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Cosmetic ingredients must be toxicologically assessed to determine their skin sensitizing potential. The in vitro human cell line activation test (h-CLAT; OECD TG 442E) addresses the activation of dermal dendritic cells by analyzing specific protein expression after exposure of THP-1 cells to the test chemical. According to the protocol, FITC-labeled antibodies are used for protein detection. However, some chemicals show strong autofluorescence at FITC-specific wavelengths so that antibody-specific signals cannot be distinguished appropriately from autofluorescence background. This leads to inconclusive or false-negative predictions. Alternative fluorochromes can be used if their equivalence with the FITC-labeled antibodies is proven. In the current paper we describe the results of a proficiency exercise, based on the proficiency chemicals listed in the guideline, with FITC-labeled antibodies as the benchmark and APC-labeled anti­bodies as an alternative detection system. APC emits fluorescence at longer wavelengths, thus avoiding interference in the FITC spectrum. Irrespective of the employed fluorochrome, all chemicals were classified correctly, and the EC150 and 200 values were in the same order of magnitude. Hence, the equivalence in performance of FITC- and APC-labeled antibodies was demonstrated, and the respective demand of the guideline was fulfilled. In a case study, we then tested a proprietary oxidative hair dye using both fluorochromes. Using APC-labeled antibodies, the hair dye was unambiguously identified as a sensitizer, whereas no classification could be made with the FITC-labeled antibodies. With APC, fluorescence interference can be circumvented and the applicability domain of the h-CLAT extended to include autofluorescent chemicals.

All cosmetic products placed onto the market must undergo a risk assessment for human health to ensure they are safe for consumers, including an assessment of skin sensitisation risk. Historically, in vivo animal test methods were used to identify and characterise skin sensitisation hazard, however non-animal and other new approach methodologies (NAMs) are now the preferred and mandated choice for use in risk assessment for cosmetic ingredients. The experience gained over the last three decades on how to conduct risk assessments based upon NAMs has allowed us to develop a non-animal, next generation risk assessment (NGRA) framework for the assessment of skin sensitisers. The framework presented here is based upon the principles published by the International Cooperation on Cosmetic Regulation (ICCR) and is human relevant, exposure led, hypothesis driven and designed to prevent harm. It is structured in three tiers and integrates all relevant information using a weight of evidence (WoE) approach that can be iterated when new information becomes available. The initial tier (TIER 0) involves a thorough review of the existing information including; identification of the use scenario/consumer exposure; characterisation of the chemical purity and structure; in silico predictions; existing data pertaining to skin sensitisation hazard (historical or non-animal); the identification of suitable read-across candidates with supporting hazard identification/characterisation information and application of exposure-based waiving. Considering all information identified in TIER 0, the next step is the generation of a hypothesis (TIER 1). All data are considered in an exposure-led WoE approach, taking into account an initial view on whether a chemical is likely to be a skin sensitiser or not, choice of defined approach (DA) and availability of read-across candidates. If existing information is insufficient for concluding the risk assessment, the generation of additional information may be required to proceed (TIER 2). Such targeted testing could involve refinement of the exposure estimation or generation of data from in vitro or in chemico NAMs. Once sufficient information is available, the final stage of the NGRA framework is the determination of a point of departure (POD), characterising uncertainty and comparing to the consumer exposure in a WoE. Thorough evaluation of the sources of uncertainty is essential to ensure transparency and build trust in new risk assessment approaches. Although significant progress has been made, industry must continue to share its experience in skin sensitisation NGRA via case studies to demonstrate that this new risk assessment approach is protective for consumers. Dialogue and collaboration between key stakeholders, i.e. risk assessors, clinicians and regulators are important to gain mutual understanding and grow confidence in new approaches.

Cosmetics Europe, the European Trade Association for the cosmetics and personal care industry, is conducting a multi-phase program to develop regulatory accepted, animal-free testing strategies enabling the cosmetics industry to conduct safety assessments. Based on a systematic evaluation of test methods for skin sensitization, five non-animal test methods (DPRA (Direct Peptide Reactivity Assay), KeratinoSensTM, h-CLAT (human cell line activation test), U-SENSTM, SENS-IS) were selected for inclusion in a comprehensive database of 128 substances. Existing data were compiled and completed with newly generated data, the latter amounting to one-third of all data. The database was complemented with human and local lymph node assay (LLNA) reference data, physicochemical properties and use categories, and thoroughly curated. Focused on the availability of human data, the substance selection resulted nevertheless resulted in a high diversity of chemistries in terms of physico-chemical property ranges and use categories. Predictivities of skin sensitization potential and potency, where applicable, were calculated for the LLNA as compared to human data and for the individual test methods compared to both human and LLNA reference data. In addition, various aspects of applicability of the test methods were analyzed. Due to its high level of curation, comprehensiveness, and completeness, we propose our database as a point of reference for the evaluation and development of testing strategies, as done for example in the associated work of Kleinstreuer et al. We encourage the community to use it to meet the challenge of conducting skin sensitization safety assessment without generating new animal data.

