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1	Alterações na superfície ocular de coelhos tratados com formulações do herbicida ácido diclorofenoxiacético (2,4 D), utilizando a graduação do teste ocular de Draize Collor, Lílian Verdolin Milo January 2007 (has links) Dissertação (mestrado)—Universidade de Brasília, Faculdade de Ciências da Saúde, 2007. / Submitted by Luanna Maia (luanna@bce.unb.br) on 2009-03-06T15:53:48Z No. of bitstreams: 1 Dissert_Lilian Collor.pdf: 2851385 bytes, checksum: 9aa50d532a9bb626e02088248ac08715 (MD5) / Approved for entry into archive by Luanna Maia(luanna@bce.unb.br) on 2009-03-06T16:09:23Z (GMT) No. of bitstreams: 1 Dissert_Lilian Collor.pdf: 2851385 bytes, checksum: 9aa50d532a9bb626e02088248ac08715 (MD5) / Made available in DSpace on 2009-03-06T16:09:23Z (GMT). No. of bitstreams: 1 Dissert_Lilian Collor.pdf: 2851385 bytes, checksum: 9aa50d532a9bb626e02088248ac08715 (MD5) / O teste de irritação ocular é requerido pelos órgãos governamentais brasileiros durante o processo de registro de um produto agrotóxico, e essencial para sua classificação toxicológica. O presente estudo avaliou as alterações macroscópicas e microscópicas na superfície do olho de coelhos após o tratamento com quatro formulações de herbicidas contendo o ácido diclorofenoxiacético (2,4-D), utilizando a escala de graduação das reações oculares proposta por Draize. Foram utilizados 84 coelhos, 21 animais por formulação, distribuídos em 7 grupos. Uma única dose de 0,1 mL ou g foi aplicada diretamente no saco conjuntival do olho esquerdo de cada animal, e o olho direito foi utilizado como controle. As alterações na córnea, íris e conjuntivas foram graduadas nos intervalos de 1, 24, 48 e 72 horas e 7, 14 e 21 dias após a aplicação. Em cada intervalo, três animais foram eutanasiados, seus olhos removidos e os tecidos preparados para exame histológico. Os resultados macroscópicos mostraram que não houve proporcionalidade entre a concentração de 2,4-D e a intensidade das alterações na córnea. Os produtos 1 e 2, com menor concentração de 2,4-D e pH próximo da neutralidade, mostraram alterações discretas e moderadas na córnea quando comparados aos produtos 3 e 4 que apresentaram na maioria alterações moderadas e acentuadas. As lesões produzidas no epitélio córneo foram, na maioria, reparadas durante o período do experimento, com exceção daquelas em um animal tratado com o produto 3 e de três animais do produto 4 que tiveram rompimento da córnea. Infiltrados de neutrófilos polimorfonucleares foram detectados microscopicamente na maioria das inflamações na córnea, íris e conjuntivas notadas nos exames clínicos. Entretanto, não houve em todos os casos relação direta entre a intensidade das alterações macro e microscópicas. Os exames micro ainda mostraram em alguns animais alterações que normalmente não são visualizadas clinicamente, como hiperplasia, queratinização e descamação do epitélio córneo, e inflamação do corpo ciliar, que podem comprometer o processo da visão. A legislação brasileira atual é conservadora, pois considera como extremamente tóxico qualquer produto que apresente opacidade na córnea, reversível ou não em 7 dias. Os resultados deste estudo indicam a necessidade da revisão da legislação em harmonia com os procedimentos utilizados internacionalmente. _____________________________________________________________________________________ ABSTRACT / Ocular irritation test is required by the Brazilian government authorities during the registration process of pesticide products, and it is essential for its toxicological classification. The present study evaluated the clinical and microscopic changes in the eye surface of rabbits after treatment with four pesticide formulations containing the herbicide 2,4-D dichlorophenoxyacetic acid (2,4-D), using the Draize scale for grading the ocular irritation. Eighty four animals were used in the experiment, being 21 for each formulation, distributed in 7 groups. A single dose of 0.1 mL or g was directly applied to the conjunctive sac of the left eye of each animal, and the right eye being used as control. Changes in the cornea, iris and conjunctives were graduated after 1, 24, 48 and 72 hours and 7, 14 and 21 days after treatment. In each interval, three animals were killed, the eyes removed and the ocular tissues prepared for the histological