As part of the EU legislation (7th amendment to the cosmetic directive) a co-culture model using Episkin® epidermis and target cells has been developed by the Dr Fabrice Nesslany’s team at Pasteur Institute (Lille) working in partnership with L’Oreal, in order to meet the predictive methods need in terms of genetic toxicology. This co-culture system is aimed to improve the exposure conditions in the in vitro genotoxicity tests in order to evaluate the risks of substances which are topically applied in human, especially cosmetic ingredients.
This test also allows to evaluate the poorly soluble substances for which the in vitro genetic toxicology tests are not very adapted indeed. This test was recognized by the ECVAM as an alternative method of genetic toxicology
The Episkin® reconstructed human skin model (RHE) is used in co-culture system in which one target cells (human lymphoblastoid TK6 cells or L5178Y mouse lymphoma cells) are cultured under the skin (Flamand et al., 2006). In this context the Episkin® model is used as a metabolically active tissue and as a physiological barrier.
The tested substances genotoxicity may be assessed by two additional tests, the comet assay carried out directly on the keratinocytes to estimate the local effect and the micronucleus assay carried out on target cells to estimate the systemic effect.
The Episkin® model is topically exposed to the test substances. The TK6 cells are cultured for a period sufficient to complete one mitosis at least.
The comet assay on keratinocytes allows to detect the DNA primary lesions such as single and double strand breaks and alkali labile sites. An image analysis software is used to quantify the Keratinocytes DNA lesions. The software allows to measure the DNA migration by calculating the parameters such as the DNA percentage generated in the comet “tail”.
The micronucleus assay allows the recognition of the numerical and structural chromosomal aberrations on the target cells and is carried out in accordance with the OECD 487 directive (2010).
Alternately the micronucleus assay may be carried out on reconstructed human epidermises, with cytochalasin B. At the end of three treatments at 24 hours interval, a fluorescent analysis of the harvested, isolated and stained cells is carried out. The cells containing one or more micronuclei are scored among the binucleated cells.
Results inerpretation
With the comet assay on Episkin® model the local genotoxic properties on the keratinocyes result in a statically significant increase of the DNA percentage in the tail versus the negative control with a dose effect relationship, and with the micronucleus assay they result in a statically significant increase of the micronuclei number in the RHE cells with at least a doubling versus the negative control and a dose effect relationship.
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