Marcadores de irritación en modelos celulares y organotípicos como alternativa a los ensayos in vivo, aplicado al estudio de tensioactivos del tipo lipoaminoácido

Résumé

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. "