Prediction of liver toxicity and mode of action using metabolomics in vitro in HepG2 cells.

Liver toxicity is a leading systemic toxicity of drugs and chemicals demanding more human-relevant, high throughput, cost effective in vitro solutions. In addition to contributing to animal welfare, in vitro techniques facilitate exploring and understanding the molecular mechanisms underlying toxicity. New 'omics technologies can provide comprehensive information on the toxicological mode of action of compounds, as well as quantitative information about the multi-parametric metabolic response of cellular systems in normal and patho-physiological conditions. Here, we combined mass-spectroscopy metabolomics with an in vitro liver toxicity model. Metabolite profiles of HepG2 cells treated with 35 test substances resulted in 1114 cell supernatants and 3556 intracellular samples analyzed by metabolomics. Control samples showed relative standard deviations of about 10-15%, while the technical replicates were at 5-10%. Importantly, this procedure revealed concentration-response effects and patterns of metabolome changes that are consistent for different liver toxicity mechanisms (liver enzyme induction/inhibition, liver toxicity and peroxisome proliferation). Our findings provide evidence that identifying organ toxicity can be achieved in a robust, reliable, human-relevant system, representing a non-animal alternative for systemic toxicology.

Knowledge sharing to facilitate regulatory decision-making in regard to alternatives to animal testing: Report of an EPAA workshop.

The European Partnership for Alternative Approaches to Animal Testing (EPAA) convened a workshop Knowledge sharing to facilitate regulatory decision-making. Fifty invited participants from the European Commission, national and European agencies and bodies, different industry sectors (chemicals, cosmetics, fragrances, pharmaceuticals, vaccines), and animal protection organizations attended the workshop. Four case studies exemplarily revealed which procedures are in place to obtain regulatory acceptance of new test methods in different sectors. Breakout groups discussed the status quo identifying the following facilitators for regulatory acceptance of alternatives to animal testing: Networking and communication (including cross-sector collaboration, international cooperation and harmonization); involvement of regulatory agencies from the initial stages of test method development on; certainty on prerequisites for test method acceptance including the establishment of specific criteria for regulatory acceptance. Data sharing and intellectual property issues affect many aspects of test method development, validation and regulatory acceptance. In principle, all activities should address replacement, reduction and refinement methods (albeit animal testing is generally prohibited in the cosmetics sector). Provision of financial resources and education support all activities aiming at facilitating the acceptance and use of alternatives to animal testing. Overall, workshop participants recommended building confidence in new methodologies by applying and gaining experience with them.

Assessing skin sensitization hazard in mice and men using non-animal test methods.

Sensitization, the prerequisite event in the development of allergic contact dermatitis, is a key parameter in both hazard and risk assessments. The pathways involved have recently been formally described in the OECD adverse outcome pathway (AOP) for skin sensitization. One single non-animal test method will not be sufficient to fully address this AOP and in many cases the use of a battery of tests will be necessary. A number of methods are now fully developed and validated. In order to facilitate acceptance of these methods by both the regulatory and scientific communities, results of the single test methods (DPRA, KeratinoSens, LuSens, h-CLAT, (m)MUSST) as well for a the simple '2 out of 3' ITS for 213 substances have been compiled and qualitatively compared to both animal and human data. The dataset was also used to define different mechanistic domains by probable protein-binding mechanisms. In general, the non-animal test methods exhibited good predictivities when compared to local lymph node assay (LLNA) data and even better predictivities when compared to human data. The '2 out of 3' prediction model achieved accuracies of 90% or 79% when compared to human or LLNA data, respectively and thereby even slightly exceeded that of the LLNA.

Mammalian erythroblast enucleation requires PI3K-dependent cell polarization.

Enucleation, the final step in terminal differentiation of mammalian red blood cells, is an essential process in which the nucleus surrounded by the plasma membrane is budded off from the erythroblast to form a reticulocyte. Most molecular events in enucleation remain unclear. Here we show that enucleation requires establishment of cell polarization that is regulated by the microtubule-dependent local activation of phosphoinositide 3-kinase (PI3K). When the nucleus becomes displaced to one side of the cell, actin becomes restricted to the other side, where dynamic cytoplasmic contractions generate pressure that pushes the viscoelastic nucleus through a narrow constriction in the cell surface, forming a bud. The PI3K products PtdIns(3,4)P2 and PtdIns(3,4,5)P3 are highly localized at the cytoplasmic side of the plasma membrane. PI3K inhibition caused impaired cell polarization, leading to a severe delay in enucleation. Depolymerization of microtubules reduced PI3K activity, resulting in impaired cell polarization and enucleation. We propose that enucleation is regulated by microtubules and PI3K signaling in a manner mechanistically similar to directed cell locomotion.

