Robert Schmucker

In vitro detection of contact allergens: Development of an optimized protocol using human peripheral blood monocyte-derived dendritic cells

Allergic contact dermatitis is a delayed T-cell mediated allergic response associated with relevant social and economic impacts. Animal experiments (e.g. the local lymph node assay) are still supplying most of the data used to assess the sensitization potential of new chemicals. However, the 7th amendment to the EU Cosmetic Directive will introduce a testing ban for cosmetic ingredients after 2013. In vitro alternative methods are thus being actively developed. Although promising results have been obtained with cell lines, their reduced functionality and inherent genomic instability led us to reinvestigate the use of peripheral blood monocyte-derived dendritic cells (PBMDCs) for the establishment of a reliable in vitro sensitization test. To solve the issues associated with the use of primary cells, the culture and exposure conditions (cytokine concentrations, incubation time, readout, pooled vs. single donors and cytotoxicity) were re-assessed and optimized. Here we propose a stable and reproducible protocol based on PBMDCs. This should allow a wider acceptance of PBMDCs as a reliable test system for the detection of human skin sensitizers and the inclusion of this protocol in an integrated testing strategy.

A Novel In Vitro Method for the Detection and Characterization of Photosensitizers

Photoactivation and binding of photoactive chemicals to proteins is a known prerequisite for the formation of immunogenic photoantigens and the induction of photoallergy. The intensive use of products and the availability of new chemicals, along with an increasing exposure to sun light contribute to the risk of photosensitizing adverse reactions. Dendritic cells (DC) play a pivotal role in the induction of allergic contact dermatitis. Human peripheral blood monocyte derived dendritic cells (PBMDC) were thus perceived as an obvious choice for the development of a novel in vitro photosensitization assay using the modulation of cell surface protein expression in response to photosensitizing agents. In this new protocol, known chemicals with photosensitizing, allergenic or non-allergenic potential were pre-incubated with PBMDCs prior to UVA irradiation (1 J/cm2). Following a 48 h incubation, the expression of the cell surface molecules CD86, HLA-DR and CD83 was measured by flow cytometry. All tested photosensitizers induced a significant and dose-dependent increase of CD86 expression after irradiation compared to non-irradiated controls. Moreover, the phototoxicity of the chemicals could also be determined. In contrast, (i) CD86 expression was not affected by the chosen irradiation conditions, (ii) increased CD86 expression induced by allergens was independent of irradiation and (iii) no PBMDC activation was observed with the non-allergenic control. The assay proposed here for the evaluation of the photoallergenic potential of chemicals includes the assessment of their allergenic, phototoxic and toxic potential in a single and robust test system and is filling a gap in the in vitro photoallergenicity test battery.

Design and application of a screening and training protocol for odour testers in the field of personal care products

The assessment of odours and in particular of human axillary odour is an integral part of the research and development of deodorant and anti‐perspirant products. One method to perform odour assessment is the odour evaluation that is carried out by experts, designated as odour testers or sniffers. Product development decisions are therefore based on human assessment. As for every scientific measurement, the influencing factors need to be standardized or regularly calibrated as effectively as possible for reasons of quality assurance. We therefore developed a screening and training concept aiming to examine the general suitability of odour testers by determining the individual odour sensitivity for relevant odours. This newly developed method is based on the national and international standards and guidelines EN 13725:2003, VDI 3882 sheet 1 and ASTM‐1207. Suitable odour testers are subsequently trained to correlate their individual odour intensity perception with an intensity calibration scale in order to achieve reproducible results. Training sessions held on a regular basis help to achieve a greater homology in the response of an existing panel. Our established screening and training protocol has already been successfully put into practice and is also subject to permanent improvement with regard to practical requirements.

Influence of cleansing on stratum corneum tryptic enzyme in human skin

Desquamation in human skin is a well‐balanced process of de novo production of corneocytes and their shedding from the skin surface. The proteolysis of corneodesmosomes is an important step in the final desquamation process. In the degradation of these adhesion molecules, the stratum corneum tryptic enzyme (SCTE) plays a key role. In initial studies with extracts of porcine epidermis, SCTE was shown to be inactivated by low concentrations of sodium lauryl ether sulphate (SLES). These in vitro findings were supported by in situ results obtained by measuring the release of fluorescent dyes coupled to trypsin‐specific substrates incubated on human skin cross‐sections. Moreover, in further studies, it could be demonstrated that the SCTE activity in the human horny layer decreases after in vivo application of cleansing products containing SLES. After repeated washing of human volunteers with tap water, a standard market cleansing product (SLES/betaine system) or a new improved cleansing product (SLES/betaine/disodium cocoyl glutamate system), the specific SCTE activity was determined in extracts from the uppermost layers of the stratum corneum. It could be shown that after application of the new formula the remaining SCTE activity was significantly higher than after use of the standard market formula. This ex vivo approach has proven to be very helpful for measuring surfactant effects on human skin enzymes. Using this assay, we developed an improved shower gel formula, which leads to a significantly higher skin enzyme activity after application, compared to a standard market formula.

Qualification of a precise and easy-to-handle sweat casting imprint method for the prediction and quantification of anti-perspirant efficacy

A time‐ and cost‐effective sweat casting method using the forearm as test site to assess the efficacy of several anti‐perspirant formulations with a low number of test subjects has been evaluated and qualified. The imprint sweat casting method is based on a 2‐component silcone‐imprint technique to measure the efficacy of more than eight products in parallel with the same test subject. In studies using aluminum chlorohydrate (ACH) formulations as test anti‐perspirants, a clear‐cut correlation could be demonstrated between sweat gland activities measured by the imprint method and gravimetric measurement of sweat gland activities. Concentration‐dependent inhibition of sweat gland activity could be observed with the imprint technique up to an ACH concentration of 15%, and all formulations containing 2% ACH or above resulted in statistically significant reduction of sweat gland activity (P < 0.001) when compared with untreated control areas. Furthermore, the SDs of individual studies using the imprint technique were in a range of ±20% of sweat gland activity, which can be regarded rather low for in vivo measurements of a complex process like sweat secretion. A group‐wise comparison between the measurements of anti‐perspirant activity as determined by the imprint protocol and the Food and Drug Administration (FDA) Guideline compliant gravimetric hot‐room protocol revealed that the test results for anti‐perspirant activity obtained with the imprint protocol are similar to those obtained with the hot‐room protocol. Moreover, the data generated with the imprint protocol have a high predictive value for the outcome of a later guideline‐compliant hot‐room test. As the imprint casting method tends to be a little more sensitive for formulations with low anti‐perspirant activity, and seems to be associated with less interassay variability than the standard gravimetric hot‐room test, the imprint casting method may select products which later fail to pass the standard gravimetric hot‐room test. Meanwhile the imprint sweat casting has proven to be a robust method useful to support efficacy‐oriented product development. Therefore, in later stages of utilization it might even evolve into an efficient claim substantiation tool.