Kerstin Reisinger

Categorization of chemicals according to their relative human skin sensitizing potency.

Although adoption of skin sensitization in vivo assays for hazard identification is likely to be successful in the next few years, this does not replace their use in potency prediction. Notably, measurement of potency of skin sensitizers in the local lymph node assay has been important. However, this local lymph node assay potency measure has not been formally assessed against a range of substances of known human sensitizing potential, because the latter is lacking. Accordingly, criteria for human data have been established that characterize 6 categories of human sensitizing potency, with 1 the most potent and 5 the least potent; category 6 represents true nonsensitizers. The literature has been searched, and 131 chemicals assigned into these categories according to their intrinsic potency judged only by the available human information. The criteria and data set generated provide a basis for examination of the capacity of nonanimal approaches for the determination of human sensitization potency.

Identifying and characterizing chemical skin sensitizers without animal testing: Colipa's research and method development program

The sensitizing potential of chemicals is usually identified and characterized using one of the available animal test methods, such as the mouse local lymph node assay. Due to the increasing public and political concerns regarding the use of animals for the screening of new chemicals, the Colipa Skin Tolerance Task Force collaborates with and/or funds research groups to increase and apply our understanding of the events occurring during the acquisition of skin sensitization. Knowledge gained from this research is used to support the development and evaluation of novel alternative approaches for the identification and characterization of skin sensitizing chemicals. At present one in chemico (direct peptide reactivity assay (DPRA)) and two in vitro test methods (cell based assays (MUSST and h-CLAT)) have been evaluated within Colipa inter-laboratory ring trials and accepted by the European Centre for the Validation of Alternative Methods (ECVAM) for pre-validation. Data from all three test methods will be used to support the development of testing strategy approaches for skin sensitizer potency prediction. The replacement of the need for animal testing for skin sensitization risk assessment is viewed as ultimately achievable and the next couple of years should set the timeline for this milestone.

Use of human in vitro skin models for accurate and ethical risk assessment: metabolic considerations.

Several human skin models employing primary cells and immortalized cell lines used as monocultures or combined to produce reconstituted 3D skin constructs have been developed. Furthermore, these models have been included in European genotoxicity and sensitization/irritation assay validation projects. In order to help interpret data, Cosmetics Europe (formerly COLIPA) facilitated research projects that measured a variety of defined phase I and II enzyme activities and created a complete proteomic profile of xenobiotic metabolizing enzymes (XMEs) in native human skin and compared them with data obtained from a number of in vitro models of human skin. Here, we have summarized our findings on the current knowledge of the metabolic capacity of native human skin and in vitro models and made an overall assessment of the metabolic capacity from gene expression, proteomic expression, and substrate metabolism data. The known low expression and function of phase I enzymes in native whole skin were reflected in the in vitro models. Some XMEs in whole skin were not detected in in vitro models and vice versa, and some major hepatic XMEs such as cytochrome P450-monooxygenases were absent or measured only at very low levels in the skin. Conversely, despite varying mRNA and protein levels of phase II enzymes, functional activity of glutathione S-transferases, N-acetyltransferase 1, and UDP-glucuronosyltransferases were all readily measurable in whole skin and in vitro skin models at activity levels similar to those measured in the liver. These projects have enabled a better understanding of the contribution of XMEs to toxicity endpoints.

A tiered approach to the use of alternatives to animal testing for the safety assessment of cosmetics: genotoxicity. A COLIPA analysis.

For the assessment of genotoxic effects of cosmetic ingredients, a number of well-established and regulatory accepted in vitro assays are in place. A caveat to the use of these assays is their relatively low specificity and high rate of false or misleading positive results. Due to the 7th amendment to the EU Cosmetics Directive ban on in vivo genotoxicity testing for cosmetics that was enacted March 2009, it is no longer possible to conduct follow-up in vivo genotoxicity tests for cosmetic ingredients positive in in vitro genotoxicity tests to further assess the relevance of the in vitro findings. COLIPA, the European Cosmetics Association, has initiated a research programme to improve existing and develop new in vitro methods. A COLIPA workshop was held in Brussels in April 2008 to analyse the best possible use of available methods and approaches to enable a sound assessment of the genotoxic hazard of cosmetic ingredients. Common approaches of cosmetic companies are described, with recommendations for evaluating in vitro genotoxins using non-animal approaches. A weight of evidence approach was employed to set up a decision-tree for the integration of alternative methods into tiered testing strategies.

International prevalidation studies of the EpiDerm™ 3D human reconstructed skin micronucleus (RSMN) assay: Transferability and reproducibility

Recently, a novel in vitro reconstructed skin micronucleus (RSMN) assay incorporating the EpiDerm 3D human skin model (Curren et al., Mutat. Res. 607 (2006) 192-204; Mun et al., Mutat. Res. 673 (2009) 92-99) has been shown to produce comparable data when utilized in three different laboratories in the United States (Hu et al., Mutat. Res. 673 (2009) 100-108). As part of a project sponsored by the European cosmetics companies trade association (COLIPA), with a contribution from the European Center for the Validation of Alternative Methods (ECVAM), international prevalidation studies of the RSMN assay have been initiated. The assay was transferred and optimized in two laboratories in Europe, where dose-dependent, reproducibly positive results for mitomycin C and vinblastine sulfate were obtained. Further intra- and inter-laboratory reproducibility of the RSMN assay was established by testing three coded chemicals, N-ethyl-N-nitrosourea, cyclohexanone, and mitomycin C. All chemicals were correctly identified by all laboratories as either positive or negative. These results support the international inter-laboratory and inter-experimental reproducibility of the assay and reinforce the conclusion that the RSMN assay in the EpiDerm 3D human skin model is a valuable in vitro method for assessment of genotoxicity of dermally applied chemicals.

