Characterisation of human skin models - stability, metabolic capacity and comparative investigations in percutaneous absorption

Doctoral thesis German OPEN
Schreiber, Sylvia (2010)
  • Publisher: Freie Universität Berlin Universitätsbibliothek, Garystr. 39, 14195 Berlin
  • Subject: percutaneous absorption | skin | 540 Chemie und zugeordnete Wissenschaften | skin metabolism | human skin models | 540 Chemistry and allied sciences | reconstructed epidermis
    • ddc: ddc:540

In recent years, the demand for alternative test methods in safety assessment of cosmetics, risk assessment of chemicals, and testing of pharmaceuticals was increasingly included in the EU directives. Thereby, alternative test methods for the determination of percutaneous absorption should achieve a more reliable in vivo prediction of the response of human skin than animal skin. Even though freshly excised human skin is considered as a preferred test matrix its routine use is often difficult due to insufficient supply. Thus, in recent years commercially available and biotechnologically manufactured human skin models have increasingly gained in importance for toxicological testing. The OECD accepts their application for the examination of percutaneous absorption, given absorption of reference chemicals by the skin models is comparable to that by human or porcine skin. To provide evidence a thorough standardisation and validation of the test method is required. For this purpose first of all the skin models were characterised more closely regarding their stability towards the conditions of a permeation experiment. Both viability and integrity tests revealed a clear toxicity of different deficiency media after 6 h incubation with reconstructed epidermis or primary cultured keratinocytes. In both matrices, the addition of 5 % BSA which is advocated by the OECD as additive to the receptor medium for percutaneous absorption studies to facilitate solubility of highly lipophilic substances let to an additional reduction of viability. It was shown that apoptosis is in fact partly involved in the reduction of keratinocyte viability provoked by deficiency media but is irrelevant for the BSA caused toxicity - the final, BSA-related cell death is a matter of necrosis. These results have to be considered when selecting appropriate receptor media for percutaneous absorption studies in which metabolism of a substance and accordingly matrix viability are of major importance. Cutaneous metabolism of caffeine is of importance because of its topical use as active ingredient in cosmetics and especially because of its exposed position as OECD reference substance. For this reason in the context of this work the metabolism of caffeine was investigated in human skin material - namely in primary keratinocytes, fibroblasts and an epidermal and full thickness skin model - for the first time. For caffeine and the xanthine theophylline which was tested in parallel a concerted HPLC-method was established because of their overlapping metabolic pathways. Both xanthines were metabolised only to a small extent (< 1 %). With respect to the following permeation experiments with caffeine an impact on penetration / permeation of the substance due to metabolism could clearly be ruled out a priori. Metabolism of the xanthines by the monolayer cultures was comparable to that by the skin cultures both qualitatively and quantitatively. Thus, we showed that metabolic capacity of both test systems is similar. Finally, first important steps towards standardisation and validation of the test method for determination of percutaneous absorption were undertaken in consideration of the previous results using the OECD reference substance caffeine and the Franz cell technique. Several aspects possibly influencing permeation were investigated experimentally. A consistent and improved experimental protocol could be developed and was then applied to comparative permeation tests with different skin matrices. Relevant permeation parameters could be clearly defined.
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