The aim of the present ring trial was to test whether two new methodological approaches for the in vitro classification of eye irritating chemicals can be reliably transferred from the developers’ laboratories to other sites. Both test methods are based on the well-established open source reconstructed 3D hemicornea models. In the first approach, the initial depth of injury after chemical treatment in the hemicornea model is derived from the quantitative analysis of histological sections. In the second approach, tissue viability, as a measure for corneal damage after chemical treatment, is analyzed separately for epithelium and stroma of the hemicornea model. The three independent laboratories that participated in the ring trial produced their own hemicornea models according to the test producer’s instructions, thus supporting the open source concept. A total of 9 chemicals with different physicochemical and eye-irritating properties were tested to assess the between-laboratory reproducibility (BLR), the predictive performance, as well as possible limitations of the test systems. The BLR was 62.5% for the first and 100% for the second method. Both methods enabled to discriminate Cat. 1 chemicals from all non-Cat. 1 substances, which qualifies them to be used in a top-down approach. However, the selectivity between No Cat. and Cat. 2 chemicals still needs optimization.

Skin sensitization is a toxicity endpoint of widespread concern, for which the mechanistic understanding and concurrent necessity for non-animal testing approaches have evolved to a critical juncture, with many available options for predicting sensitization without using animals. Cosmetics Europe and the National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods collaborated to analyze the performance of multiple non-animal data integration approaches for the skin sensitization safety assessment of cosmetics ingredients. The Cosmetics Europe Skin Tolerance Task Force (STTF) collected and generated data on 128 substances in multiple in vitro and in chemico skin sensitization assays selected based on a systematic assessment by the STTF. These assays, together with certain in silico predictions, are key components of various non-animal testing strategies that have been submitted to the Organization for Economic Cooperation and Development as case studies for skin sensitization. Curated murine local lymph node assay (LLNA) and human skin sensitization data were used to evaluate the performance of six defined approaches, comprising eight non-animal testing strategies, for both hazard and potency characterization. Defined approaches examined included consensus methods, artificial neural networks, support vector machine models, Bayesian networks, and decision trees, most of which were reproduced using open source software tools. Multiple non-animal testing strategies incorporating in vitro, in chemico, and in silico inputs demonstrated equivalent or superior performance to the LLNA when compared to both animal and human data for skin sensitization.

In the two years since the last workshop report, the environment surrounding the prediction of skin sensitisation hazards has experienced major change. Validated non-animal tests are now OECD Test Guidelines. Accordingly, the recent cross sector workshop focused on how to use in vitro data for regulatory decision-making. After a review of general approaches and six case studies, there was broad consensus that a simple, transparent stepwise process involving non-animal methods was an opportunity waiting to be seized. There was also strong feeling the approach should not be so rigidly defined that assay variations/additional tests are locked out. Neither should it preclude more complex integrated approaches being used for other purposes, e.g. potency estimation. All agreed the ultimate goal is a high level of protection of human health. Thus, experience in the population will be the final arbiter of whether toxicological predictions are fit for purpose. Central to this is the reflection that none of the existing animal assays is perfect; the non-animal methods should not be expected to be so either, but by integrated use of methods and all other relevant information, including clinical feedback, we have the opportunity to continue to improve toxicology whilst avoiding animal use.

Significant progress has been made in the development and validation of non-animal test methods for skin sensitization assessment. At present, three of the four key events of the Adverse Outcome Pathway (AOP) are assessable by OECD-accepted in vitro methods. The fourth key event describes the immunological response in the draining lymph node where activated dendritic cells present major histocompatibility complex-bound chemically modified peptides to naive T cells, thereby priming the proliferation of antigen-specific T cells. Despite substantial efforts, modelling and assessing this adaptive immune response to sensitizers with in vitro T cell assays still represents a challenge. The Cosmetics Europe Skin Tolerance Task Force organized a workshop, bringing together academic researchers, method developers, industry representatives and regulatory stakeholders to review the scientific status of T cell-based assays, foster a mutual scientific understanding and conceive new options to assess T cell activation. Participants agreed that current T cell assays have come a long way in predicting immunogenicity, but that further investment and collaboration is required to simplify assays, optimize their sensitivity, better define human donor-to-donor variability and evaluate their value to predict sensitizer potency. Furthermore, the potential role of T cell assays in AOP-based testing strategies and subsequent safety assessment concepts for cosmetic ingredients was discussed. It was agreed that it is currently difficult to anticipate uses of T cell assay data for safety assessment and concluded that experience from case studies on real-life risk assessment scenarios is needed to further consider the usefulness of assessing the fourth AOP key event.