Comet assay in reconstructed 3D human epidermal skin models – investigation of intra- and inter-laboratory reproducibility with coded chemicals

Reconstructed 3D human epidermal skin models are being used increasingly for safety testing of chemicals. Based on EpiDerm™ tissues, an assay was developed in which the tissues were topically exposed to test chemicals for 3h followed by cell isolation and assessment of DNA damage using the comet assay. Inter-laboratory reproducibility of the 3D skin comet assay was initially demonstrated using two model genotoxic carcinogens, methyl methane sulfonate (MMS) and 4-nitroquinoline-n-oxide, and the results showed good concordance among three different laboratories and with in vivo data. In Phase 2 of the project, intra- and inter-laboratory reproducibility was investigated with five coded compounds with different genotoxicity liability tested at three different laboratories. For the genotoxic carcinogens MMS and N-ethyl-N-nitrosourea, all laboratories reported a dose-related and statistically significant increase (P < 0.05) in DNA damage in every experiment. For the genotoxic carcinogen, 2,4-diaminotoluene, the overall result from all laboratories showed a smaller, but significant genotoxic response (P < 0.05). For cyclohexanone (CHN) (non-genotoxic in vitro and in vivo, and non-carcinogenic), an increase compared to the solvent control acetone was observed only in one laboratory. However, the response was not dose related and CHN was judged negative overall, as was p-nitrophenol (p-NP) (genotoxic in vitro but not in vivo and non-carcinogenic), which was the only compound showing clear cytotoxic effects. For p-NP, significant DNA damage generally occurred only at doses that were substantially cytotoxic (>30% cell loss), and the overall response was comparable in all laboratories despite some differences in doses tested. The results of the collaborative study for the coded compounds were generally reproducible among the laboratories involved and intra-laboratory reproducibility was also good. These data indicate that the comet assay in EpiDerm™ skin models is a promising model for the safety assessment of compounds with a dermal route of exposure.

Characterization of enzyme activities of Cytochrome P450 enzymes, Flavin-dependent monooxygenases, N-acetyltransferases and UDP-glucuronyltransferases in human reconstructed epidermis and full-thickness skin models

With the perspective to use human reconstructed skin models for genotoxicity testing which require metabolic activation of xenobiotics, this study aimed to characterize activities of biotransforming enzymes within two human reconstructed skin models, the epidermis model EpiDerm™ (MatTek) and the Phenion® Full-Thickness skin model Phenion®FT (Henkel). According to existing gene expression profiles, Cytochrome P450 (CYP) enzymes, Flavin-dependent monooxygenases (FMO), N-acetyltransferases (NAT) and UDP-glucuronyltransferases (UDP-GT) were investigated in S9 or microsomal fractions. CYP-catalyzed monooxygenation was assayed using 7-ethoxyresorufin, pentoxyresorufin and benzyloxyresorufin as substrates. FMO activity was tested using benzydamine. Conjugating activities of NAT and UDP-GT were determined by acetylation of p-aminobenzoic acid or glucuronation of 4-methylumbelliferone, respectively. Although CYPs were detected by expression profiling, no CYP activity was detected in either the epidermal nor the full-thickness reconstructed skin model while expression and activity of FMO, UDP-GT and NAT were demonstrated in both.

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.

Dermal Xenobiotic Metabolism: A Comparison between Native Human Skin, Four in vitro Skin Test Systems and a Liver System

The xenobiotic metabolism of 4 in vitro human skin test systems (2D and 3D) was compared with that of the native human skin samples from which the skin test systems had been produced. In total 3 skin samples were investigated, each from a different donor to exclude variability due to gender, donor or tissue supplier. In addition, the skin cultures were compared with a surrogate of the liver. Basal and induced phase I and phase II enzymes were analyzed regarding gene/protein expression as well as enzyme activity. The distinctions between the different test systems and the two dermal compartments (epidermis and dermis) were more noticeable than any donor variability. The 3D models of skin and liver mirrored the in vivo situation more realistically than did the monolayer cultures. Phase I metabolism was more pronounced in the hepatic model, whereas phase II metabolism was more prominent in the reconstructed skin. These results show that reconstructed skin models are a valuable tool for organ-specific safety assessment with regard to xenobiotic metabolism.

Eye irritation potential: Usefulness of the HET-CAM under the Globally Harmonized System of Classification and Labeling of Chemicals (GHS)

Extensive research has been conducted over the past decades to develop alternatives to the rabbit eye irritation test (Draize test) used in a regulatory context to assess eye irritation potentials. Although no single in vitro test has emerged as being completely acceptable for full replacement, various tests are considered to be suitable and are regularly used to assess certain aspects. Amongst these, the Hens Egg Test Chorioallantoic Membrane (HET-CAM) has gained regulatory acceptance in various countries to classify severe eye irritants. In this retrospective study, historical eye irritation data (in vivo and in vitro) from 137 samples (approx. 75% non-irritants; 25% (severe) irritants) tested both in the HET-CAM and Draize eye test was compared with regard to the predicted eye irritation classes under the GHS and the traditional EU classification system (DSD).The overall concordance was in the range of 80-90%. A high specificity (96-98%, depending on the classification system and the chosen discrimination) but rather low sensitivity (48-65%) was observed. The study indicates that HET-CAM results are useful as part of weight-of-evidence assessments or in tiered approaches to assess eye irritation potentials rather than as stand-alone classification method.