Testing – Co-culture assessment

IDEA Group and the Pasteur Institute of Lille have developed a non-animal genetic toxicology test using the Episkin reconstructed skin model. It improves exposure conditions in in vitro genotoxicity tests and allows the evaluation of poorly soluble substances

The in vitro department of the IDEA Group in partnership with the Pasteur Institute of Lille has developed a non-animal genetic toxicology test using the Episkin reconstructed skin model. Fabrice Nesslany reports

Ten years ago this month, in February 2003, the European Union (EU) passed the 7th Amendment to the Cosmetics Directive (2003/15/EC – Amendment VII to Directive 76/768/EEC), introducing new provisions for the phasing out of animal tests on finished cosmetic products and ingredients.

As part of the introduction process of the legislation, the European Reference Laboratory on Alternatives to Animal Testing (EURL EVCAM) funded a range of studies looking at alternative, non-animal tests. Among them was a co-culture model using Episkin epidermis and target cells for the testing of skin irritation. The model was developed by a team led by Dr Fabrice Nesslany at the Pasteur Institute in Lille working in partnership with L’Oréal’s Safety Research Department in Aulnay-sous-Bois, France and the IDEA Group.

The system is aimed at improving exposure conditions in in vitro genotoxicity tests in order to evaluate the risks of substances that are topically applied to human skin, in particular cosmetic ingredients. It also allows the evaluation of poorly soluble substances, for which in vitro genetic toxicology tests are not very well adapted.

The Episkin reconstructed human skin model (RHE) is used in a co-culture system in which target cells (human lymphoblastoid TK6 cells or L5178Y mouse lymphoma cells) are cultured under the skin.1 In this context the model was used as a metabolically active tissue and as a physiological barrier. It was topically exposed to the test substances and the TK6 cells cultured for a period long enough to complete at least one mitosis.


Figure 1 In the co-culture system target cells are cultured under the Episkin RHE model


The substances’ genotoxicity was also assessed using two additional tests: a comet assay carried out directly on the keratinocytes to estimate the local effect and a micronucleus assay carried out on target cells to estimate the systemic effect (figure 1).

The comet assay on keratinocytes enables the detection of DNA primary lesions such as single and double strand breaks and alkali labile sites. Image analysis software is used to quantify the keratinocytes’ DNA lesions. This enables DNA migration to be measured by calculating parameters such as the percentage of DNA generated in the comet ‘tail’ or the ‘olive tail moment’ (OTM), ie the product of the percentage of DNA in the tail and the tail length.

The micronucleus assay enables the recognition of the numerical and structural chromosomal aberrations on the target cells and is carried out in accordance with OECD Test Guideline 487 (2010) (OECD guideline for the testing of chemicals: in vitro mammalian cell micronucleus test).

With the comet assay on the Episkin model, the local genotoxic properties on the keratinocytes resulted in a statistically significant increase in the percentage of DNA in the tail versus the negative control, with a dose effect relationship.

The micronucleus assay showed a statistically significant increase in the number of micronuclei in the TK6 cells, at least doubling in comparison with the negative control, and with a dose effect relationship.

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