Skin models as an alternative to animal testing

The overall mission of the EU is to ensure that people’s health and safety at work are properly safeguarded. However, the information on which regulatory decisions regarding human health are based comes in part from studies conducted in experimental animals. The 7th Amendement of the Cosmetics Directive is transposed into national law in September 2004. It is a new Directive that pertains to testing of finished cosmetic products and ingredients. The most important rules for the near future are:

  • As of 11 March 2009; a ban on animal testing of finished cosmetic ingredients within the EU.
  • From March 2009; a ban for all human health effects with the exception of repeated-dose toxicity, reproductive toxicity and toxicokinetics. For these specific health effects the marketing ban will apply step by step as soon as alternative methods are validated and adopted in EU legislation with due regard to the OECD validation process, but with a maximum cut-off date of 10 years after entry into force of the Directive, i.e., 11 March 2013, irrespective of the availability of alternative non-animal tests.

All together this clearly shows that there is a need for validated in vitro methods for toxicity testing of chemicals, e.g. skin sensitizers. Many years ago, researchers have started the search for alternative methods. The key steps in the skin sensibilization process, (allergen detection, uptake and processing, cytokine signaling, migration/maturation of antigen-presenting cells (DC’s), activation and proliferation of T cells), have been used as read-out parameters in these tests. Although some tests look very promising, they are still far from being perfect.

HSEs are being used to test compounds for skin irritation, skin corrosion or skin sensibilisation (A). The epidermis detaches from the dermal part when 2% of SLS is topically applied onto the HSE (B). The HSE contains a competent barrier, shown is a Thin Layer Chromatography profile of the lipid composition of HSEs and native skin (C)

Figure 4: HSEs are being used to test compounds for skin irritation, skin corrosion or skin sensibilisation (A). The epidermis detaches from the dermal part when 2% of SLS is topically applied onto the HSE (B). The HSE contains a competent barrier, shown is a Thin Layer Chromatography profile of the lipid composition of HSEs and native skin (C).

Although the skin models reproduce to a large extent the barrier properties of normal human skin, still some small differences are present. These differences might affect the predictability of a tested compound. In a collaborative project conducted at the LACDR we aim to further improve the barrier properties of the skin models by modifying lipids composition in the stratum corneum. In previous studies we have shown that LEMs are reliable and producible epidermal models that are suitable for the screening of potential skin irritants. The test substances can be applied topically and their irritant potential can be evaluated using various endpoints, such as the induction of tissue damage or the release of various pro-inflammatory mediators, changes in protein and mRNA expression profiles. Similar approach can be followed for testing other compounds such as corrosive compounds. Studies with HSEs can therefore contribute to our knowledge on the basic biochemical mechanisms underlying irritant reactions, and can be used to understand the structural features of molecules, which may be responsible for eliciting an irritant reaction. In addition, generation of epidermal equivalents populated with both keratinocytes and melanocytes makes it possible to study the regulation of melanogenesis, melanocyte-keratinocyte interactions, and how these processes are affected by UV irradiation. Such a model can also be used for testing the phototoxic or photo-protective potentials of various compounds and sunscreens. In another collaborative project with the RIVM we anticipate to identify biomarkers that can be used to discriminate skin sensitizers from skin irritants in order to develop simple and reliable test systems. For this purpose we will use different cell types including cell lines, primary keratinocytes, dendritic cells and/or epidermal skin models. In addition, signal transduction pathways that are known to play an important role in skin sensitization, such as the KEAP1/Nrf2 pathway will be studied in depth to better understand the skin sensitization process.  

Projects:

  • LACDR: "Improvement of the barrier properties in reconstructed human skin models”. In collaboration with Prof.dr.J.Bouwstra.
  • Nederlands Toxicologisch Centrum (NTC): “Validation of molecular markers of skin sensitization by gene silencing in human keratinocytes and 3D reconstructed skin models”. In collaboration with Prof.Dr H van Loveren (RIVM).