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1	Estratégia para a avaliação do potencial de irritação ocular de ingredientes cosméticos utilizando métodos alternativos à experimentação animal / Strategy for the evaluation of ocular irritation potencial of cosmetic ingredients using alternative methods to animal testing Sá, Larissa de Lima 09 May 2017 (has links) Considerando que os produtos cosméticos são de livre acesso ao consumidor e alguns podem acidentalmente entrar em contato com os olhos, eles devem ser aprovados em testes de irritação ocular. Durante muitos anos, essa avaliação foi feita pelo teste de Draize utilizando animais de experimentação. Entretanto, devido a questões éticas envolvendo o uso desses animais, atualmente há uma busca por métodos alternativos para essa avaliação. Assim, o objetivo deste projeto foi propor uma estratégia para a avaliação do potencial de irritação ocular de alguns ingredientes cosméticos, como os tensoativos, dodecil sulfato de sódio (SDS), lauril éter sulfato de sódio (LESS), cocoilglutamato de sódio (CGS), polissorbato 20, capriloil/caproil metil glucamida (CCMG), cocoamidopropilbetaína (CAB), bem como os silicones, dimeticone e ciclopentasiloxano, utilizados na fabricação de produtos cosméticos para cabelo (shampoos), área dos olhos e rosto (demaquilantes e cremes). Para isso, foram utilizados métodos alternativos à experimentação animal, tais como Hen\'s Egg Test - Chorioallantoic Membrane (HET-CAM) e ensaio de Permeabilidade e Opacidade de Córnea Bovina (BCOP) e análise histopatológica das córneas. O primeiro ensaio avalia os eventos vasculares, hiperemia, hemorragia e coagulação, que a substância teste possa causar na CAM, mimetizando, com isso, a conjuntiva ocular. Já o BCOP avalia as alterações na opacidade e na permeabilidade provocada pela substância teste. A análise histopatológica é uma ferramenta complementar ao ensaio de BCOP, avaliando o grau e a profundidade da lesão ocasionada pela substância teste na córnea bovina. A maioria dos tensoativos foi considerada irritante severo no ensaio de HET-CAM, com exceção do polissorbato 20 (10%) classificado como irritante moderado, enquanto que os silicones não foram irritantes. Já no ensaio de BCOP, a maioria dos tensoativos foi classificada como sem predição, exceto o polissorbato 20 (10%), o CCMG 1,5% e os silicones, os quais foram classificados como não irritantes. Na análise histopatológica, os tensoativos sem predição no BCOP, foram classificados como irritantes severos: SDS 10%, CAB 3% e 1,5% e CCMG 3%; irritantes moderados: LESS 10% e 7%; irritantes leves: SDS 1% e CGS 3%; e não irritantes: CGS 1,5% e CCMG 1,5%. Os silicones não causaram nenhum dano na córnea. Diante dos resultados obtidos no presente trabalho, a seguinte estratégia foi proposta para a avaliação do potencial de irritação ocular de ingredientes cosméticos: em um primeiro nível deve-se utilizar o ensaio de HET-CAM, o qual avalia os eventos vasculares, mimetizando a conjuntiva ocular. Em seguida, no 2º nível, com o ensaio de BCOP, avaliam-se os efeitos do ingrediente sobre a córnea (opacidade e permeabilidade). Se o ingrediente for classificado como irritante severo ou não irritante nos dois ensaios, ele não necessita de mais avaliações. Entretanto, se esse produto apresentar algum potencial irritante no HET-CAM e for classificado como sem predição ou sem categoria no BCOP, deve-se prosseguir para o 3º nível, a avaliação histopatológica. Nessa etapa o ingrediente é avaliado quanto ao grau e a profundidade da lesão que ele provoca na córnea bovina, possibilitando, até mesmo, inferir sobre a reversibilidade da lesão. Portanto, a estratégia proposta para avaliar o potencial de irritação ocular de ingredientes e produtos cosméticos é bastante promissora, pois é possível avaliar os eventuais danos que eles podem causar na conjuntiva ocular e na córnea por diferentes mecanismos toxicológicos, predizendo, assim, a toxicidade ocular desses produtos. / Cosmetic products are freely available to the consumer and may accidentally come into contact with the eyes; thus they must be approved by eye irritation tests. For many years, these evaluations were performed in experimental animals (Draize test). However, due to ethical issues involving the use of animals, there is a search for alternative methods for this evaluation. Thus, the aim of this project was to measure the ocular irritation potential of some cosmetic ingredients, such as sodium dodecyl sulfate (SDS), sodium cocoylglutamate (SCG), polysorbate 20, capryloyl /caproyl methyl glucamide (CCMG), cocoamidopropyl betaine (CAPB), as