Effect of estrogenic binary mixtures in the yeast estrogen screen (YES).

Endocrine disrupting compounds (EDCs) of natural or synthetic origin can interfere with the balance of the hormonal system, either by altering hormone production, secretion, transport, or their binding and consequently lead to an adverse outcome in intact animals. An important aspect is the prediction of effects of combined exposure to two or more EDCs at the same time. The yeast estrogen assay (YES) is a broadly used method to assess estrogenic potential of chemicals. Besides exhibiting good predictivity to identify compounds which interfere with the estrogen receptor, it is easy to handle, rapid and therefore allows screening of a large number of single compounds and varying mixtures. Herein, we applied the YES assay to determine the potential combination effects of binary mixtures of two estrogenic compounds, bisphenol A and genistein, as well as one classical androgen that in vitro also exhibits estrogenic activity, trenbolone. In addition to generating data from combined exposure, we fitted these to a four-parametric logistic dose-response model. As all compounds tested share the same mode of action dose additivity was expected. To assess this, the Loewe model was utilized. Deviations between the Loewe additivity model and the observed responses were always small and global tests based on the whole dose-response data set indicated in general a good fit of the Loewe additivity model. At low concentrations concentration additivity was observed, while at high concentrations, the observed effect was lower than additivity, most likely reflecting receptor saturation. In conclusion, our results suggest that binary combinations of genistein, bisphenol A and trenbolone in the YES assay do not deviate from expected additivity.

Non-animal test methods for predicting skin sensitization potentials.

Contact allergies are complex diseases, and it is estimated that 15-20 % of the general population suffers from contact allergy, with increasing prevalence. Evaluation of the sensitization potential of a substance is usually carried out in animal models. Nowadays, there is much interest in reducing and ultimately replacing current animal tests. Furthermore, as of 2013, the EU has posed a ban on animal testing of cosmetic ingredients that includes skin sensitization. Therefore, predictive and robust in vitro tests are urgently needed. In order to establish alternatives to animal testing, the in vitro tests must mimic the very complex interactions between the sensitizing chemical and the different parts of the immune system. This review article summarizes recent efforts to develop in vitro tests for predicting skin sensitizers. Cell-based assays, in chemico methods and, to a lesser extent, in silico methods are presented together with a discussion of their current status. With considerable progress having been achieved during the last years, the rationale today is that data from different non-animal test methods will have to be combined in order to obtain reliable hazard and potency information on potential skin sensitizers.

Putting the parts together: combining in vitro methods to test for skin sensitizing potentials.

Allergic contact dermatitis is a common skin disease and is elicited by repeated skin contact with an allergen. In the regulatory context, currently only data from animal experiments are acceptable to assess the skin sensitizing potential of substances. Animal welfare and EU Cosmetic Directive/Regulation call for the implementation of animal-free alternatives for safety assessments. The mechanisms that trigger skin sensitization are complex and various steps are involved. Therefore, a single in vitro method may not be able to accurately assess this endpoint. Non-animal methods are being developed and validated and can be used for testing strategies that ensure a reliable prediction of skin sensitization potentials. In this study, the predictivities of four in vitro assays, one in chemico and one in silico method addressing three different steps in the development of skin sensitization were assessed using 54 test substances of known sensitizing potential. The predictivity of single tests and combinations of these assays were compared. These data were used to develop an in vitro testing scheme and prediction model for the detection of skin sensitizers based on protein reactivity, activation of the Keap-1/Nrf2 signaling pathway and dendritic cell activation.

A testing strategy for the identification of mammalian, systemic endocrine disruptors with particular focus on steroids.

