Transferability and reproducibility of the EpiSkin™ Micronucleus Assay.

A novel in vitro 3D micronucleus assay was developed in China using the EpiSkin™ 3D human skin model. This EpiSkin™ Micronucleus Assay showed good predictivity and reproducibility during internal validation and is expected to contribute to in vitro genotoxicity testing as a follow-up for positive results from 2D micronucleus assay. Having developed the assay in one laboratory, further work focused on the transferability and inter-laboratory reproducibility in two additional Chinese authority laboratories (Guangdong Provincial Center for Disease Control and Prevention and Zhejiang Institute for Food and Drug Control). Formal training was provided for both laboratories, which resulted in good transferability based on the results of two positive compounds, such as mitomycin C and vinblastine. Independent experiments were then performed, and inter-laboratory reproducibility was checked using 2-acetylaminofluorene, 5-fluorouracil, 2,4-dichlorophenol, and d-limonene. The dose-responses of the positive control chemical, mitomycin C, were similar to those of the developing laboratory, and all test chemicals were correctly classified by all laboratories. Overall, there was a good transferability as well as intra- and inter-laboratory reproducibility of the EpiSkin™ Micronucleus Assay. This study further confirmed the assay's robustness and provided confidence to enter following validation stages for scientific acceptance.

A new 3D model for genotoxicity assessment: EpiSkin™ Micronucleus Assay.

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.

How to facilitate the implementation of 3D models in China by applying good in vitro method practice for regulatory use.

The Organization for Economic Co-operation and Development (OECD) Guidance Document No. 34 and No. 286 on Good In Vitro Method Practices (GIVIMPs) for the development and implementation of in vitro methods for regulatory use in human safety assessment have been endorsed. Considering that China is accelerating the development of alternative approaches in both research and acceptance, early application of these principles is beneficial to the implementation and acceptance of in vitro alternative methods in China. To promote the replacement of animal testing for regulatory use, L'Oréal initiated the EpiSkin™ skin irritation test (SIT) implementation program in China. More than 50 external scientists participated, and the method has been established in 34 organizations including authorities, industries, and testing service laboratories. Taking two collaborations with Guangdong CDC and Shanghai SGS for in vitro SIT as examples, we demonstrated a method implementation process in good alignment with the OECD principles. The current study illustrated the practical way in which both OECD Guidance documents assisted in the transfer and establishment of in vitro approaches and further promoted the future scientific recognition and acceptance of new OECD-accepted alternative testing methodologies in China.

A ready-to-use integrated in vitro skin corrosion and irritation testing strategy using EpiSkin™ model in China.

The need of in vitro alternative methods has been increasing in toxicology research as well as in cosmetic industry in China recently. Following the establishment of China EpiSkin™ skin corrosion and irritation testing methods, both as stand-alone in vitro tests according to Organization for Economic Co-operation and Development (OECD) TG 431 and TG 439, the present study aims to evaluate the use of these two methods within the Integrated Approach on Testing and Assessment (IATA). The IATA, adopted by OECD as Guidance Document 203, provides guidance on the integration of existing and new data in a modular approach for classification and labelling of chemicals according to Globally Harmonized System of classification and labeling of chemicals (GHS) issued by the United Nations (UN). By applying bottom-up and top-down integrated testing strategies to a set of 60 chemicals representing various chemicals classes (organic acid/base/neutral, inorganic acid/base/salt, and surfactant) and physical states (liquid and solid), the results demonstrated that both strategies reached a high overall accuracy of 83.3% to distinguish non-classified, Category 2, Category 1B/1C and Category 1A according to UN GHS, identically. In conclusion, the integration of China EpiSkin™ skin corrosion and irritation testing data into either bottom-up or top-down strategy allows accurate assessment of potential skin hazard of chemicals. It brings a future extension of application of alternative methods and implementation of alternative testing strategies in China.

A way forward of alternative methods in China: Implementation of skin corrosivity potential using in vitro reconstructed human epidermis.

