal stratification, homeostasis, inflammatory response and wound healing. In this study, we compare in-house developed models of Reconstructed Human Epidermis (i.e. USP-RHE) and full thickness skin (i.e. USP-FTS) regarding their response when submitted to skin corrosion assays, based on Guideline 431 (OECD). The results show that both models correctly classified the four substances tested (2-phenylethyl bromide, benzylacetone, lactic acid, octanoic acid) as corrosive or non-corrosive. Furthermore, we have demonstrated higher cell viability of the USP-FTS model compared to the USP-RHE model, a sign of its improved barrier function, following the exposure to the substances test on the corrosion assay. This emphasizes the importance of employing in vitro models that are more physiologically relevant and that better mimic the in vivo situation for the toxicological screening of substances., (Copyright © 2018 Elsevier B.V. All rights reserved.) more...

Allergic and irritant skin reactions caused by topical exposure to permanent hair dyes are a common problem. For regulatory and ethnical purposes, it is required to perform chemical safety assessment following the replacement, reduction, and refinement of animal testing (3Rs). Permanent hair dyes are formed by a mixture of ingredients that vary from low to extreme skin sensitizing potency and that inter-react to form unknown by-products. Because of the complex reaction, this cytotoxic mechanism has not yet been elucidated and is the subject of this study. Here, we topically exposed p-phenylenediamine (PPD), Resorcinol (RES), Hydrogen Peroxide (H 2 O 2 ) alone or as a mixture to RhE and evaluated parameters related to skin irritation such as epidermal viability, keratinocytes damage, barrier loss and IL-1 α. Our data indicates that ingredients tested alone did not lead to an increase of cytotoxic parameters related to skin irritation. However, when the mixture of PPD/H 2 O 2 /RES and PPD/H 2 O 2 was applied to the RhE, some of the parameters such as morphological changes including the presence of apoptotic cells, barrier loss and increased IL- 1 α release were observed. The results indicate that the mixture of ingredients used in permanent hair dyes have an irritant effect in RhE while the ingredients alone not., (Copyright © 2017 Elsevier Ltd. All rights reserved.) more...

Different models of reconstructed human epidermis (RHE) are currently validated to assess skin irritation in vitro and ultimately to the animal replacement of the Draize test. The development of a new RHE model is a challenge for many laboratories, representing a potential gain of autonomy and improvement of technological knowledge. The Organization for Economic Co-operation and Development (OECD) encourages the development of new models and, for this purpose, offers a thorough guideline on quality control parameters (OECD TG 439 performance standards). This work aimed to develop an RHE model (i.e. USP-RHE) for in vitro skin irritation assays, following the OECD TG 439. The developed model presents a well-differentiated epidermis similar to the Validated Reference Methods (VRM) and to native human epidermis. Quality parameters, i.e. optical density of negative control, tissue integrity and barrier function, were similar to VRM and in accordance with OECD TG 439. Moreover, the USP-RHE model was shown to have 85,7% of specificity (6/7), 100% of sensitivity (6/6) and 92,3% of accuracy (12/13) when compared to in vivo UN GHS classification. The within-laboratory reproducibility was 92.3% (12/13). Thus, we demonstrated that USP-RHE model attends to all OECD TG 439 performance standards and is ready to be used by private and public laboratories and companies for future validation studies., (Copyright © 2017 Elsevier Ltd. All rights reserved.) more...

In this study, we detected Leishmania (Viannia) braziliensis infection in equids living in endemic regions of cutaneous leishmaniasis. To determine the role of these animals in the Leishmania cycle, we used two approaches: serological and molecular methods. Antibodies to the parasite were assayed using the Enzyme Linked Immunosorbent Assay (ELISA). Blood samples were collected and tested by polymerase chain reaction (PCR), and the positive products were sequenced. The results showed that 11.0% (25/227) of the equids were seropositive for Leishmania sp, and 16.3% (37/227) were PCR positive. Antibodies were detected in 20 horses, 3 donkeys, and 2 mules, and the parasite DNA was detected in 30 horses, 5 donkeys, and 2 mules. Sequencing the amplified DNA revealed 100% similarity with sequences for Viannia complex, corroborating the results of PCR for L. braziliensis. Our results show that equids are infected with L. braziliensis, which could be food sources for phlebotomines in the peridomiciliary environment and consequently play a role in the cutaneous leishmaniasis cycle. more...