The present thesis provides an in-depth study of the barrier function of the skin by investigating the penetration of two toxicologically important substance classes: polycyclic aromatic hydrocarbons (PAH) and the metal allergens nickel, cobalt and palladium. Furthermore, the method of tape stripping was scrutinized to reveal the removal rate of stratum corneum (s.c.) with each tape strip. Skin contact to harmful substances can occur through consumer products containing them. Harmful substances are added to consumer products either by design — for example, as additives in plastic or certain metals in alloys — or inadvertently as contaminants. PAH were found to contaminate those plastics that are dyed with carbon black or contain mineral oils used as plasticizers. Nickel, cobalt and palladium, known to cause allergic contact dermatitis, are added into alloys in jewelry to attain certain characteristics such as color or corrosion resistance.
Skin penetration studies rely on the well-established in vitro Franz diffusion cell (FDC) assay. A skin sample or model is fixed over a receptor chamber containing an aqueous medium. The substance of interest is applied onto the skin in a matrix, for example, in solvents, petrolatumor in the consumer products themselves. After a certain incubation time, the compartments are analyzed to reveal the penetration rates of that substance into the skin.
In order to quantify the amount of substance in separate s.c. layers, the technique of tape stripping plays an important role. A tape strip is pressed onto the skin and removes a layer of the s.c. when taken off. However, the amount of s.c. removed with each tape strip is still subject of scientific debate. In a histological study presented here, the s.c. was removed by tape stripping of pigskin and the remaining s.c. was quantified microscopically. The study revealed a linear decrease in s.c. in terms of thickness as well as number of cell layers up to 20 removed tape strips. Each tape strip removed about one cell layer, corresponding to 0.4 μm.
In this thesis, the penetration of PAH through the s.c. was extensively studied. The influence of lipophilicity — expressed by the octanol-water partition coefficient (logP) — on the permeability of these toxic compoundswas demonstrated. Applying 24 different PAH(152–302 g/mol, logP: 3.9–7.3) in acetonitrile in an FDC assa, revealed that more lipophilic PAH exhibit lower penetration rates. Furthermore, the penetration was investigated for time intervals ranging from 2 h to 48 h. Highly lipophilic, six-ringed PAH like the dibenzopyrenes did not permeate the skin at all, whereas the less lipophilic, three-ringed PAH like acenaphthene permeate after 2 h already. The evaluation of FDC assays using human and pigskin allowed for the comparison of these two skin types. Human s.c. was shown to retain a greater share of the PAH that partition into the skin than porcine s.c.
The dependence on lipophilicity of PAH skin penetration is further supported by experiments that aimed to determine partition and diffusion coefficients of five selected PAH from a polypropylene surrogate (squalane) into porcine s.c. The PAH content in the skin compartments was quantified by gas chromatography coupled to tandem mass spectrometry. The use of tape stripping resulted in a concentration profile along the s.c. This profile was fitted to a solution of Fick’s second law, allowing the calculation of partition and diffusion coefficients. The partition coefficients positively correlated with the logP, while the diffusion coefficient was highest for naphthalene and about the same for the other PAH.
Nickel and cobalt are, contrary to palladium, part of the standard patch test used as diagnostic tool to detect contact allergies. Therefore, fewer allergies against palladium are diagnosed than those against nickel and cobalt. Nonetheless, Pd2+ exhibited higher frequencies of CD154+ in an in vitro activated marker assay in human blood samples thanNi2+ and Co2+. A reason for the lower number of diagnosed palladium allergies next to fewer tests could be a lower penetration rate of Pd2+ as compared to Ni2+ or Co2+. To test this hypothesis, FDC assays using pigskin were employed. Patch test preparations used for clinical diagnosis (the respective metal salt dispersed in petrolatum) were given onto the skin and incubated for 48 h, which corresponds to the time frame of a patch test. Additionally, FDC assays with the three ions in an aqueous solution were carried out to evaluate penetration rates independent of ion solubility. The recoveries in the skin compartments were quantified by inductively coupled plasma mass spectrometry. Pd2+ showed lower penetration rates than Ni2+ and Co2+, regardless of application in petrolatum or aqueous solution, thus confirming the hypothesis.
Collectively, these studies contribute to the understanding and analysis of the s.c. barrier function and the factors influencing the dermal penetration of two distinct classes of toxicologically relevant substances.
