Estratégia associando métodos substitutivos à experimentação animal para avaliação da toxicidade ocular, categorização e rotulagem de formulações agrotóxicas comercializadas no Brasil
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2018-11-20
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Universidade Federal de Goiás
Resumo
Introduction: Different strategies for categorization and labeling (C & L) of ingredients and finished products have been considered by the Regulatory Agencies. Regarding agrochemical formulations, the lack of global harmonization for C & L is the main problem for the implementation of substitute methods. On the other hand, in vitro and ex vivo methods are successfully adopted to assess the potential toxicity of chemicals. The standard Bovine Cornea Opacity and Permeability (BCOP) test provides information on permeability and opacity, but histomorphometric analysis of the cornea has been recommended as an additional parameter to better categorize irritant products for the eyes. Objective: To suggest a platform, associating in vitro and ex vivo methods, based on mechanistic parameters of ocular toxicity evaluation for C & L of agrochemical formulations commercialized in Brazil. As well as measuring the depth of bovine corneal injury induced by eye irritants from all categories of the United Nations Globally Harmonized System for Classification and Labeling of Chemicals (UN GHS) and associating it with the severity of the damage. Material and Methods: For the platform, 86 agrochemical formulations of different categories of ocular toxicity were selected in the categorization system of the National Agency of Sanitary Surveillance (ANVISA). First, the potential for ocular toxicity was assessed through the cytotoxicity endpoint using the Short Time Exposure (STE) test. In the second step, the non-classified samples and those classified as non-irritating by the STE were evaluated for the opacity, permeability and histopathological changes of the cornea through the BCOP test. The third step was to evaluate the potential for induction of vascular damage through coagulation and hemorrhage outcomes using the Hen egg Chorion-allantoic Membrane test (HET-CAM). For proficiency, 12 substances belonging to the categories UN GHS (Category 1, 2A and 2B) were selected. The BCOP assay was performed to determine the in vitro irritation index (IVIS). After the fluorescein permeability assay, the corneas used were sectioned in a 5 μm thick cryostat, stained with Hoechst 33342 and analyzed by fluorescence microscopy. Five measurements of depth of permeation of fluorescein were obtained in relation to the total thickness of the epithelium. Results and Discussion: From the palataform, of the 86 formulations of agrochemicals, after the STE test, 68 were highly cytotoxic and were categorized as Category 1 by UN GHS; 13 not classified and 5 non-irritating. These 18 agrochemical formulations were evaluated at the second level of the platform by the BCOP test followed by histopathological analysis, and the irritation profile of these agrochemical formulations was confirmed by the HET-CAM assay. Thus, 7 presented opacity, damage to the cornea in the epithelial portion and in the stroma, coagulation and hemorrhage, and therefore were categorized in Category 2 A of the UN GHS. 11 caused only coagulation and hemorrhage and were categorized into Category 2 B of the UN GHS. In the proficiency, the results showed that the permeation depth of fluorescein was directly proportional to the degree of irritation of the substances tested. Conclusion: Since C&L of agrochemical formulations in Brazil is not based on the UN GHS, this tier platform could be useful to harmonize the categorization of agrochemical formulations in Brazil, providing regulatory subsidies for ANVISA. As well as, refinement of BCOP by analysis of lesion depth through epithelial permeation of fluorescein can be considered a useful quantitative parameter to better categorize eye irritants.
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Palavras-chave
Agrotóxicos, Métodos alternativos à experimentação animal, Irritação ocular, Teste de Draize, Agência nacional de vigilância sanitária, Agrochemical, Alternative methods to animal experimentation, Eye irritation, Draize test, National health surveillance agency
Citação
ANDRADE, W. M. Estratégia associando métodos substitutivos à experimentação animal para avaliação da toxicidade ocular, categorização e rotulagem de formulações agrotóxicas comercializadas no Brasil. 2018. 111 f. Tese (Doutorado em Inovação Farmacêutica em Rede) - Universidade Federal de Goiás, Goiânia, 2018.