well as silicones, dimethicone and cyclopentalosiloxane, used to manufacture cosmetic products for hair (shampoos), eye and face area (make-up removers and creams). For that purpose, alternative methods of animal experimentation were used, such as Hen\'s Egg Test - Chorioallantoic Membrane (HET-CAM) and Bovine Corneal Permeability and Opacity (BCOP) test and histopathological analysis of the corneas. The first assay evaluates the vascular events, hyperemia, hemorrhage and coagulation, which a test substance can cause in the CAM, simulating endpoints that occur in ocular conjunctiva. BCOP evaluates alterations in opacity and permeability caused by test substance on the cornea. The histopathological analysis is a complementary tool to BCOP test, evaluating the degree and depth of the lesion caused by the test substance in the bovine cornea. Most of the surfactants were non-irritant on HET-CAM, except polysorbate 20 (10%) that was classified as moderate irritant, while silicones were non-irritant. In the BCOP assay, most surfactants were classified as unpredicted, except polysorbate 20 (10%) and CCMG 1.5%, and silicones, which were classified as no category (non-irritant). In the histopathological analysis, unpredictive surfactants in BCOP were better classified as severe irritants: SDS10%, CAB 3% and 1.5% and CCMG 3%; moderate irritants: LESS 10% and 7%; mild irritants: SDS 1% and CGS 3%; and non-irritant: CGS 1.5% and CCMG 1.5%. Silicones did not cause any damage on the cornea. Considering the results obtained in the present study, the following tiered strategy was proposed for the evaluation of the ocular irritation potential of cosmetic ingredients: the first tier should be the HET-CAM assay, which evaluates the vascular events, simulating the conjunctiva. Then, the second tier, with the BCOP assay, the effects of the ingredient on the cornea (opacity and permeability) are evaluated. If the ingredient is classified as a severe irritant or non-irritant in both tests, it does not require further evaluation. However if that ingredient or product is considered as a potential irritant in HET-CAM and in BCOP, i.e. it is classified as unpredicted or no category, it is necessary to proceed to the third tier, the histopathological evaluation. At this stage, the ingredient is evaluated for the degree and depth of the lesion that it provoked in the bovine cornea, allowing even to infer about the reversibility of the lesion. Finally, the proposed strategy for evaluating the potential of eye irritation is very promising to evaluate ingredients and cosmetic products, since it is possible to evaluate the possible damages that they can cause in the conjunctiva and in the cornea by different toxicological mechanisms, thereby predicting the ocular safety of those products. alternative methods BCOP BCOP cosméticos cosmetics eye irritation HET-CAM HET-CAM histhopatology histopatologia irritação ocular métodos alternativos
2	Development of Sensitive In Vitro Assays to Assess the Ocular Toxicity Potential of Chemicals and Ophthalmic Products McCanna, David January 2009 (has links) The utilization of in vitro tests with a tiered testing strategy for detection of mild ocular irritants can reduce the use of animals for testing, provide mechanistic data on toxic effects, and reduce the uncertainty associated with dose selection for clinical trials. The first section of this thesis describes how in vitro methods can be used to improve the prediction of the toxicity of chemicals and ophthalmic products. The proper utilization of in vitro methods can accurately predict toxic threshold levels and reduce animal use in product development. Sections two, three and four describe the development of new sensitive in vitro methods for predicting ocular toxicity. Maintaining the barrier function of the cornea is critical for the prevention of the penetration of infections microorganisms and irritating chemicals into the eye. Chapter 2 describes the development of a method for assessing the effects of chemicals on tight junctions using a human corneal epithelial and canine kidney epithelial cell line. In Chapter 3 a method that uses a primary organ culture for assessing single instillation and multiple instillation toxic effects is described. The ScanTox system was shown to be an ideal system to monitor the toxic effects over time as multiple readings can be taken of treated bovine lenses using the nondestructive method of assessing for the lens optical quality. Confirmations of toxic effects were made with the utilization of the viability dye alamarBlue. Chapter 4 describes the development