We have developed a new in vitro skin irritation test based on an open source reconstructed epidermis (OS-REp) with openly accessible protocols for tissue production and test performance. Due to structural, mechanistic and procedural similarity, a blinded catch-up validation study for skin irritation according to OECD Performance Standards (PS) was conducted in three laboratories to promote regulatory acceptance, with OS-REp models produced at a single production site only. While overall sensitivity and predictive capacity met the PS requirements, overall specificity was only 57%. A thorough analysis of the test results led to the assumption that some of the false-positive classifications could have been evoked by volatile skin-irritating chemicals tested in the same culture plate as the non-irritants falsely predicted as irritants. With GC/MS and biological approaches the cross-contamination effect was confirmed and the experimental set-up adapted accordingly. Retesting of the affected chemicals with the improved experimental set-up and otherwise identical protocol resulted in correct classifications as non-irritants. Taking these re-test results into account, 93% overall sensitivity, 70% specificity and 82% accuracy was achieved, which is in accordance with the OECD PS. A sufficient reliability of the method was indicated by a within-laboratory-reproducibility of 85–95% and a between-laboratory-reproducibility of 90%.

The process of skin aging is a combination of an extrinsic and intrinsic aspect, and knowing the molecular changes underlying both is a prerequisite to being able to effectively counter it. However, despite its importance for a deeper understanding of skin aging as a whole, the process of intrinsic skin aging in particular has barely been investigated. In this study, the molecular changes of intrinsic skin aging were analyzed by applying 'Serial Analysis of Gene Expression' (SAGE(TM)) to skin biopsies of young and aged donors. The analysis resulted in several hundred differentially expressed genes with varying statistical significance. Of these, several genes were identified that either have never been described in skin aging before (e.g. APP) or have no identified function, e.g. EST sequences. This is the first time that intrinsic skin aging has been analyzed in such a comprehensive manner, offering a new and partially unexpected set of target genes that have to be analyzed in more detail in terms of their contribution to the skin aging process.

In many cultures, a youthful look is strictly linked to strong and healthy hair. Source of the hair fibre is the hair follicle, a highly specialized skin appendage. Biological alterations because of intrinsic or extrinsic stimuli can destabilize this perfectly organized system, thus effecting hair growth or metabolism. Also, ageing could be characterized as a disturbance in this well-balanced machinery. Albeit the predominant symptom of hair ageing, greying, is addressed in a plurality of research activities, further age-related changes, e.g. related to hair structure, remain obscure. Therefore, we characterized hair follicles of two volunteer panels (below 25 years, above 50 years) on the molecular level, especially focussing on alterations influencing gene expression of keratins and keratin-associated proteins. We showed that concordantly to other biological systems the hair follicle undergoes several modifications during the ageing process associated among others with a significant decline in these structural proteins. Providing strategies to fight against these age-related changes is a challenge for hair science.

The alpha3Abeta1 integrin is a laminin receptor with a broad specificity for different laminin isoforms. Furthermore, it regulates the function of other integrins, like alpha2beta1, alpha5beta1 and alpha6Abeta1. In a yeast two hybrid screen of a human placenta cDNA library, we identified cDNAs coding for four different proteins that strongly interact with the conserved region of the cytoplasmic domain of the alpha3A integrin subunit. In addition to the cDNA for nucleotide exchange factor Mss4 and the putative tumour suppressor protein BIN1, two novel cDNAs were identified. Association analysis with different integrin subunits revealed them as cDNAs that encode binding proteins which react with a broad spectrum of alpha subunits. The conserved membrane proximal region of the alpha3A chain was identified as the binding site for all four proteins. They, therefore, may be involved in the regulation of general functions of integrins.

