RESUMEN
The European Regulation on Cosmetics (no. 1223/2009) has prohibited the use of animals in safety testing since March 2009 for ingredients used in cosmetics. Irreversible events at the chromosome level (clastogenesis and aneugenesis) are commonly evaluated by scoring either micronuclei or chromosome aberrations using cell-based genotoxicity assays. Like most in vitro genotoxicity assays, the 2D in vitro micronucleus assay exhibits a poor specificity and does not mimic the dermal route. To address these limitations, the current project aims to develop and validate a 3D micronucleus assay using the EpiSkin™ model. This project is scientifically supported by the Cosmetics Europe Genotoxicity Task Force. In a first step, two key criteria for the development of micronucleus assay, namely, the sufficient yield of cells from the EpiSkin™ model and an acceptable proliferation rate of the basal layer, were assessed and demonstrated. Subsequently, six chemicals (vinblastine, n-ethylnitrosourea, ß-butyrolactone, 2-acetylaminofluorene, 2,4-dichlorophenoland d-limonene) were evaluated in the EpiSkin™ Micronucleus Assay. At least two independent experiments using 48- and 72-h incubations were performed for each chemical. Results showed good inter-experimental reproducibility, as well as the correct identification of all six tested chemicals. The metabolism of 2-acetylaminofluorene on the EpiSkin™ model was also investigated and confirmed by the formation of an intermediate metabolite (2-aminofluorene). These preliminary results from the EpiSkin™ Micronucleus Assay indicate that it is a promising in vitro assay for assessing genotoxicity. The availability and suitability of this test method contribute significantly to the development of non-animal testing methods in China and its impact on the worldwide field.
Asunto(s)
Bioensayo/métodos , Daño del ADN , Laboratorios/normas , Pruebas de Micronúcleos/métodos , Mutágenos/efectos adversos , Piel/patología , Humanos , Piel/efectos de los fármacos , Piel/metabolismoRESUMEN
The European Cosmetic Toiletry and Perfumery Association (COLIPA), along with contributions from the European Centre for the Validation of Alternative Methods (ECVAM), initiated a multi-lab international prevalidation project on the reconstructed skin micronucleus (RSMN) assay in EpiDerm™ for the assessment of the genotoxicity of dermally applied chemicals. The first step of this project was to standardize the protocol and transfer it to laboratories that had not performed the assay before. Here we describe in detail the protocol for the RSMN assay in EpiDerm™ and the harmonized guidelines for scoring, with an atlas of cell images. We also describe factors that can influence the performance of the assay. Use of these methods will help new laboratories to conduct the assay, thereby further increasing the database for this promising new in vitro genotoxicity test.
Asunto(s)
Pruebas de Micronúcleos/métodos , Pruebas de Irritación de la Piel/métodos , Pruebas de Irritación de la Piel/normas , Guías como Asunto , Humanos , Pruebas de Micronúcleos/normas , Pruebas de Micronúcleos/estadística & datos numéricos , Mutágenos/toxicidad , Piel , Ingeniería de Tejidos/métodos , Estudios de Validación como AsuntoRESUMEN
Recently, a novel in vitro reconstructed skin micronucleus (RSMN) assay incorporating the EpiDerm 3D human skin model (Curren et al., Mutat. Res. 607 (2006) 192-204; Mun et al., Mutat. Res. 673 (2009) 92-99) has been shown to produce comparable data when utilized in three different laboratories in the United States (Hu et al., Mutat. Res. 673 (2009) 100-108). As part of a project sponsored by the European cosmetics companies trade association (COLIPA), with a contribution from the European Center for the Validation of Alternative Methods (ECVAM), international prevalidation studies of the RSMN assay have been initiated. The assay was transferred and optimized in two laboratories in Europe, where dose-dependent, reproducibly positive results for mitomycin C and vinblastine sulfate were obtained. Further intra- and inter-laboratory reproducibility of the RSMN assay was established by testing three coded chemicals, N-ethyl-N-nitrosourea, cyclohexanone, and mitomycin C. All chemicals were correctly identified by all laboratories as either positive or negative. These results support the international inter-laboratory and inter-experimental reproducibility of the assay and reinforce the conclusion that the RSMN assay in the EpiDerm 3D human skin model is a valuable in vitro method for assessment of genotoxicity of dermally applied chemicals.