RESUMO
The ocular irritation potential of products that may come into contact with the eyes should be assessed by the combination of different in vitro alternative methods to determine different mechanisms of toxicity previously evaluated by the Draize in vivo assay. Thus, this study proposed to apply two strategies for the prediction of the eye irritation potential of different concentrations of surfactants and silicones, the first one involving evaluation Hen's Egg Test - Chorioallantoic Membrane (HET-CAM), and the other one using Bovine Corneal Opacity and Permeability (BCOP) followed by histopathological. HET-CAM was considered important in assessing the ocular irritation potential and, despite classifying almost all surfactants as "severe irritants", it could discriminate moderate and slight irritant SLES concentrations as well as Cocoamidopropyl Betaine as a severe irritant, when the coagulation score was taken into consideration. The BCOP assay alone also did not offer a good prediction of the irritant potential of surfactants, since almost all of them were classified as "no prediction can be made". However, the histopathological evaluation of the BCOP corneas was very important for establishing the degree and depth of damage related to reversibility. The present study also showed those strategies are sensitive to small variations in the studied anionic, cationic amphoteric surfactant concentrations and can be used for predicting their toxicity in the final product and can be used depending on the focus of the analysis.
Assuntos
Opacidade da Córnea , Cosméticos , Alternativas aos Testes com Animais/métodos , Animais , Bovinos , Galinhas , Opacidade da Córnea/patologia , Cosméticos/toxicidade , Olho , Feminino , Irritantes/toxicidade , Silicones/farmacologia , Tensoativos/toxicidadeRESUMO
The hen's egg test on chorioallantoic membrane (HET-CAM) is one of the most frequently used alternative tests for prediction of ocular irritation of cosmetic products. There are different HET-CAM protocols widely accepted, but there is no information about which of the protocols better correlates with the results obtained in product use clinical study under the conditions of use. Two Fix Time Methods (FTM) -Lüepke and the ICCVAM guideline - and two Reaction Time Methods (RTM) -ECVAM DBALM Prot. No. 47 and No. 96- were employed to test 18 cosmetic products. Simultaneously, they were evaluated by an ophthalmological clinical test. A unified classification system was used, and products were classified into four irritation levels: non-irritant, weak, moderate and severe irritant. The duration of use (rinse-off or leave-on), and the concentration and type of surfactants were taken into account in the analysis. All the products that were classified as non-irritant by any HET-CAM protocols were also safe in the product use clinical study. The product that was found to be non-safe in the product use clinical evaluation was also unsuitable by most of the HET-CAM protocols. These results were employed to develop an algorithm that allows selecting the appropriate HET-CAM protocol for each type of product to be tested.
Assuntos
Alternativas aos Testes com Animais , Membrana Corioalantoide/química , Cosméticos/toxicidade , Oftalmopatias/induzido quimicamente , Tensoativos/toxicidade , Animais , Embrião de Galinha , Humanos , Irritantes/toxicidade , Tensoativos/químicaRESUMO
Historically, the ocular toxicity of manufactured consumer materials has been evaluated using the rabbit in vivo Draize rabbit eye test. The animal data obtained were used by the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS) to define the classification and labelling (C&L) for eye damage/irritation endpoint. However, the Draize test, a method which was never formally validated, has been widely criticized because of its technical limitations. In addition, ethical and economic issues and advances in scientific knowledge, and political and public pressures have made animal experimentation unsustainable. This scenario has consequently led to the development of nonanimal testing and protocols/approaches with considerable predictive value and relevance for humans. It is widely accepted that one single nonanimal method cannot cover all the criteria of damage/inflammation assessed by regulatory adopted in vivo animal testing. Thus, integrated testing strategies (ITS) have been proposed, including a tiered testing approach combining different nonanimal testing with different endpoints, which have been used for regulatory purposes, on a case-by-case basis and within integrated approaches to testing and assessment (IATA), to identify materials according to their ability to trigger eye damage. In particular, the top-down and bottom-up approaches have been recommended for the C&L of materials, which cause serious eye damage or eye irritation, respectively. This chapter describes detailed protocols for eye irritation testing based on cells (Short Time Exposure-STE, OECD No. 491/2017), a vascularized membrane (the Hen's Egg Test-Chorioallantoic Membrane-HET-CAM) and corneal tissue (Bovine Corneal Opacity and Permeability-BCOP, OECD No. 437/2017), which can be applied using top-down or bottom-up approaches. In addition, it suggests making a corneal histomorphometric evaluation as an additional parameter in the BCOP method to differentiate materials that cause serious eye tissue damage (UN GHS Cat. 1) from materials that have reversible eye irritation effects (UN GHS Cat. 2).