of sensitive in vitro assays for detecting ocular toxicity by measuring the effects of chemicals on the mitochondrial integrity of bovine cornea, bovine lens epithelium and corneal epithelial cells, using fluorescent dyes. The goal of this research was to develop an in vitro test battery that can be used to accurately predict the ocular toxicity of new chemicals and ophthalmic formulations. By comparing the toxicity seen in vivo animals and humans with the toxicity response in these new in vitro methods, it was demonstrated that these in vitro methods can be utilized in a tiered testing strategy in the development of new chemicals and ophthalmic formulations. in vitro Alternative Methods alamarBlue rhodamine tight junctions bovine lens viability toxicity mitochondria mitochondria integrity benzalkonium chloride sodium dodecyl sulphate sodium lauryl sulfate scanning electron microscopy Draize Draize maximal average scores zonula occludens cosmetic directive PETA humane society animal league ICCVAM ECVAM JaCVAM BCOP bovine cornea ocular irritants ocular irritation ocular toxicity human corneal epithelial cells vitro confocal confocal microscopy metabolic activity optical quality reactive oxygen species contact lens contact lens care solutions barrier function microbial keratitis mulitipurpose solutions tight junction cell damage claudin ZO-1 occludin MDCK Madin Madin-Darby canine kidney cells cornea human cornea human corneal epithelium epithelial cell line human corneal epithelial cell line sodium fluorescein sodium fluorescein permeability fluorescein permeability ocular surface cell monolayer Araki-Sasaki cell physiology risk assessment safety assessment ScanTox scanning laser lens epithelium corneal cells in vitro model ophthalmic formulations multiple instillation back vertex animal testing rabbit testing alternatives to animal testing cultured bovine lens toxicity of chemicals irritation irritants laser scanner organ culture delayed toxicity toxins hydrogen peroxide cornea toxicity cornea toxicity models prediction of human toxicity no observable adverse effect level lowest observable adverse effect level NOAEL LOAEL toxicity thresholds safety factors cornea uncertainty factors preservatives disinfectants ophthalmic products preclinical preclinical testing epithelial barrier drug penetration clinical confocal microscopy animal rights rabbit cornea human cornea human clinical effects toxic animal rights activist sensitive measures toxic effect toxicity threshold agar overlay agar diffusion agar overlay method agar diffusion method cytochrome cytochrome c apoptosis necrotic apoptotic necrosis caspase rhodamine 123 resazuran resorufin cell death cell viability metabolic dye microsomal microsomal enzymes cytotoxicity cytotoxic cytotoxic effect MTT XTT WST-1 plasma membrane mitochondrial mitochondrial morphology ocular toxicity potential ocular toxicity human corneal epithelial cell line confocal analysis corneal epithelium cell fluorescence alamarBlue assay rhodamine dye animal welfare toxic injury degraded mitochondria epithelial monolayer disinfectants membrane integrity eye toxicity eye viability dye toxicity in humans human toxicity effects on the mitochondria mitochondrial toxicity in vitro battery in vitro test battery ophthalmic eye drop direct contact product safety cytotoxicity potential molecular molecular biology refine reduce replace sensitivity and relevance sensitivity relevance rabbit ocular irritation test product development cytotoxicity models cytotoxicity alternative methods replacements for animal testing three r's beagle tiered testing tiered testing strategy replacements animal testing mechanistic toxicity cornea mitochondria dose response threshold Vision Science and Biology
3	Development of Sensitive In Vitro Assays to Assess the Ocular Toxicity Potential of Chemicals and Ophthalmic Products McCanna, David January 2009 (has links) The utilization of in vitro tests with a tiered testing strategy for detection of mild ocular irritants can reduce the use of animals for testing, provide mechanistic data on toxic effects, and reduce the uncertainty associated with dose selection for clinical trials. The first section of this thesis describes how in vitro methods can be used to improve the prediction of the toxicity of chemicals and ophthalmic products. The proper utilization of in vitro methods can accurately predict toxic threshold levels and reduce animal use in product development. Sections two, three and four describe the development of new sensitive in vitro methods for predicting ocular toxicity. Maintaining the barrier