Huntington's disease (HD) is a neurodegenerative disorder caused by a (CAG)>37 repeat expansion in a novel gene of unknown function. Although the huntingtin gene is expressed in neuronal and non-neuronal tissues, the disease affects nerve cells of selected regional areas of the central nervous system. To gain insight into the regulation of the HD gene we analysed 1348 bp of the rat huntingtin promoter region. This region lacks a TATA and a CAAT box, is rich in GC content and has several consensus sequences for binding sites for SP1, PEA3, Sif and H2A. The stretch between nucleotides -56 and -206 relative to the first ATG is highly conserved between human and rodents and it harbours several potential binding sites for transcription factors. We analysed deletion mutants fused with the chloramphenicol acetyltransferase reporter gene in transfected, HD-expressing neuronal (NS20Y, NG108-15) and non-neuronal Chinese hamster ovary cell lines. Hence these cells should contain the required trans-acting factors necessary for HD gene expression. Partial deletion of the evolutionarily conserved part of the promoter significantly decreases the activity in both neuronal and non-neuronal cells, indicating that the core promoter activity is located between nucleotides -332 and -15. DNase I footprinting and electrophoretic mobility-shift assays were used to define the nucleotide positions and binding affinity of DNA–protein interactions.

Synapsin II is a neuron-specific phosphoprotein that selectively binds to small synaptic vesicles in the presynaptic nerve terminal. Here we report the cloning and sequencing of the 5′-flanking region of the human synapsin II gene. This sequence is very GC-rich and lacks a TATA or CAAT box. Two major transcriptional start sites were mapped. A hybrid gene consisting of the Escherichia coli chloramphenicol acetyltransferase gene under the control of 837 base pairs of the synapsin II 5′-upstream region was transfected into neuronal and non-neuronal cells. While reporter gene expression was low in neuroblastoma and non-neuronal cells, high chloramphenicol acetyltransferase activities were monitored in PC12 pheochromocytoma cells. However, there was no correlation between reporter gene expression in the transfected cells and endogenous synapsin II immunoreactivity. Using DNA-protein binding assays we showed that the transcription factors zif268/egr-1, polyoma enhancer activator 3 (PEA3), and AP2 specifically contact the synapsin II promoter DNA in vitro. Moreover, the zif268/egr-1 protein as well as PEA3 were shown to stimulate transcription of a reporter gene containing synapsin II promoter sequences. In the nervous system, zif268/egr-1 functions as a “third messenger” with a potential role in synaptic plasticity. PEA3 is expressed in the brain and its activity is regulated by proteins encoded from non-nuclear oncogenes. We postulate that zif268/egr-1 and PEA3 couple extracellular signals to long-term responses by regulating synapsin II gene expression.

In many cultures, a youthful look is strictly linked to strong and healthy hair. Source of the hair fibre is the hair follicle, a highly specialized skin appendage. Biological alterations because of intrinsic or extrinsic stimuli can destabilize this perfectly organized system, thus effecting hair growth or metabolism. Also, ageing could be characterized as a disturbance in this well-balanced machinery. Albeit the predominant symptom of hair ageing, greying, is addressed in a plurality of research activities, further age-related changes, e.g. related to hair structure, remain obscure. Therefore, we characterized hair follicles of two volunteer panels (below 25 years, above 50 years) on the molecular level, especially focussing on alterations influencing gene expression of keratins and keratin-associated proteins. We showed that concordantly to other biological systems the hair follicle undergoes several modifications during the ageing process associated among others with a significant decline in these structural proteins. Providing strategies to fight against these age-related changes is a challenge for hair science.

Skin inflammation is an often-occurring phenomenon in a wide range of skin pathologies. In order to understand the basics of the underlying molecular mechanisms, early responses such as gene expression changes in this tissue are the focus of numerous studies. The prerequisite for gene expression analysis is the isolation of relevant tissue samples and the subsequent preparation of high quality RNA.

