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Previous issue date: 2008 / Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde / A sociedade científica mundial vem sofrendo pressão para que seja efetuada a substituição de ensaios in vivo por ensaios in vitro, sendo o teste de irritação ocular de Draize um dos testes in vivo mais fortemente combatidos pelos setores ativistas que lutam pela promoção e preservação do bem estar animal. Como a substituição do teste de Draize por metodologia alternativa se mostrou tarefa mais difícil do que se imaginava inicialmente, sobretudo devido ao grande número de desfechos que esta metodologia quantifica, as agências internacionais de validação de metodologia alternativas adotaram como conduta a busca por uma bateria de ensaios in vitro que possam servir de triagem para avaliação do potencial irritante ocular de produtos e ingredientes. Neste caso, o uso de animais só é recomendado, quando os resultados obtidos in vitro sugerem um baixo potencial irritante. Neste contexto, o presente estudo objetivou avaliar o grau de preditibilidade do método de Quantificação de Proteínas Totais (QPT) na avaliação do potencial citotóxico de tensoativos (N=6) e produtos cosméticos acabados (xampus e condicionadores, N=19). Todos os produtos avaliados no presente estudo foram previamente analisados quanto ao seu potencial irritante pela metodologia in vivo. O teste in vitro foi realizado utilizando linhagem celular proveniente de fibroblasto de embrião de camundongo (3T3) e o corante Azul Brilhante de Coomassie R-250. A avaliação do coeficiente de variação do teste de QPT em células 3T3 mostrou um valor de CV por cento global de 30,00 por cento. Para a comparação entre os resultados in vivo (MEM) e in vitro (IC50) utilizamos o coeficiente de correlação de Pearson. / The world scientific community is under pressure to find effective ways to substitute in vivo by in vitro tests. The Draize Eye Test is one of the most disapproved in vivo tests by the animal welfare activists. Since the substitution of the Draize Eye Test by an alternative method turned out to be a more difficult issue than it was first thought, mainly due to the great number of endpoints that this assay is able to quantify, the international validation agencies for alternative methods adopted, as a conduct, the search for a set of in vitro assays, which might work as a screening for the evaluation of the ocular irritant potential of products and ingredients. The use of animals would only be recommended if the results obtained with the in vitro screening tests indicate a low irritant potential for a determined substance. In this context, the main goal of the present study was to evaluate the degree of predictability of the Total Protein Quantification Methodology (QPT) in the evaluation of the citotoxic potential of surfactants (n=6) as well as in cosmetics (shampoos and hair conditioners, n=19). All the evaluated products in this study were previously analyzed in vivo, regarding their irritant potential. The in vitro test was performed using a cellular lineage derived from embryo mouse fibroblast (3T3) and the dye Blue Brilhant of Coomassie – R 250. The evaluation of the test variation coefficient (CV%) in 3T3 cells demonstrated a global CV% of 30%. In order to compare the in vivo (MMS) results with the ones obtained in vitro (IC50), we applied the Pearson´s coefficient of correlation. The value obtained for the correlation between the two methods was –0.420 (p=0.022). The analysis of the IC50 and MMS values of the different ocular structures showed a better correlation of the in vitro method with the injuries on the conjunctive, than with the damage found on the iris or cornea in vivo. Furthermore, an enhanced correlation of the in vitro versus in vivo method was observed for surfactants, when compared to whole products. In order to evaluate the predictability of the in vitro test in regard to the in vivo method a cut-off point was established, where substances with values of IC50<1 mg/ml were determined as irritant and substances with levels of IC50 higher then 1mg/ml were considered as non-irritant. Hence, under the experimental conditions of the present study, the assay accuracy was of 80%, the sensibility was 94% and the specificity was of 43%. Based on the results obtained we conclude that the QPT method, using Dye Blue Brilliant of Comassie R-250 and the cellular lineage 3T3, shows a better ability to predict the irritant potential of isolated products (surfactants) than of entire products. Even though the global CV% value found with our in vitro method, according to WHO, is considered suitable for assays performed with cell lines, the comparison of our results with the published data shows that the SIRC cell line is more interesting once considering inter-assay accuracy. We also conclude that the proposed assay has a better capacity to predict the irritant effect on the conjunctive than on the other ocular structures. At last, it is worth pointing out that this method shows a high sensibility degree and moderate level of specificity, and the application of the QPT assay may be useful to add the screening set assays for the evaluation of toxicity induced by products under the scope of Sanitary Surveillance.
| Identifer | oai:union.ndltd.org:IBICT/oai:www.arca.fiocruz.br:icict/9270 |
| Date | January 2008 |
| Creators | Abreu, Clarice Lima do Canto |
| Contributors | Nogueira, Ana Cristina Martins de Almeida, Presgrave, Rosaura de Faria, Paixão, Rita Leal, Delgado, Isabella Fernandes, Delgado, Isabella Fernandes |
| Source Sets | IBICT Brazilian ETDs |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/masterThesis |
| Source | reponame:Repositório Institucional da FIOCRUZ, instname:Fundação Oswaldo Cruz, instacron:FIOCRUZ |
| Rights | info:eu-repo/semantics/openAccess |
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