examination. Clinical results showed no proportionality between concentration of 2,4-D in the formulation and severity of corneal damage. The products 1 and 2, with lower 2,4-D concentration and pH near the neutrality, caused discrete and moderate changes in the cornea when compared with the products 3 and 4, which caused moderate and accentuated changes. Lesions produced in the corneal epithelium were repaired during the experiment, with exception of those in one animal treated with product 3 and three animals treated with product 4 which had corneal perforation. Infiltrates of polymorphonuclear neutrophil cells were observed microscopically in the majority of the inflammatory events clinically noted on the cornea, iris and conjunctives. However, not always the intensity of the changes observed macro and microscopically were correlated. Microscopy showed corneal hyperplasia, queratinization and desquamation and inflammation of the ciliary body of many animals as a result of the treatment, changes that can affect the vision but are not normally observed during the clinical examination. The current Brazilian legislation is conservative, as it considers any product that shows corneal opacity, reversible or not after 7 days, as extremely toxic. The present work indicates the need to update this legislation according to the international procedures. Irritação ocular Agrotóxicos Teste de Draize
2	Avaliação de metodologia alternativa in vitro ao teste de irritação ocular de Draize / The evaluation of alternative methods in vitro to the Draize eye test Azevedo, Janice Campos de 28 August 1998 (has links) Para a avaliação do potencial irritante de substâncias aplicadas topicamente tem-se utilizado metodologias que baseiam nos estudos de Draize, onde preconiza-se o uso de animais. Devido à forte oposição a estes testes, intensa tem sido a busca por métodos alternativos visando diminuir ou eliminar a utilização de animais. Este estudo avaliou o potencial irritante de diversas substâncias através de duas metodologias: o teste in vivo , empregando metodologia oficial do Food and Drug Administration (FDA), onde utiliza-se coelhos e o teste de citotoxicidade in vitro - método de difusão em camada de agar sobreposta à monocamada de células - empregando as linhagens celulares He La e NCTC L 929 e o corante vital vermelho neutro. As amostras de produto acabado abrangeram colírios, testados sem diluição e xampus de uso adulto e infantil testados puros e diluídos a 25%, 5%, 1% e 0,1%. As matérias-primas compreenderam tensoativos aniônicos, não-iônicos e anfóteros testados após diluição. O material de acondicionamento foi representado por frascos de polietileno para colírios, testados através dos seus extratos em solução de NaCI 0,9%. Os resultados obtidos com as amostras de colírios e frascos de acondicionamento foram negativos quanto à irritação ocular em coelhos e comprovaram ausência de citotoxicidade nas duas linhagens celulares. As amostras de xampus apresentaram boa correlação dos resultados quando testadas sem diluição ou diluídas a 1,0% e 0,1 %. Foi calculada a correlação de Spearman entre os dados obtidos com as duas linhagens celulares sendo o valor do coeficiente r = 0,950 indicando uma correlação direta entre os resultados obtidos com as duas células. Os resultados demonstram a possibilidade de desenvolvimento de metodologias alternativas, sujeitas à validação, que resultem em procedimentos sensíveis, reprodutíveis, não sujeitos a critérios subjetivos de interpretação, visando assim, num primeiro estágio diminuir o uso de animais pelo emprego destas metodologias em avalições prévias do potencial irritante de substâncias químicas. / To evaluate the irritability potential of toppically laid on substances, some methodologies based on Draize\'s study where the use of animals is demanded, have been used. Due to the strong opposition of society and scientific comunity, the search for alternative methods having in view the reduction or even the elimination of animals have been intense, either through the use of volunteers or of in vitro methodologies. The present study has evaluated the irritability of several substances including finished products, raw materials and packagíng materials through two methodologies: the in vivo test which makes use of official methodology from Food and Drug Administration (FDA), where rabbits