IFSCC Magazine, 11 (2008) (1) 37–42 Ubiquinones are the most widespread and therefore best investigated bioquinones. Due to their hydrophobic isoprenoid side chain, ubiquinones can be solubilized in organic solvents or lipids but are insoluble in water. Using a specific emulsifier system it has been possible to deliver positive effects to biological systems also from aqueous formulations. Ubiquinone-50, also referred to as coenzyme Q10, is well known in cosmetic science and especially in skin care because of its antioxidant activity. It is found in the membranes of peroxisomes, lysosomes, vesicles, the endoplasmic reticulum and notably in the inner membrane of the mitochondrion, where it is an important part of the electron transport chain. Using coenzyme Q10 in the proposed emulsifier system we could show that the molecule not only has relevance as an anti-aging bioactive in skin care but also has positive effects on the human hair follicle. The hair follicle is a complex mini organ and synthesis of hair keratin, the major component of hair fibers, is an essential prerequisite for the growth of strong and healthy hair. But like all biological systems the hair follicle, the biologically active part of the hair, also undergoes an aging process associated among other things with a decline in certain hair keratins. Due to this age-related shift in basic structural proteins of the hair shaft, mature hair often becomes fragile and difficult to manage. Therefore it is a challenge for cosmetic science to provide bioactives to fight age-related changes and maintain a youthful appearance of hair. Using cultivated hair follicle keratinocytes we identified coenzyme Q10 as a potent bioactive that stimulates the gene expression of different hair keratins, especially those which are reduced during aging processes in hair follicles. These results led us to investigate a shampoo and a tonic formulation enriched with coenzyme Q10 in a placebo-controlled panel study. In a left/right comparison a group of healthy volunteers older than 40 years of age applied the formulations daily for 4 days. Throughout the test period the gene expression of different hair keratins from plucked hair follicles was determined using quantitative polymerase chain reaction techniques. Subsequent statistical analysis revealed an increase in age-relevant hair keratins in human hair roots treated with coenzyme Q10, thus pointing out the striking benefits of coenzyme Q10 in hair care formulations. We conclude that coenzyme Q10 is an ideal ingredient for hair care formulations, providing anti-aging properties through activation of specific keratins aligned with the needs of mature hair.

Using immunoprecipitation and phosphotyrosine detection by Western blotting, intracellular signaling intermediates were analyzed in human primary dermal fibroblasts, either seeded as monolayers on collagen I coats (2D) or seeded within three-dimensional collagen I lattices (3D). Previous results demonstrated that integrin activation in these systems resulted in a cascade of protein tyrosine phosphorylation, including focal adhesion kinase (D. Roeckel and T. Krieg, 1994, Exp. Cell Res. 211, 42-48). Further downstream signaling events are now shown to include coordinate activation of ERK1 and ERK2 at 2 h after cell-collagen contact, irrespective of 2D or 3D culture conditions. Applying U-73122, an inhibitor of PLC, inhibits collagen lattice contraction in a dose-dependent fashion. Immunoprecipitation identified the isoform PLCgamma-1 as playing a role as signaling intermediate in fibroblast-collagen interactions. PLCgamma-1 becomes phosphorylated within 10 min after culture initiation and declines after 2 h. So far, no qualitative differences in signaling intermediates between 2D and 3D cultures have been identified.

Synaptobrevin II is a small integral membrane protein of synaptic vesicles that plays a key role in exocytosis. The 5'-flanking region of the human synaptobrevin II gene is very (G+C)-rich and contains a 13-bp motif that includes overlapping binding sites for the zinc finger transcription factors Sp1 and zif268/egr-1. To test whether Sp1 and zif268/egr-1 interact with this motif, gel retardation assays were performed. These assays revealed that both transcription factors bind to the (G+C)-rich motif of the synaptobrevin II gene in vitro. The binding of Sp1 was additionally confirmed by supershift analysis with antibodies specific for Sp1. To determine whether zif268/egr-1 plays a role in controlling synaptobrevin II gene expression, a plasmid was constructed containing the (G+C)-rich motif of the synaptobrevin II gene upstream of a minimal promoter and the Escherichia coli chloramphenicol acetyltransferase (CAT) gene as a reporter. This plasmid was transfected into CHO-K1 cells together with an expression vector encoding zif268/egr-1. Zif268/egr-1 failed to activate transcription from this reporter gene, although it transactivated a reporter gene containing an identical (G+C)-rich motif derived from the human synapsin I promoter. Overexpression of Sp1, however, clearly activated transcription of a reporter gene under the control of the synaptobrevin II promoter (G+C)-rich sequence in Drosophila SL2 cells, which provided an Sp1-deficient background. Furthermore, a glutathione S-transferase protein containing the DNA-binding domain of Sp1 was shown to function as a dominant negative form of Sp1, reducing transcription of the synaptobrevin II promoter-CAT reporter gene in mammalian cells to basal levels. From these data, we conclude that the zif268/egr-1-binding site in the synaptobrevin II promoter is not functionally active. Instead, an overlapping Sp1-binding site in this (G+C)-rich region clearly mediates constitutive transcriptional activation.