Assuntos
Alternativas aos Testes com Animais , Bioensaio , Membrana Corioalantoide/irrigação sanguínea , Membrana Corioalantoide/efeitos dos fármacos , Córnea/efeitos dos fármacos , Irritantes/toxicidade , Neuropatia Óptica Tóxica , Testes de Toxicidade , Animais , Bovinos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Embrião de Galinha , Córnea/patologia , CoelhosRESUMO
Considering the successful employment of alternative methods for eye toxicity assessment of products for regulatory purposes, and the recent advances in Brazilian legislative scenario, which adopted the UN GHS classification system for agrochemical formulations toxicity assessment, there is an emerging demand for strategies that allow the evaluation of such products. Based on this, the present study aimed to address the applicability of a mechanistic-based defined approach for eye toxicity assessment of agrochemical formulations. It was investigated the opacity/permeability, depth and location of corneal injury in bovine cornea, and vascular events in chorioallantoic membrane induced for different Brazilian agrochemicals using a Sequential Testing Strategy (STS). Cytotoxicity induced by the agrochemical formulations was evaluated by Short Time exposure (STE) (OECD TG 491) assay (step 1), corneal injury was investigated by standard Bovine Corneal Opacity and Permeability (BCOP) (OECD TG 437) followed by histopathological evaluation (step 2), and Hen Chorionic-allantoic Membrane test (HET-CAM) was used to evaluate vascular injury (step 3). The results demonstrated that the proposed defined approach enabled a classification corresponding UN GHS classification of agrochemical formulations while minimizing the use of live animals. Therefore, this approach may be useful for categorization of agrochemicals in Brazil according to the new regulatory scenario.
Assuntos
Agroquímicos/toxicidade , Alternativas aos Testes com Animais , Membrana Corioalantoide/efeitos dos fármacos , Córnea/efeitos dos fármacos , Irritantes/toxicidade , Animais , Bioensaio , Brasil , Bovinos , Galinhas , Membrana Corioalantoide/irrigação sanguínea , Córnea/metabolismo , Córnea/patologia , Opacidade da Córnea/induzido quimicamente , Permeabilidade , Testes de Toxicidade Aguda/métodosRESUMO
In vitro eye toxicity assessment using reconstructed corneal epithelial models has emerged highlighting its applicability domain for Classification and Labeling of products and chemicals. However, due to bureaucratic issues, such models are not commercially available in Brazil and Latin America. In this work, we developed, characterized and evaluated the applicability of a new corneal epithelial biomimetic model using a cell lineage for in vitro eye toxicity assessment. The reconstructed tissue was obtained through the cultivation of HaCaT cells in an air-liquid interface, which presented morphology and biomarkers expression such as cytokeratin, CD44, and Ki-67 similar to human tissue. Furthermore, tissue viability was evaluated after exposure of the epithelial model to isolated chemicals from different Globally Harmonized System (GHS) eye irritation categories, and it has been demonstrated to be a suitable endpoint for classification of test materials, allowing discrimination between irritant and non-irritant chemicals. Furthermore, the model showed suitability for testing "real-life mixtures", once it identified irritant products between the analyzed eyebrow henna samples commercially labeled as non-irritants. This reproducible and low-cost epithelial corneal model presents features very important for Brazil and South America for R&D&I with no unnecessary animal experimentation.
Assuntos
Epitélio Corneano/efeitos dos fármacos , Irritantes/toxicidade , Testes de Toxicidade/métodos , Alternativas aos Testes com Animais , Biomimética , Técnicas de Cultura de Células , Linhagem Celular , Humanos , Modelos BiológicosRESUMO
The Bovine Corneal Opacity and Permeability (BCOP) assay is an alternative method used to ocular toxicity potential assessment of chemicals and mixtures. The standard BCOP test provides information about permeability and opacity, however, corneal histopathological analysis has been recommended as an additional parameter to better categorize eye irritants. Moreover, such analysis associated with depth of substance-induced corneal injury analysis may provide additional scientific measurement for the refinement of BCOP test. The aim of this study was to measure the depth of injury into the bovine cornea induced by eye irritants and associate it with the damage severity. For this purpose, BCOP assay was performed for 12 substances from different Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS) categories and, additionally, corneal sections of 5⯵m thickness were obtained and analyzed by fluorescence microscopy. The results showed that the fluorescein permeation depth was directly proportional to the substances irritation degree. Severe irritants promoted highest rates of permeation followed by moderate and mild irritants, while non-irritants showed similar permeation indexes to the negative control. The refinement of BCOP by the depth of injury analysis through epithelial permeation of fluorescein can be considered a useful quantitative parameter to better categorize eye irritants.