are employed and the citotoxicity in vitro test - method of diffusion in agar layer added to the monolayer of cells - using He La and NCTC L 929 cells lines and neutral red. The samples of the finished products embraced eye drops, tested without dilution, and shampoos for adults and children use both concentrated and diluted to 25%, 5%, 1% and 0.1 %. Raw materiais included anionis, non-anionic and amphoteric surfactants tested after dilution. The packaging material, represented by eye drops polyethylene bottles, was tested through its extracts in a NaCI 0,9% solution. The results obtained with the samples of eye drops and packaging materials were negative as for ocular irritation in rabbits and confirmed the absence of cytotoxicity in both cells lines. The samples of shampoos presented good correlation of results when tested without dilution or diluted to 1% and 0.1 %. It has been calculated Spearman correlation considering dates obtained with two cells lines, with value of coefficient r = 0,950 showing a direct correlation between results obtained with two cells. The results obtained with this study are likely to lead to sensitive and reproductive procedures, which won\'t arise subjective criteria of interpretation. Thus, the elimination of the use of animals will be possible at a first stage of previous evalution, and once a posterior validation of this methodology is done, the rational use of a smaller number of animais will be a reality. Cell culture Citotoxicidade Cosmetic Cosmético Cultura celular Cytotoxicity Draize Draize test Eye iritation Irritação ocular Medicamento
3	Teste de citotoxicidade \'in vitro\' como alternativa ao teste \'in vivo\' de Draize na avaliação de produtos cosméticos / In vitro citotoxicity test as an alternative to in vivo Draize test for the evaluation of cosmetic Cruz, Aurea Silveira 30 July 2003 (has links) Os procedimentos descritos por Draize são a base dos testes de irritação ocular e cutânea adotados internacionalmente para avaliar produtos e substâncias. Entretanto, eles têm sido criticados por motivos éticos, devido à crueldade com os animais e, por isso, metodologias alternativas têm sido estudas para avaliar a toxicidade de produtos que entram em contato com o ser humano. Sendo assim, um estudo comparativo foi realizado entre testes de irritação ocular, cutânea e mucosa oral usando coelhos e testes in vitro pelos métodos de difusão em ágar e de captura do vermelho neutro, ambos usando as linhagens celulares NCTC clone 929, FPC-IAL e SIRC. Os cosméticos avaliados foram batons, bases faciais, pós compactos, blushes, sombras para os olhos, máscaras para os cílios, lápis ou delineadores para os olhos, e sabonetes líquidos de uso adulto e infantil. Os testes in vivo e as linhagens celulares utilizadas no método de difusão em ágar foram escolhidas de acordo com o local de uso dos produtos, com exceção da linhagem NCTC clone 929 que foi utilizada na avaliação de todas as amostras. Das 204 amostras, 141 foram avaliadas pelo teste de irritação cutânea e linhagem FPC-IAL, 80 pelo teste de irritação ocular e linhagem SIRC e 78 pelo teste de irritação de mucosa oral e linhagem FPC-IAL. Somente as amostras de sabonetes líquidos foram avaliadas também pelo método de captura do vermelho neutro com as três linhagens celulares. Na avaliação in vitro as amostras foram analisadas em diferentes concentrações e o parâmetro observado foi viabilidade celular com o corante vermelho neutro. Os resultados obtidos permitiram observar que as amostras positivas no método de difusão em ágar que apresentaram até índice de zona 3 ou seja, halo de morte celular variando de 0,5 a 1,0 cm a partir da amostra, segundo a Farmacopéia Americana, não apresentaram irritação ocular, cutânea ou de mucosa oral. As amostras que apresentaram índice de zona 4, apresentaram diferentes graus de irritação ocular e cutânea com exceção de 2 sabonetes líquidos de uso infantil que não apresentaram reações in vivo, embora apresentaram citotoxicidade até a concentração de 10%. Este método apresentou correlação significante com o teste de irritação ocular, inclusive para os valores individuais da córnea e conjuntiva. Quanto ao método de captura do vermelho neutro obteve-se correlação significante para o teste de irritação cutânea e ocular, podendo inferir que quando as amostras de sabonetes líquidos apresentarem