We have constructed a vector, pHIVTATA-CAT, that contains the Escherichia coli cat gene, encoding chloramphenicol acetyltransferase, under the control of a minimal promoter consisting of the HIV TATA box and the adenovirus major late promoter initiator element. Putative transcriptional elements can be inserted either directly upstream from the TATA box or downstream from the reporter gene in an enhancer position. Transcription can be monitored enzymatically or by RNase protection mapping. An analysis of mRNAs' generated from pHIVTATA-CAT constructs revealed that transcription starts at the transcription start point and no read-through transcripts are generated.

zif268/egr-1 is an immediate early response gene that is involved in regulation of growth and differentiation. Its mRNA encodes a sequence-specific transcriptional activator containing three zinc fingers that act as the DNA-binding domain. Although zif268/egr-1 is expressed in the nervous system during neuronal excitation, no target gene has yet been identified. Here we report that the zif268/egr-1 protein bound in vitro to two sites in the proximal regulatory region of the human synapsin I gene. The zif268/egr-1 protein was also shown to stimulate transcription from this control region in transactivation assays. Additionally, the presence of a putative neural-restrictive silencer element next to one of the zif268/egr-1-binding sites interfered with transactivation in a tissue-independent manner. An analysis of the temporal expression pattern of zif268/egr-1 and synapsin I during neuronal differentiation of P19 embryonal carcinoma cells revealed that zif268/egr-1 mRNA was induced on day 5 and synapsin I mRNA on day 8 after retinoic acid treatment. From this data we conclude that the synapsin I gene is a target of the zif268 transcription factor; however, intermediate factors may also be involved in the activation.

Plant secondary metabolites such as flavonoids, isoflavones and phytosterols have been proposed as cosmetic ingredients displaying anti-aging effects. On the cellular level, however, the activity profiles of these ingredients are only partially understood. In this study we analyzed the effects of apple seed phytosterols on age-related structural and functional parameters using cell biochemical, molecular biological and bioengineering techniques. The expression of age-related genes was studied using skin equivalents and cDNA microarrays. Incubation of skin equivalents with apple seed phytosterols had significant consequences: (i) differential regulation of a set of genes associated with keratinocyte proliferation and differentiation, (ii) stimulation of hyaluronic acid synthesis, and (iii) increase of epidermal thickness. In vivo studies revealed that apple seed phytosterols improve skin elasticity and decrease skin roughness. In conclusion, apple seed phytosterols display distinct biological effects and significantly improve the structure and function of mature skin.

The need for non-animal data to assess skin sensitisation properties of substances, especially cosmetics ingredients, has spawned the development of many in vitro methods. As it is widely believed that no single method can provide a solution, the Cosmetics Europe Skin Tolerance Task Force has defined a three-phase framework for the development of a non-animal testing strategy for skin sensitisation potency prediction. The results of the first phase – systematic evaluation of 16 test methods – are presented here. This evaluation involved generation of data on a common set of ten substances in all methods and systematic collation of information including the level of standardisation, existing test data, potential for throughput, transferability and accessibility in cooperation with the test method developers. A workshop was held with the test method developers to review the outcome of this evaluation and to discuss the results. The evaluation informed the prioritisation of test methods for the next phase of the non-animal testing strategy development framework. Ultimately, the testing strategy – combined with bioavailability and skin metabolism data and exposure consideration – is envisaged to allow establishment of a data integration approach for skin sensitisation safety assessment of cosmetic ingredients.

Integrated approaches using different in vitro methods in combination with bioinformatics can (i) increase the success rate and speed of drug development; (ii) improve the accuracy of toxicological risk assessment; and (iii) increase our understanding of disease. Three-dimensional (3D) cell culture models are important building blocks of this strategy which has emerged during the last years. The majority of these models are organotypic, i.e., they aim to reproduce major functions of an organ or organ system. This implies in many cases that more than one cell type forms the 3D structure, and often matrix elements play an important role. This review summarizes the state of the art concerning commonalities of the different models. For instance, the theory of mass transport/metabolite exchange in 3D systems and the special analytical requirements for test endpoints in organotypic cultures are discussed in detail. In the next part, 3D model systems for selected organs – liver, lung, skin, brain – are presented and characterized in dedicated chapters. Also, 3D approaches to the modeling of tumors are presented and discussed. All chapters give a historical background, illustrate the large variety of approaches, and highlight up- and downsides as well as specific requirements. Moreover, they refer to the application in disease modeling, drug discovery and safety assessment. Finally, consensus recommendations indicate a roadmap for the successful implementation of 3D models in routine screening. It is expected that the use of such models will accelerate progress by reducing error rates and wrong predictions from compound testing.