function of the cornea is critical for the prevention of the penetration of infections microorganisms and irritating chemicals into the eye. Chapter 2 describes the development of a method for assessing the effects of chemicals on tight junctions using a human corneal epithelial and canine kidney epithelial cell line. In Chapter 3 a method that uses a primary organ culture for assessing single instillation and multiple instillation toxic effects is described. The ScanTox system was shown to be an ideal system to monitor the toxic effects over time as multiple readings can be taken of treated bovine lenses using the nondestructive method of assessing for the lens optical quality. Confirmations of toxic effects were made with the utilization of the viability dye alamarBlue. Chapter 4 describes the development of sensitive in vitro assays for detecting ocular toxicity by measuring the effects of chemicals on the mitochondrial integrity of bovine cornea, bovine lens epithelium and corneal epithelial cells, using fluorescent dyes. The goal of this research was to develop an in vitro test battery that can be used to accurately predict the ocular toxicity of new chemicals and ophthalmic formulations. By comparing the toxicity seen in vivo animals and humans with the toxicity response in these new in vitro methods, it was demonstrated that these in vitro methods can be utilized in a tiered testing strategy in the development of new chemicals and ophthalmic formulations. in vitro Alternative Methods alamarBlue rhodamine tight junctions bovine lens viability toxicity mitochondria mitochondria integrity benzalkonium chloride sodium dodecyl sulphate sodium lauryl sulfate scanning electron microscopy Draize Draize maximal average scores zonula occludens cosmetic directive PETA humane society animal league ICCVAM ECVAM JaCVAM BCOP bovine cornea ocular irritants ocular irritation ocular toxicity human corneal epithelial cells vitro confocal confocal microscopy metabolic activity optical quality reactive oxygen species contact lens contact lens care solutions barrier function microbial keratitis mulitipurpose solutions tight junction cell damage claudin ZO-1 occludin MDCK Madin Madin-Darby canine kidney cells cornea human cornea human corneal epithelium epithelial cell line human corneal epithelial cell line sodium fluorescein sodium fluorescein permeability fluorescein permeability ocular surface cell monolayer Araki-Sasaki cell physiology risk assessment safety assessment ScanTox scanning laser lens epithelium corneal cells in vitro model ophthalmic formulations multiple instillation back vertex animal testing rabbit testing alternatives to animal testing cultured bovine lens toxicity of chemicals irritation irritants laser scanner organ culture delayed toxicity toxins hydrogen peroxide cornea toxicity cornea toxicity models prediction of human toxicity no observable adverse effect level lowest observable adverse effect level NOAEL LOAEL toxicity thresholds safety factors cornea uncertainty factors preservatives disinfectants ophthalmic products preclinical preclinical testing epithelial barrier drug penetration clinical confocal microscopy animal rights rabbit cornea human cornea human clinical effects toxic animal rights activist sensitive measures toxic effect toxicity threshold agar overlay agar diffusion agar overlay method agar diffusion method cytochrome cytochrome c apoptosis necrotic apoptotic necrosis caspase rhodamine 123 resazuran resorufin cell death cell viability metabolic dye microsomal microsomal enzymes cytotoxicity cytotoxic cytotoxic effect MTT XTT WST-1 plasma membrane mitochondrial mitochondrial morphology ocular toxicity potential ocular toxicity human corneal epithelial cell line confocal analysis corneal epithelium cell fluorescence alamarBlue assay rhodamine dye animal welfare toxic injury degraded mitochondria epithelial monolayer disinfectants membrane integrity eye toxicity eye viability dye toxicity in humans human toxicity effects on the mitochondria mitochondrial toxicity in vitro battery in vitro test battery ophthalmic eye drop direct contact product safety cytotoxicity potential molecular molecular biology refine reduce replace sensitivity and relevance sensitivity relevance rabbit ocular irritation test product development cytotoxicity models cytotoxicity alternative methods replacements for animal testing three r's beagle tiered testing tiered testing strategy replacements animal testing mechanistic toxicity cornea mitochondria dose response threshold Vision Science and Biology

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