IC50 maior que 0,085% não irão induzir irritação cutânea e ocular nos coelhos. Não foi observada relação entre a origem das linhagens celulares usadas e o tecido alvo utilizado no teste in vivo, sendo que todas as linhagens mostraram correlação significante com a linhagem NCTC clone 929. De acordo com os dados, conclui-se que os métodos in vitro são mais sensíveis e que o método de difusão em ágar usando a graduação da Farmacopéia Americana, pode ser adotado como ensaio de triagem na avaliação de cosméticos. Isto resulta de sua capacidade de predizer a irritação, contribuindo significativamente para a redução dos testes que utilizam animais. / The procedures described by Draize are the basis of both ocular and cutaneous irritation tests and have been adopted internationally for in vivo evaluation of products and substances. However, they have been criticized for ethical reasons, due to their cruelty towards animals. Therefore, alternative methodologies are being studied to evaluate the toxicity of products, which have contact with human beings. Thus, a comparative study was performed between ocular, cutaneous and oral mucosa irritation tests using rabbits and in vitro tests through agar diffusion method and neutral red uptake method, both with the use of NCTC clone 929, FPC-IAL and SIRC cell lines. Lipsticks, makeup base, face powder, blushes, eye shadows, mascara, pencils and eyeliners, as well as liquid soap for children and adult use were evaluated. The in vivo tests and the cell lines used in the agar diffusion test were chosen according to the place where the products are used. Only the NCTC clone 929 linage was used in the evaluation of all the samples. From the 204 analyzed samples, 141 were evaluated through the cutaneous irritation test and FPC-IAL lineage, 80 through the ocular irritation test and SIRC lineage, and 78 through the oral mucosa irritation test and FPC-IAL lineage. Only the samples of liquid soap were also evaluated through the neutral red uptake method with the three cells lines. The samples submitted to the in vitro evaluation were analyzed in different concentrations and the observed parameter was cellular viability with the use of neutral red. The results obtained revealed that the agar diffusion test positive samples which presented up to degree 3 zone rate, that is cellular death halo varying from 0,5 to 1,0cm from the samples, according to USP 25, didnt provoke ocular, cutaneous or oral mucosa irritation. The samples which presented degree 4 zone also showed different degrees of ocular and cutaneous irritation, except to two units of liquid soap for children use which didnt present in vivo reactions, although citotoxicity up to 10% concentration. This method showed significant correlation with the ocular irritation test and also concerning the individual values of cornea and conjunctiva. As for the neutral red uptake test, it presented significant correlation in the ocular and cutaneous irritation test, what make it possible to infer that when the liquid soap samples present IC50 above 0,085% they wont cause ocular and cutaneous irritations in rabbits. No relation was observed between the origin of the cell lines and the target tissue used in the in vivo test, having all the cellular lines shown significant correlation with the NCTC clone 929 line. According to the data, the in vitro methods are more sensitive and the diffusion agar method, using the American Pharmacopeia graduation, can be adopted as a sorting procedure in the evaluation of cosmetics. This is a result of its capacity of predicting irritation, what largely contributes for the reduction of animal using tests. Citotoxicidade Cosmetic Cosméticos Cultura celular Cytotoxicity Draize test Irritação in vivo Irritation Testes de Draize