The purpose of present study was to investigate the applicability of reconstructed human epidermis model to identify skin corrosive UN GHS Categories 1A, 1B/1C and non-corrosive chemicals in China. By using a commercialized reconstructed human epidermis model, China EpiSkin™ which had been proven to be applicable as a stand-alone test method to predict skin irritation in previous study, the predictive capacity of corrosion was assessed with 76 chemicals that included 30 reference chemicals recommended by OECD TG 431 in this study. The latter reference chemicals were tested in three runs, the within-laboratory reproducibility reached 100%, the accuracy was 90% for distinguishing corrosive and non-corrosive chemicals and 80% for sub-categorization (Cat. 1A vs Cat. 1B/1C vs non corrosive). Additional 46 chemicals were also tested, and the overall accuracy for sub-categorization of all 76 tested chemicals was 80.3% with 91.7% sensitivity for Category 1A, 82.1% sensitivity for category 1B/1C and 75% specificity which met all required predictive capacity by OECD. The present study results show that China EpiSkin™ model can be applied to predict sub-categorization 1A and 1B/1C of corrosive chemicals. The availability of skin corrosion in vitro test method provides the applicability of in vitro non-animal testing method for chemicals widely used in various industries, and will further support the implementation and promotion of alternative methods in China.

In vitro skin irritation assessment becomes a reality in China using a reconstructed human epidermis test method.

The in vitro EpiSkin™ test method was validated in 2007 by the European Union Reference Laboratory for alternatives to animal testing (EURL ECVAM) as a full replacement method for the Draize acute skin irritation test and adopted in the OECD Test Guideline 439 in 2009. Based on the EpiSkin™ technology, the production of a reconstructed epidermis model has been established and standardized in China. The evaluation of the in vitro skin irritation test method using this EpiSkin™ model produced in China was performed on a set of 45 chemicals. Good predictive capacity was obtained with 94% (n=17) for sensitivity, 75% (n=28) for specificity and 82% for accuracy. The accuracy of the included 20 OECD reference chemicals also met the OECD acceptance criteria, indicating that this testing method based on the EpiSkin™ model produced in China can be used as a stand-alone test method to predict skin irritation. The availability and validity of in vitro epidermis model and testing method are of great significance for extending the applications of non-animal alternative testing methods in China.

Deficiency of kinase suppressor of Ras1 prevents oncogenic ras signaling in mice.

In Drosophila and Caenorhabditis elegans, kinase suppressor of ras (KSR) positively modulates Ras/Raf-mitogen-activated protein kinase (MAPK) signaling. The precise signaling mechanism of mammalian KSR1 and its role in Ras-mediated transformation, however, remain uncertain. To gain insight into KSR1 function in vivo, we generated mice homozygous null for KSR1. ksr1-/- mice are viable and without major developmental defects. However, an unusual disorganized hair follicle phenotype manifest in epidermal growth factor receptor knockout mice is recapitulated in ksr1-/- mice, providing genetic support for the notion that epidermal growth factor receptor, Ras, and KSR1 are on the same signaling pathway in mammals. Furthermore, ksr1-/- mice allow for the definition of KSR1-dependent and -independent mechanisms of c-Raf-1 activation. In embryonic fibroblasts, epidermal growth factor and 12-O-tetradecanoylphorbol-13-acetate activated the MAPK cascade to a similar extent, yet only c-Raf-1 activation by epidermal growth factor depended on KSR1. Moreover, whereas the genesis of polyomavirus middle T antigen (MT)-driven mammary cancer appears independent of KSR1, KSR1 is obligate for v-Ha-ras-mediated skin tumor formation. The growth of MT-driven mammary tumor was moderately slowed in ksr1-/- mice, however, consistent with a decreased rate of proliferation of ksr1-/- cells (T cells and embryonic fibroblasts). Nonetheless, all ksr1-/- animals succumbed to mammary cancer. In contrast, papilloma formation in Tg.AC mice, resulting from skin-specific v-Ha-ras expression, was completely abrogated in the ksr1-/- background. Hence, MT-driven mammary tumor genesis, which is signaled through src and phosphatidylinositol 3'-kinase, appears KSR1 independent, whereas v-Ha-ras-mediated skin cancer, signaled through the Raf-1/MAPK cascade, requires KSR1. These results suggest KSR1 may represent a therapeutic target for Ras/MAPK signaling of human tumorigenesis.