There has been a continuous effort at EU level to find alternative methods to avoid testing on laboratory animals.The development of strategies to use fewer animals, cause less harm to animals or replace animals is supported if possible. In all relevant EU legislations the “3Rs Principle” [replacement, reduction and refinement of animal use (1)] is mandatory (2).

In this context reconstructed human epidermis models can be used as in vitro methods for different applications. Three dimensional human epidermis equivalents are derived from normal human keratinocytes and form a multilayered, highly differentiated tissue that mimics biochemical and physiological properties of the epidermis. During cultivation the tissue cultures are lifted to the air-liquid interphase to induce differentiation, epithelial stratification and cornification. The cellular structure of reconstructed human epidermis closely resembles the human epidermis including proliferating keratinocytes and a stratum corneum with intact barrier function (Fig. 1).

Fig. 1 Reconstructed human epidermis (RhE), a highly differentiated model of the human epidermis. (left) Cryosection of epiCS (8µm, H&E staining) after 14d culture at air-liquid interphase. (right) Schematic drawing of the corresponding cellular layers of the human epidermis compared to the epiCS morphology.

In addition to reconstructed human epidermis models comprising keratinocytes, models containing different cell types have been developed. For example, reconstructed human pigmented epidermis models like epiCS-M, which consist of keratinocytes and melanocytes. Major applications are tanning and lightening studies.

For which applications can reconstructed human epidermis (RhE) models be used?

Reconstructed human epidermis models are widely used to assess skin corrosivity or skin irritation in vitro.

In the past, the regulatory classification of a single substance or mixture to cause skin corrosion was based on the rabbit skin test. To replace existing animal based in vivo models the new in vitro models need to provide sufficient levels of sensitivity and specificity. Today four different reconstructed human epidermis models are validated for the assessment of skin corrosion (epiCS®, EpiDerm™, Episkin and SkinEthic RHE™).

In 1944 the Draize rabbit test for eye and dermal irritation was established to evaluate skin irritation potential of xenobiotics. The intense research by academia, industry and publicly funded research programs led to the development of in vitro tissue constructs based on human keratinocytes. In 2009 three commercially available reconstructed human epidermis models were validated by the ECVAM (European Centre for Validation of Alternative Methods) for the determination of hazardous (skin irritant) properties of single substances and mixtures in regards to the UN-GHS compliant rules for classification and labelling (3).

Further applications in the arena of toxicity testing are phototoxicity, genotoxicity and recently skin sensitisation.

However, these test methods were not formally validated and accepted by regulatory authorities.

For the determination of skin sensitisers and non-sensitisers, three non-animal test methods, the Direct Peptide Reactivity Assay (DPRA), the KeratinosensTM and the human Cell Line Activation Test (hCLAT) are under formal validation at EURL-ECVAM for their ability to predict skin sensitisation potential.
A recent study shows that reconstructed human epidermis models can be used to identify skin sensitising potential of chemicals as well as their potency in an "Integrated Testing Strategy (ITS)" approach. (4)(5).
Due to the complex mechanisms which lead to skin allergy in humans a battery of test systems (including those mentioned above) is combined to be used in an Integrated Testing Strategy (ITS) approach under the supervision of ECVAM and international partners. The ITS aims to identify and classify skin sensitisers according to the Globally Harmonised System of Classification and Labeling for chemicals (GHS) and will be an important tool to assess potency of skin sensitisers in vitro.