4	Teste de citotoxicidade \'in vitro\' como alternativa ao teste \'in vivo\' de Draize na avaliação de produtos cosméticos / In vitro citotoxicity test as an alternative to in vivo Draize test for the evaluation of cosmetic Aurea Silveira Cruz 30 July 2003 (has links) Os procedimentos descritos por Draize são a base dos testes de irritação ocular e cutânea adotados internacionalmente para avaliar produtos e substâncias. Entretanto, eles têm sido criticados por motivos éticos, devido à crueldade com os animais e, por isso, metodologias alternativas têm sido estudas para avaliar a toxicidade de produtos que entram em contato com o ser humano. Sendo assim, um estudo comparativo foi realizado entre testes de irritação ocular, cutânea e mucosa oral usando coelhos e testes in vitro pelos métodos de difusão em ágar e de captura do vermelho neutro, ambos usando as linhagens celulares NCTC clone 929, FPC-IAL e SIRC. Os cosméticos avaliados foram batons, bases faciais, pós compactos, blushes, sombras para os olhos, máscaras para os cílios, lápis ou delineadores para os olhos, e sabonetes líquidos de uso adulto e infantil. Os testes in vivo e as linhagens celulares utilizadas no método de difusão em ágar foram escolhidas de acordo com o local de uso dos produtos, com exceção da linhagem NCTC clone 929 que foi utilizada na avaliação de todas as amostras. Das 204 amostras, 141 foram avaliadas pelo teste de irritação cutânea e linhagem FPC-IAL, 80 pelo teste de irritação ocular e linhagem SIRC e 78 pelo teste de irritação de mucosa oral e linhagem FPC-IAL. Somente as amostras de sabonetes líquidos foram avaliadas também pelo método de captura do vermelho neutro com as três linhagens celulares. Na avaliação in vitro as amostras foram analisadas em diferentes concentrações e o parâmetro observado foi viabilidade celular com o corante vermelho neutro. Os resultados obtidos permitiram observar que as amostras positivas no método de difusão em ágar que apresentaram até índice de zona 3 ou seja, halo de morte celular variando de 0,5 a 1,0 cm a partir da amostra, segundo a Farmacopéia Americana, não apresentaram irritação ocular, cutânea ou de mucosa oral. As amostras que apresentaram índice de zona 4, apresentaram diferentes graus de irritação ocular e cutânea com exceção de 2 sabonetes líquidos de uso infantil que não apresentaram reações in vivo, embora apresentaram citotoxicidade até a concentração de 10%. Este método apresentou correlação significante com o teste de irritação ocular, inclusive para os valores individuais da córnea e conjuntiva. Quanto ao método de captura do vermelho neutro obteve-se correlação significante para o teste de irritação cutânea e ocular, podendo inferir que quando as amostras de sabonetes líquidos apresentarem IC50 maior que 0,085% não irão induzir irritação cutânea e ocular nos coelhos. Não foi observada relação entre a origem das linhagens celulares usadas e o tecido alvo utilizado no teste in vivo, sendo que todas as linhagens mostraram correlação significante com a linhagem NCTC clone 929. De acordo com os dados, conclui-se que os métodos in vitro são mais sensíveis e que o método de difusão em ágar usando a graduação da Farmacopéia Americana, pode ser adotado como ensaio de triagem na avaliação de cosméticos. Isto resulta de sua capacidade de predizer a irritação, contribuindo significativamente para a redução dos testes que utilizam animais. / The procedures described by Draize are the basis of both ocular and cutaneous irritation tests and have been adopted internationally for in vivo evaluation of products and substances. However, they have been criticized for ethical reasons, due to their cruelty towards animals. Therefore, alternative methodologies are being studied to evaluate the toxicity of products, which have contact with human beings. Thus, a comparative study was performed between ocular, cutaneous and oral mucosa irritation tests using rabbits and in vitro tests through agar diffusion method and neutral red uptake method, both with the use of NCTC clone 929, FPC-IAL and SIRC cell lines. Lipsticks, makeup base, face powder, blushes, eye shadows, mascara, pencils and eyeliners, as well as liquid soap for children and adult use were evaluated. The in vivo tests and the cell lines used in the agar diffusion test were chosen according to the place where the products are used. Only the NCTC clone 929 linage was used in the evaluation of all the samples. From the 204 analyzed samples, 141 were evaluated through the cutaneous irritation test and FPC-IAL lineage, 80 through the ocular irritation test and SIRC lineage, and 78 through the oral mucosa irritation test and FPC-IAL lineage. Only the samples of liquid soap were also evaluated through the neutral red uptake method with the three cells lines. The samples submitted to the in vitro evaluation were analyzed in different concentrations and the observed parameter was cellular viability with the use