Most endocrine disruptors interact with hormone receptors or steroid biosynthesis and metabolism, thereby modifying the physiological function of endogenous hormones. Here, we present an alternative testing paradigm for detection of endocrine modes of action that replace and reduce animal testing through refinement. Receptor mediated endocrine effects were assessed using the yeast-based receptor-mediated transcriptional activation YES/YAS assays and effects on steroid hormone biosynthesis were assessed using the human cell line H295R in the steroidogenesis assay. In our testing paradigm we propose to complement the in vitro assays with a single in vivo repeated dose study in which plasma samples are analyzed for their metabolome profile in addition to classical parameters such as histopathology. The combination of these methods does not only contribute to refinement and reduction of animal testing, but also has significantly increased the efficient allocation of resources and allows for a sound assessment of the endocrine disruption potential of compounds. Thus, this proposal constitutes a potentially attractive alternative to EPA's Endocrine Disruptor Screening Program to identify mammalian, systemic endocrine modes of action. Data on 14 reference substances for which the in vitro YES/YAS and steroidogenesis assays and the in vivo metabolome analysis were performed to assess their putative endocrine modes of action are presented here.

Histone deacetylase 2 is required for chromatin condensation and subsequent enucleation of cultured mouse fetal erythroblasts.

BACKGROUND: During the final stages of differentiation of mammalian erythroid cells, the chromatin is condensed and enucleated. We previously reported that Rac GTPases and their downstream target, mammalian homolog of Drosophila diaphanous 2 (mDia2), are required for enucleation of in vitro cultured mouse fetal liver erythroblasts. However, it is not clear how chromatin condensation is achieved and whether it is required for enucleation. DESIGN AND METHODS: Mouse fetal liver erythroblasts were purified from embryonic day 14.5 pregnant mice and cultured in erythropoietin-containing medium. Enucleation was determined by flow-cytometry based analysis after treatment with histone deacetylase inhibitors or infection with lentiviral short hairpin RNA. RESULTS: We showed that histone deacetylases play critical roles in chromatin condensation and enucleation in cultured mouse fetal liver erythroblasts. Enzymatic inhibition of histone deacetylases by trichostatin A or valproic acid prior to the start of enucleation blocked chromatin condensation, contractile actin ring formation and enucleation. We further demonstrated that histone deacetylases 1, 2, 3 and 5 are highly expressed in mouse fetal erythroblasts. Short hairpin RNA down-regulation of histone deacetylase 2, but not of the other histone deacetylases, phenotypically mimicked the effect of trichostatin A or valproic acid treatment, causing significant inhibition of chromatin condensation and enucleation. Importantly, knock-down of histone deacetylase 2 did not affect erythroblast proliferation, differentiation, or apoptosis. CONCLUSIONS: These results identify histone deacetylase 2 as an important regulator, mediating chromatin condensation and enucleation in the final stages of mammalian erythropoiesis.

S-adenosyl-L-methionine counteracts mitotic disturbances and cytostatic effects induced by sodium arsenite in HeLa cells.

Aneuploidy represents a serious problem for human health. Toxicological data have shown that aneuploidy can be caused by exposure to chemical agents known as mitotic spindle poisons, since they arrest cell cycle in mitosis through their interaction with tubulin. Among these agents is arsenic. In previous reports, we demonstrated that the aneugenic events induced by sodium arsenite can be abolished by the exogenous addition of S-adenosyl-l-methionine (SAM). Nevertheless, the mechanisms involved are still unknown. The aim of the present work was to study the influence of SAM on the mitotic disturbances caused by sodium arsenite. To achieve this goal, we analyzed microtubule (MT) polymerization by immunolocalization and live cell microscopy of mitotic cells. Our findings indicate that sodium arsenite alters the dynamics of MT polymerization, induces centrosome amplification and delays mitosis. Furthermore, SAM reduces the alterations on MT dynamics, as well as centrosome amplification, and therefore diminishes the formation of multipolar spindles in treated HeLa cells. In addition, SAM decreases the progression time through mitosis. Taking these data together, we consider that the mechanism by which SAM reduces the frequency of aneuploid cells must be related to the modulation of the dynamics and organization of MT, suggesting a role of SAM on chromosome segregation, which should be further investigated in primary cells.

Prevention of aneuploidy by S-adenosyl-methionine in human cells treated with sodium arsenite.