Assuntos
Córnea/efeitos dos fármacos , Irritantes/toxicidade , Alternativas aos Testes com Animais/métodos , Animais , Bioensaio/métodos , Bovinos , Córnea/metabolismo , Córnea/patologia , Opacidade da Córnea/induzido quimicamente , Opacidade da Córnea/metabolismo , Opacidade da Córnea/patologia , Fluoresceína/metabolismo , Permeabilidade , Testes de Toxicidade/métodosRESUMO
Snakes belonging to the genus Naja (Elapid family), also known as "spitting cobras", can spit venom towards the eyes of the predator as a defensive strategy, causing painful and potentially blinding ocular envenoming. Venom ophthalmia is characterized by pain, hyperemia, blepharitis, blepharospasm and corneal erosions. Elapid venom ophthalmia is not well documented and no specific treatment exists. Furthermore, accidental ejection of venom by non-spitting vipers, as Bothrops, also occurs. The Ex vivo Eye Irritation Test model (EVEIT) has enabled important progress in the knowledge of chemical ocular burns. Considering the lack of experimental animal model, we adapted the EVEIT to study venom ophthalmia mechanisms. Ex vivo rabbit corneas were exposed to venoms from spitting (Naja mossambica, Naja nigricollis) and non-spitting (Naja naja, Bothrops jararaca and Bothrops lanceolatus) snakes, and rinsed or not with water. The corneal thickness and the depth of damage were assessed using high-resolution optical coherence tomography (HR-OCT) imaging and histological analysis. All Naja venoms induced significant corneal edema, collagen structure disorganization and epithelial necrosis. Corneas envenomed by African N. mossambica and N. nigricollis venoms were completely opaque. Opacification was not observed in corneas treated with venoms from non-spitting snakes, such as the Asian cobra, N. naja, and the vipers, B. jararaca and B. lanceolatus. Moreover, Bothrops venoms were able to damage the epithelium and cause collagen structure disorganization, but not edema. Immediate water rinsing improved corneal status, though damage and edema could still be observed. In conclusion, the present study shows that the EVEIT model was successfully adapted to set a new experimental ex vivo animal model of ophthalmia, caused by snake venoms, which will enable to explore new therapies for venom ophthalmia.
Assuntos
Córnea/efeitos dos fármacos , Venenos Elapídicos/toxicidade , Testes de Toxicidade/métodos , Animais , Elapidae , CoelhosRESUMO
Snakes belonging to the genus Naja (Elapid family), also known as "spitting cobras", can spit venom towards the eyes of the predator as a defensive strategy, causing painful and potentially blinding ocular envenoming. Venom ophthalmia is characterized by pain, hyperemia, blepharitis, blepharospasm and corneal erosions. Elapid venom ophthalmia is not well documented and no specific treatment exists. Furthermore, accidental ejection of venom by non-spitting vipers, as Bothrops, also occurs. The Ex vivo Eye Irritation Test model (EVEIT) has enabled important progress in the knowledge of chemical ocular burns. Considering the lack of experimental animal model, we adapted the EVEIT to study venom ophthalmia mechanisms. Ex vivo rabbit corneas were exposed to venoms from spitting (Naja mossambica, Naja nigricollis) and non-spitting (Naja naja, Bothrops jararaca and Bothrops lanceolatus) snakes, and rinsed or not with water. The corneal thickness and the depth of damage were assessed using high-resolution optical coherence tomography (HR-OCT) imaging and histological analysis. All Naja venoms induced significant corneal edema, collagen structure disorganization and epithelial necrosis. Corneas envenomed by African N. mossambica and N. nigricollis venoms were completely opaque. Opacification was not observed in corneas treated with venoms from non-spitting snakes, such as the Asian cobra, N. naja, and the vipers, B. jararaca and B. lanceolatus. Moreover, Bothrops venoms were able to damage the epithelium and cause collagen structure disorganization, but not edema. Immediate water rinsing improved corneal status, though damage and edema could still be observed. In conclusion, the present study shows that the EVEIT model was successfully adapted to set a new experimental ex vivo animal model of ophthalmia, caused by snake venoms, which will enable to explore new therapies for venom ophthalmia.
RESUMO
A new molecule, LQFM048, originally designed through molecular hybridization using green chemistry approach, is in development as a photoprotective agent. Eye irritation, skin toxicity and genotoxicity evaluations are mandatory for predicting health risks. In this context, the purpose of this study was to investigate the eye irritation potential of LQFM048 by combining Short Time Exposure (STE), Bovine Corneal Opacity and Permeability (BCOP) associated with corneal histomorphometry and Hen's Egg Test-Chorioallantoic Membrane (HET-CAM). Additionally, skin toxicity was evaluated by interleukin-18 production in the HaCaT keratinocyte, Local Lymph Node Assay (LLNA:BrdU-ELISA) method, 3T3 Neutral red uptake (NRU) assay and in vivo phototoxicity test. Genotoxic potential of LQFM048 was also analyzed by cytokinesis-block micronucleus assay (MNvit test-cytoB) in HepG2 cells. Our results showed that LQFM048 did not induce eye irritation and it was classified as UN GHS No Category for both STE and BCOP assays and non-irritating for HET-CAM test. LQFM048 showed non-potential skin sensitization with stimulation index (SI=0.7) in the LLNA:BrdU-ELISA method. Corroborating in vivo tests, it did not promote significant cytotoxicity in HaCaT cells and it showed similar levels of IL-18 when compared to control. Furthermore, LQFM048 induced non-phototoxic potential with photo-irritation factor (PIF) and mean photo effect (MPE) of 1 and -0.138, respectively, for 3T3 cells. Similarly, it was not phototoxic for in vivo testing with or without exposure to UVA, showing SI values of 1 and 1.2, respectively. The micronucleus test showed that LQFM048 was not genotoxic, under the conditions tested.In conclusion, LQFM048, a heterocyclic compound obtained through an environmentally acceptable simple synthetic route, seems to be safe for human use, especially for the development of a new sunscreen product, since it is neither an eye irritant, nor a contact allergen, nor mutagenic and nor phototoxic.