of neutral red. The results obtained revealed that the agar diffusion test positive samples which presented up to degree 3 zone rate, that is cellular death halo varying from 0,5 to 1,0cm from the samples, according to USP 25, didnt provoke ocular, cutaneous or oral mucosa irritation. The samples which presented degree 4 zone also showed different degrees of ocular and cutaneous irritation, except to two units of liquid soap for children use which didnt present in vivo reactions, although citotoxicity up to 10% concentration. This method showed significant correlation with the ocular irritation test and also concerning the individual values of cornea and conjunctiva. As for the neutral red uptake test, it presented significant correlation in the ocular and cutaneous irritation test, what make it possible to infer that when the liquid soap samples present IC50 above 0,085% they wont cause ocular and cutaneous irritations in rabbits. No relation was observed between the origin of the cell lines and the target tissue used in the in vivo test, having all the cellular lines shown significant correlation with the NCTC clone 929 line. According to the data, the in vitro methods are more sensitive and the diffusion agar method, using the American Pharmacopeia graduation, can be adopted as a sorting procedure in the evaluation of cosmetics. This is a result of its capacity of predicting irritation, what largely contributes for the reduction of animal using tests. Citotoxicidade Cosméticos Cultura celular Irritação in vivo Testes de Draize Cosmetic Cytotoxicity Draize test Irritation
5	Avaliação de metodologia alternativa in vitro ao teste de irritação ocular de Draize / The evaluation of alternative methods in vitro to the Draize eye test Janice Campos de Azevedo 28 August 1998 (has links) Para a avaliação do potencial irritante de substâncias aplicadas topicamente tem-se utilizado metodologias que baseiam nos estudos de Draize, onde preconiza-se o uso de animais. Devido à forte oposição a estes testes, intensa tem sido a busca por métodos alternativos visando diminuir ou eliminar a utilização de animais. Este estudo avaliou o potencial irritante de diversas substâncias através de duas metodologias: o teste in vivo , empregando metodologia oficial do Food and Drug Administration (FDA), onde utiliza-se coelhos e o teste de citotoxicidade in vitro - método de difusão em camada de agar sobreposta à monocamada de células - empregando as linhagens celulares He La e NCTC L 929 e o corante vital vermelho neutro. As amostras de produto acabado abrangeram colírios, testados sem diluição e xampus de uso adulto e infantil testados puros e diluídos a 25%, 5%, 1% e 0,1%. As matérias-primas compreenderam tensoativos aniônicos, não-iônicos e anfóteros testados após diluição. O material de acondicionamento foi representado por frascos de polietileno para colírios, testados através dos seus extratos em solução de NaCI 0,9%. Os resultados obtidos com as amostras de colírios e frascos de acondicionamento foram negativos quanto à irritação ocular em coelhos e comprovaram ausência de citotoxicidade nas duas linhagens celulares. As amostras de xampus apresentaram boa correlação dos resultados quando testadas sem diluição ou diluídas a 1,0% e 0,1 %. Foi calculada a correlação de Spearman entre os dados obtidos com as duas linhagens celulares sendo o valor do coeficiente r = 0,950 indicando uma correlação direta entre os resultados obtidos com as duas células. Os resultados demonstram a possibilidade de desenvolvimento de metodologias alternativas, sujeitas à validação, que resultem em procedimentos sensíveis, reprodutíveis, não sujeitos a critérios subjetivos de interpretação, visando assim, num primeiro estágio diminuir o uso de animais pelo emprego destas metodologias em avalições prévias do potencial irritante de substâncias químicas. / To evaluate the irritability potential of toppically laid on substances, some methodologies based on Draize\'s study where the use of animals is demanded, have been used. Due to the strong opposition of society and scientific comunity, the search for alternative methods having in view the reduction or even the elimination of animals have been intense, either through the use of volunteers or of in vitro methodologies. The present study has evaluated the irritability of several substances including