Alterations in methyl group's metabolism affect availability of S-adenosyl-L-methionine (SAM); these modifications can be originated by enzyme polymorphisms, nutritional deficiencies, and exposure to chemical agents. There are several types of chemicals that interfere with methyl groups, among them is arsenic. It deserves special attention because it modifies a number of cell functions that influence the development of diseases such as cancer. Since part of arsenic's toxicity is influenced by changes on SAM availability, in a previous study we investigated whether exogenous addition of SAM to cells treated with sodium arsenite (NaAsO(2)) has an effect on its genotoxicity. Results demonstrated that SAM reduces the frequency of cells presenting micronuclei (MN) and tubulin-cytoskeleton defects after treatment with NaAsO(2). MN are fragments of the cell nucleus that may contain whole chromosomes or chromosome fragments depending on whether they derive either from the aneugenic or from the clastogenic action of chemicals. Therefore one question generated by these results was whether SAM reduced only the frequency MN resulting from aneugenic damage. To answer this question, in the present work we used an all-centromere DNA probe to distinguish the type of MN reduced by SAM after treatment with NaAsO(2) and vinblastine. In addition, the capacity of SAM to reduce clastogenicity was also evaluated. Results show that SAM decreases the frequency of cells with MN containing whole chromosomes in cultures treated either with NaAsO(2) or with vinblastine; however, induction of double-strand breaks by NaAsO(2) was not prevented by SAM.

Non-disjunction events induced by albendazole in human cells.

Albendazole (ABZ), a benzimidazole carbamate used for the treatment of several human helminthiases has high affinity for tubulin, which results in an inhibition of microtubule polymerization, blocking several vital processes in the parasites, such as motility and nutrient uptake. The ability of ABZ to act as mitotic spindle poison leads to a potential risk for aneuploidy induction in exposed human beings. ABZ, as well as albendazole sulphoxide (ABZSO), its main metabolite, induce micronuclei in human cells in a dose-dependent manner. Despite recognition that ABZ and ABZSO increase micronucleus frequency, their potential as inducers of non-disjunction in human cells, an event considered more frequent than chromosome loss, and one of the main mechanisms involved in aneuploidy induction, has not been evaluated. In the present work, we investigated the ability of ABZ and ABZSO to induce non-disjunction in cultured human lymphocytes. Non-disjunction was scored by chromosome-specific FISH using a classical or alpha satellite probe for chromosomes 1 and 7, respectively. Significant increase in non-disjunction events that involved either chromosome were observed in cells treated with ABZ or ABZSO. Both ABZ and ABZSO induced non-disjunction at lower concentrations than those at which MN were observed.

A multi-laboratory evaluation of microelectrode array-based measurements of neural network activity for acute neurotoxicity testing.

There is a need for methods to screen and prioritize chemicals for potential hazard, including neurotoxicity. Microelectrode array (MEA) systems enable simultaneous extracellular recordings from multiple sites in neural networks in real time and thereby provide a robust measure of network activity. In this study, spontaneous activity measurements from primary neuronal cultures treated with three neurotoxic or three non-neurotoxic compounds was evaluated across four different laboratories. All four individual laboratories correctly identifed the neurotoxic compounds chlorpyrifos oxon (an organophosphate insecticide), deltamethrin (a pyrethroid insecticide) and domoic acid (an excitotoxicant). By contrast, the other three compounds (glyphosate, dimethyl phthalate and acetaminophen) considered to be non-neurotoxic ("negative controls"), produced only sporadic changes of the measured parameters. The results were consistent across the different laboratories, as all three neurotoxic compounds caused concentration-dependent inhibition of mean firing rate (MFR). Further, MFR appeared to be the most sensitive parameter for effects of neurotoxic compounds, as changes in electrical activity measured by mean frequency intra burst (MFIB), and mean burst duration (MBD) did not result in concentration-response relationships for some of the positive compounds, or required higher concentrations for an effect to be observed. However, greater numbers of compounds need to be tested to confirm this. The results obtained indicate that measurement of spontaneous electrical activity using MEAs provides a robust assessment of compound effects on neural network function.

Intra- and inter-laboratory reproducibility and accuracy of the LuSens assay: A reporter gene-cell line to detect keratinocyte activation by skin sensitizers.

Several non-animal methods are now available to address the key events leading to skin sensitization as defined by the adverse outcome pathway. The KeratinoSens assay addresses the cellular event of keratinocyte activation and is a method accepted under OECD TG 442D. In this study, the results of an inter-laboratory evaluation of the "me-too" LuSens assay, a bioassay that uses a human keratinocyte cell line harboring a reporter gene construct composed of the rat antioxidant response element (ARE) of the NADPH:quinone oxidoreductase 1 gene and the luciferase gene, are described. Earlier in-house validation with 74 substances showed an accuracy of 82% in comparison to human data. When used in a battery of non-animal methods, even higher predictivity is achieved. To meet European validation criteria, a multicenter study was conducted in 5 laboratories. The study was divided into two phases, to assess 1) transferability of the method, and 2) reproducibility and accuracy. Phase I was performed by testing 8 non-coded test substances; the results showed a good transferability to naïve laboratories even without on-site training. Phase II was performed with 20 coded test substances (performance standards recommended by OECD, 2015). In this phase, the intra- and inter-laboratory reproducibility as well as accuracy of the method was evaluated. The data demonstrate a remarkable reproducibility of 100% and an accuracy of over 80% in identifying skin sensitizers, indicating a good concordance with in vivo data. These results demonstrate good transferability, reliability and accuracy of the method thereby achieving the standards necessary for use in a regulatory setting to detect skin sensitizers.