Assuntos
Córnea/efeitos dos fármacos , Irritantes/toxicidade , Pele/efeitos dos fármacos , Protetores Solares/toxicidade , Células 3T3 , Animais , Bovinos , Linhagem Celular Transformada , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Sobrevivência Celular/efeitos da radiação , Galinhas , Córnea/fisiologia , Córnea/efeitos da radiação , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Feminino , Células Hep G2 , Humanos , Irritantes/administração & dosagem , Camundongos , Camundongos Endogâmicos BALB C , Testes de Mutagenicidade/métodos , Distribuição Aleatória , Pele/metabolismo , Pele/efeitos da radiação , Raios Ultravioleta/efeitos adversosRESUMO
In an attempt to build the evaluation strategies to assess the human eye irritation, a reassessment of some in vitro tests is necessary, particularly concerning the non-irritants, mild and moderate irritants. Thus, the correlations between results obtained from the Draize test with the in vitro methods HET-CAM (Hens Egg Test-Chorion Allantonic Membrane) and RBC haemolysis assay to assess the ocular irritancy potential of 20 eye drops were examined. Parameters such as accuracy (%) and specificity (%) were determined. All results were correlated with RBC correctly with the results obtained in the Draize test. The HET-CAM presented four false-positive results, showing a tendency of data overestimation. Despite the high specificity provided mainly by the RBC, it would be necessary to test a wider range of products representing all the scales of irritation to confirm its ability to be used as a first alternative to test products that could be presumptive non-irritants.
RESUMO
Introducción: el estudio realizado previo al mercadeo de un cosmético para satisfacer la necesidad de obtener el registro sanitario, contribuye a garantizar su seguridad y eficacia para prevenir un problema de salud pública. Objetivo: describir el riesgo ocular latente por un champú mediante pruebas in vivo. Métodos: se realizó un estudio clínico hermenéutico y reflexivo, bajo la observación directa macroscópica en términos de efectos clínicos; determinación de los efectos promedios en 6 conejos mediante la escala de valores según la OCDE y estudio histopatológico de una muestra de tejido del ojo. Resultados: el 50 % de los animales mostraron el nivel 1 de lesión ocular; el 16,67 % el nivel 2 y el 33,33 % el nivel 3. Ninguno de los animales mostró el nivel 4 (lesión máxima). El análisis del promedio de los efectos clínicos y el análisis histopatológico confirman la sensibilidad del conejo como referente experimental aplicable para los bebés. Conclusiones: el champú podría producir efectos oculares leves, pero el posible riesgo ocular debe ser advertido hasta que no se demuestre su inocuidad.
Introduction: the study that is performed for health registration before marketing any cosmetic product assures its safety and efficacy to prevent a public health problem. Objective: to describe the possible eye risk caused by a shampoo for babies through in vivo testing. Methods: a clinical exploratory and hermeneutic study based on direct macroscopic observation of clinical effects; determination of average effects in 6 rabbits by the OECD value scale and a histopathological study of a sample of eye tissue. Results: fifty percent of rabbits showed level 1 ocular injury; 16.67 % suffered level 2 whereas 33.33 % had level 3. None of the animals reached level 4 (maximum level in injuries). The analysis of the average clinical effects and the histopathological study confirmed the sensitivity of the rabbit as experimental reference that may be applicable to babies. Conclusions: the shampoo might cause mild eye effects; however the possible ocular risk must be stated until such time as the safety of the product is proven.
RESUMO
Aqueous solutions of the molluscicidal latex of Euphorbia splendens are irritant to the rabbit eye in concentrations higher than 0.35% and to the rabbit skin in concentrations higher than 0.5%. Although this irritant potential does not proclude its use as a molluscicide, special precautions are recommended for hanbdling and application of the product and the hazard of skin tumor-promoting potencial should be carefully investigated before its use for schistosomiasis vector control.