finished products, raw materials and packagíng materials through two methodologies: the in vivo test which makes use of official methodology from Food and Drug Administration (FDA), where rabbits are employed and the citotoxicity in vitro test - method of diffusion in agar layer added to the monolayer of cells - using He La and NCTC L 929 cells lines and neutral red. The samples of the finished products embraced eye drops, tested without dilution, and shampoos for adults and children use both concentrated and diluted to 25%, 5%, 1% and 0.1 %. Raw materiais included anionis, non-anionic and amphoteric surfactants tested after dilution. The packaging material, represented by eye drops polyethylene bottles, was tested through its extracts in a NaCI 0,9% solution. The results obtained with the samples of eye drops and packaging materials were negative as for ocular irritation in rabbits and confirmed the absence of cytotoxicity in both cells lines. The samples of shampoos presented good correlation of results when tested without dilution or diluted to 1% and 0.1 %. It has been calculated Spearman correlation considering dates obtained with two cells lines, with value of coefficient r = 0,950 showing a direct correlation between results obtained with two cells. The results obtained with this study are likely to lead to sensitive and reproductive procedures, which won\'t arise subjective criteria of interpretation. Thus, the elimination of the use of animals will be possible at a first stage of previous evalution, and once a posterior validation of this methodology is done, the rational use of a smaller number of animais will be a reality. Citotoxicidade Cosmético Cultura celular Draize Irritação ocular Medicamento Cell culture Cosmetic Cytotoxicity Draize test Eye iritation
6	Marcadores de irritación en modelos celulares y organotípicos como alternativa a los ensayos in vivo, aplicado al estudio de tensioactivos del tipo lipoaminoácido Martínez Ocaña, Verónica 26 January 2007 (has links) Los tensioactivos son unos compuestos químicos que debido a sus propiedades fisicoquímicas pueden provocar irritación. Por tanto, es interesante desarrollar nuevos compuestos que sean más biocompatibles que los tensioactivos comerciales que se utilizan actualmente. Por otro lado, los ensayos de Draize, basados en la utilización de animales, son los únicos métodos aceptados oficialmente para valorar la irritación antes de la comercialización de nuevos compuestos. No obstante, por motivos éticos y científicos existe la necesidad de disponer de métodos que permitan remplazar los animales de experimentación en esos ensayos toxicológicos. Por ese motivo, se ha estudiado el potencial efecto irritante ocular y dérmico de nuevos tensioactivos derivados de aminoácidos utilizando distintos métodos alternativos a la experimentación animal. Para valorar la irritación ocular se ha utilizado, en primer lugar, el ensayo de hemólisis basado en un modelo celular. Según este ensayo, los lipoaminoácidos son menos irritantes oculares que los tensioactivos comerciales utilizados como referencia. También se ha valorado la irritación ocular mediante los ensayos HET-CAM y CAM-TBS que se basan en un modelo organotípico, la membrana corioalantoidea del huevo de gallina. A efectos comparativos también se realizó el ensayo de Draize ocular con algunos de los compuestos. Los resultados del ensayo in vivo han revelado que la membrana corioalantoidea del huevo de gallina es un modelo demasiado sensible a los tensioactivos por lo que se descarta su utilización para valorar ese tipo de compuestos. Para valorar la irritación dérmica se ha utilizado el cultivo en monocapa de líneas celulares y se han analizado diversos marcadores de irritación. En primer lugar, se valoró la citotoxicidad inducida por los compuestos mediante dos ensayos (NRU y MTT) y se calcularon las concentraciones de tensioactivo que inhibían un 50 % la viabilidad celular (CI50). A partir de esos resultados, se ha propuesto un modelo de predicción en la línea de fibroblastos 3T3: compuestos con una CI50 inferior a 50 µg/ml presentan un potencial irritante elevado, compuestos con una CI50 entre 50 y 100 µg/ml son irritantes moderados, y compuestos con una CI50 superior a 100 µg/ml presentan un potencial irritante bajo. También, se ha estudiado la producción de interleucina 1 alfa, un