Combination treatment of glioblastoma multiforme cell lines with the anti-malarial artesunate and the epidermal growth factor receptor tyrosine kinase inhibitor OSI-774.

New drugs and combination modalities for otherwise non-responsive brain tumors are urgently required. The anti-malarial artesunate (ART) and the EGFR tyrosine kinase inhibitor OSI-774 reveal profound cytotoxic activity. The effectiveness of a combination treatment and the underlying molecular determinants of cellular response are unknown. In the present investigation, we studied ART and OSI-774 in glioblastoma multiforme (GBM) cell lines. Supra-additive inhibition of cell growth was observed in U-87MG.DeltaEGFR cells transduced with a deletion-mutant constitutively active EGFR gene, while additive effects were present in cells transduced with wild-type EGFR (U-87MG.WT-2N), kinase-deficient EGFR (U-87MG.DK-2N), mock vector controls (U-87MG.LUX), or non-transduced parental U-87MG cells. Among nine other non-transduced GBM cell lines, supra-additive effects were found in two cell lines (G-210GM, G-599GM), while ART and OSI-774 acted in an additive manner in the other seven cell lines (G-211GM, G-750GM, G-1163GM, G-1187GM, G-1265GM, G-1301GM, and G-1408GM). Sub-additive or antagonistic effects were not observed. Genomic gains and losses of genetic material in the non-transduced cell lines as assessed by comparative genomic hybridization were correlated with the IC(50) values for ART and OSI-774 and subsequently subjected to hierarchical cluster analysis and cluster image mapping. A genomic profile of imbalances was detected that predicted cellular response to ART and OSI-774. The genes located at the genomic imbalances of interest may serve as candidate resistance genes of GBM cells towards ART and OSI-774. In conclusion, the combination treatment of ART and OSI-774 resulted in an increased growth inhibition of GBM cell lines as compared to each drug alone.

Specific patterns of DNA copy number gains and losses in eight new glioblastoma multiforme cell lines.

Eight cell lines newly established from glioblastoma multiforme were examined by comparative genomic hybridization for their patterns of genomic imbalance. The total number of DNA copy number alterations (CNAs) found in the eight cell lines varied between 15 and 24. This characterized the examined cell lines (or the tumors they were derived from) as distinctly progressed in karyotypic evolution. The most frequent CNAs were gains of the entire chromosome 6 or, at least, parts of it, and of 7p22, which were found in all eight cell lines. Other changes present in seven of the eight cell lines were gains of 3q26qter and the entire chromosome 7 and losses of segments on chromosome 4q (e.g., 4q34q35) and of the short arm of chromosome 10. Enh(3q21q25), dim(4q22q33) and dim(4qter), dim(13q22), enh(15q14), and enh(18q22q23) were found in six of the eight cell lines. Several other CNAs [e.g., dim(9p21)] were found in common in five or less of the eight lines. Using a hierarchical cluster analysis, the specific patterns of genomic imbalance allowed various groupings of the examined cell lines. Although a close relation could be confirmed among all examined lines on the basis of shared CNAs, two main groups could be roughly differentiated. Among those there were also more or less closely related subgroups. However, also alterations which were restricted to one single cell line each were found, e.g., dim(1q41qter), dim(2q22qter), enh(4p), dim(5p), dim(4p13pter), dim(8q21qter), enh(9p), dim(9q), dim(11p14pter), enh(12q15q23), enh(13q21), dim(14q21qter), dim(15q21qter), dim(19q), and enh(22q). The comparison of the obtained data on gains and losses of DNA copy numbers in specific chromosomal segments with the data on localization of genes possibly associated with the biology of glioblastoma multiforme additionally shows high conformity but also disparity of the examined cell lines among each other, as well as compared to primary glioblastoma multiforme. Eventually, each of the cell lines could be characterized by its specific pattern of genomic imbalance.