mediador proinflamatorio, en la línea de queratinocitos NCTC 2544. Según los resultados, los lipoaminoácidos tienen una menor capacidad para inducir la neosíntesis de la citocina que los tensioactivos comerciales, por tanto, presentan una menor capacidad para desencadenar una respuesta inflamatoria. Por último, se ha analizado la acumulación de lípidos neutros intracelulares como posible marcador de irritación dado que desempeñan un papel muy importante en la reparación de la membrana celular cuando esta sufre algún tipo de agresión. Se ha estudiado en la línea celular NCTC 2544 tras el tratamiento con los diferentes tensioactivos. Para la detección de los lípidos neutros se utilizó el colorante rojo de nilo que es específico de lípidos neutros y se realizó el análisis mediante citofluorometría y microscopia de fluorescencia. Los tensioactivos indujeron incrementos de fluorescencia con un perfil concentración dependiente. Además, los incrementos de fluorescencia se detectaron antes de observar incrementos en la actividad lactato deshidrogenasa en el medio de cultivo, un marcador de daños sobre la membrana. Ese hecho implica que la acumulación de lípidos neutros es un marcador que indica daños sobre la membrana previo a la muerte celular. Finalmente, a efectos comparativos también se ha realizado el ensayo de Draize dérmico con algunos de los compuestos. Según este ensayo los lipoaminoácidos se clasificaron como ligeramente irritantes. Además, los resultados revelaron que los ensayos in vitro son más sensibles y permiten detectar pequeñas diferencias en el potencial irritante de los compuestos, sobretodo cuando se trata de compuestos con un potencial irritante ligero y moderado. / Surfactants are among the most versatile and frequently used excipients in cosmetic and pharmaceutical products. However, the application of pharmaceutical or cosmetic preparations containing these compounds may result in eye and skin irritation. Therefore, it is of great interest to identify surfactants with low irritant properties and it is also necessary to have rapid assays to assess potentially damaging effects. The evaluation of the irritant potential of chemicals in vivo is traditionally conducted on rabbits using the Draize test method. However, due to increasing concern over animal use and in lights of its potential ban in the near future, in vitro alternative methods should now be encouraged. The purpose of this work was to evaluate the potential eye or skin irritation of new amino acid-based surfactants using different in vitro assays. For predicting eye irritation the red blood cell test was used. According to the results of this assay, the new surfactants were less toxic than the commercial surfactants employed as a reference. Two methods based on the chorioallantoic membrane were also used. The results showed that these methods are oversensitive to this class of compounds, therefore, their utilization for predicting the eye irritation potential of surfactants is not recommended. For predicting skin irritation, different cell lines (fibroblasts 3T3 and 3T6, keratinocytes NCTC 2544 and HaCaT) were employed taking into account different endpoints, such as cell viability, IL-1alpha production and lipid droplet accumulation. Cell viability was measured using two different assays (NRU and MTT) and we proposed a prediction model taking into account the IC50 values. The release and production of the cytokine IL-1alpha was measured in the NCTC 2544 cell line. The results revealed that new surfactants are less potent than the commercial surfactants to stimulate the synthesis of the cytokine. Lipid droplet accumulation was also studied in the NCTC 2544 cell line. After the surfactant exposure, cells were treated with Nile Red. The results of the fluorescence measurements showed a concentration dependent profile for all the compounds. Furthermore, fluorescence increases were detected before significant lactate deshidrogenase release into the culture medium was observed. Accordingly, the accumulation of lipid droplets could be used to detect membrane injuries prior to cell death. Assaigs de Draize Biocompatibilitat Toxicologia Tensioactius Ciències de la Salut 612
7	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
8	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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