S-adenosyl-L-methionine is able to reverse micronucleus formation induced by sodium arsenite and other cytoskeleton disrupting agents in cultured human cells.

Deficiencies of folic acid and methionine, two of the major components of the methyl metabolism, correlate with an increment of chromosome breaks and micronuclei. It has been proposed that these effects may arise from a decrease of S-adenosyl-L-methionine (SAM), the universal methyl donor. Some xenobiotics, such as arsenic, originate a reduction of SAM levels, and this is believed to alter some methylation processes (e.g. DNA methylation). The aim of the present work was to analyze the effects of exogenous SAM on the micronucleus (MN) frequency induced by sodium arsenite in human lymphocytes treated in vitro and to investigate whether these effects are related to DNA methylation. Results showed a reduction in the MN frequency in cultures treated with sodium arsenite and SAM compared to those treated with arsenite alone. To understand the mechanism by which SAM reduced the number of micronucleated cells, its effect on MN induced by other xenobiotics was also analyzed. Results showed that SAM did not have any effect on the increase in MN frequency caused by alkylating (mitomycin C or cisplatin) or demethylating agents (5-azacytidine, hydralazine, ethionine and procainamide), but it reduced the number of micronucleated cells in those treated with agents that inhibit microtubule polymerization (albendazole sulphoxide and colcemid). Since albendazole sulphoxide and colcemid inhibit microtubule polymerization, we decided to evaluate the effect of SAM on microtubule integrity. Data obtained from these evaluations showed that sodium arsenite, albendazole sulphoxide, and colcemid affect the integrity and organization of microtubules and that these effects are significantly reduced when cultures were treated at the same time with SAM. The data taken all together point out that the positive effects of SAM could be due to its ability to protect microtubules through an unknown mechanism.

LuSens: a keratinocyte based ARE reporter gene assay for use in integrated testing strategies for skin sensitization hazard identification.

Allergic contact dermatitis can develop following repeated exposure to allergenic substances. To date, hazard identification is still based on animal studies as non-animal alternatives have not yet gained global regulatory acceptance. Several non-animal methods addressing key-steps of the adverse outcome pathway (OECD, 2012) will most likely be needed to fully address this effect. Among the initial cellular events is the activation of keratinocytes and currently only one method, the KeratinoSens™, has been formally validated to address this event. In this study, a further method, the LuSens assay, that uses a human keratinocyte cell line harbouring a reporter gene construct composed of the antioxidant response element (ARE) of the rat NADPH:quinone oxidoreductase 1 gene and the luciferase gene. The assay was validated in house using a selection of 74 substances which included the LLNA performance standards. The predictivity of the LuSens assay for skin sensitization hazard identification was comparable to other non-animal methods, in particular to the KeratinoSens™. When used as part of a testing battery based on the OECD adverse outcome pathway for skin sensitization, a combination of the LuSens assay, the DPRA and a dendritic cell line activation test attained predictivities similar to that of the LLNA.

State-of-the-art of 3D cultures (organs-on-a-chip) in safety testing and pathophysiology.

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.

Vinclozolin: a case study on the identification of endocrine active substances in the past and a future perspective.

In the late 1980s vinclozolin was tested for prenatal developmental toxicity in rats for registration purposes in USA. At 1000mg/kgbw, 95% of all fetuses were female upon visual inspection (ano-genital distance determination). Anti-androgenic effects (AA) were also noted in a subsequent 2-generation study. These findings triggered mechanistic investigations at BASF and at US-EPA. Results published by the latter were the starting point of the endocrine disruption (ED) discussion in the 1990s. AA effects of vinclozolin are mediated by two metabolites, which have an antagonistic effect on the androgen receptor. Currently, determination of ED has become a major end-point in toxicology testing and the US-EPA has set up an elaborated testing paradigm to fulfill this requirement. Future screening for ED can be improved making use of new technologies. ED modes of action can be determined by three alternative (3R) methods. Steroid synthesis in H295R cells (1), androgen-receptor binding in modified yeast (2) and metabolomics (3). Using vinclozolin as a case study, results indicate: (1) an effect on steroid synthesis in vitro, (2) an antagonistic effect on the androgen receptor and (3) that the metabolome profile of vinclozolin is similar to that of other receptor mediated anti-androgens (e.g. flutamide).