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Identification of neurotoxic drugs and environmental chemicals is an important challenge. However, only few tools to address this topic are available. The aim of this study was to develop a neurotoxicity/developmental neurotoxicity (DNT) test system, using the pluripotent mouse embryonic stem cell line CGR8 (ESCs). The test system uses ESCs at two differentiation stages: undifferentiated ESCs and ESC-derived neurons. Under each condition, concentration-response curves were obtained for three parameters: activity of the tubulin alpha 1 promoter (typically activated in early neurons), activity of the elongation factor 1 alpha promoter (active in all cells), and total DNA content (proportional to the number of surviving cells). We tested 37 compounds from the ESNATS test battery, which includes polypeptide hormones, environmental pollutants (including methylmercury), and clinically used drugs (including valproic acid and tyrosine kinase inhibitors). Different classes of compounds showed distinct concentration-response profiles. Plotting of the lowest observed adverse effect concentrations (LOAEL) of the neuronal promoter activity against the general promoter activity or against cytotoxicity, allowed the differentiation between neurotoxic/DNT substances and non-neurotoxic controls. Reporter activity responses in neurons were more susceptible to neurotoxic compounds than the reporter activities in ESCs from which they were derived. To relate the effective/toxic concentrations found in our study to relevant in vivo concentrations, we used a reverse pharmacokinetic modeling approach for three exemplary compounds (teriflunomide, geldanamycin, abiraterone). The dual luminescence reporter assay described in this study allows high-throughput, and should be particularly useful for the prioritization of the neurotoxic potential of a large number of compounds.

Identification of neurotoxic drugs and environmental chemicals is an important challenge. However, only few tools to address this topic are available. The aim of this study was to develop a neurotoxicity/developmental neurotoxicity (DNT) test system, using the pluripotent mouse embryonic stem cell line CGR8 (ESCs). The test system uses ESCs at two differentiation stages: undifferentiated ESCs and ESC-derived neurons. Under each condition, concentration-response curves were obtained for three parameters: activity of the tubulin alpha 1 promoter (typically activated in early neurons), activity of the elongation factor 1 alpha promoter (active in all cells), and total DNA content (proportional to the number of surviving cells). We tested 37 compounds from the ESNATS test battery, which includes polypeptide hormones, environmental pollutants (including methylmercury), and clinically used drugs (including valproic acid and tyrosine kinase inhibitors). Different classes of compounds showed distinct concentration-response profiles. Plotting of the lowest observed adverse effect concentrations (LOAEL) of the neuronal promoter activity against the general promoter activity or against cytotoxicity, allowed the differentiation between neurotoxic/DNT substances and non-neurotoxic controls. Reporter activity responses in neurons were more susceptible to neurotoxic compounds than the reporter activities in ESCs from which they were derived. To relate the effective/toxic concentrations found in our study to relevant in vivo concentrations, we used a reverse pharmacokinetic modeling approach for three exemplary compounds (teriflunomide, geldanamycin, abiraterone). The dual luminescence reporter assay described in this study allows high-throughput, and should be particularly useful for the prioritization of the neurotoxic potential of a large number of compounds.

The use of laboratory animals for toxicity testing in chemical safety assessment meets increasing ethical, economic and legislative constraints. The development, validation and application of reliable alternatives for in vivo toxicity testing are therefore urgently needed. In order to use toxicity data obtained from in vitro assays for risk assessment, in vitro concentration-response data need to be translated into in vivo dose-response data that are needed to obtain points of departure for risk assessment, like a benchmark dose (BMD). In the present study, we translated in vitro concentration-response data of the retinoid all-trans-retinoic acid (ATRA), obtained in the differentiation assay of the embryonic stem cell test, into in vivo dose-response data using a physiologically based kinetic model for rat and human that is mainly based on kinetic model parameter values derived using in vitro techniques. The predicted in vivo dose-response data were used for BMD modeling, and the obtained BMDL10 values [lower limit of the 95 % confidence interval on the BMD at which a benchmark response equivalent to a 10 % effect size (BMR10) is reached (BMD10)] for rat were compared with BMDL10 values derived from in vivo developmental toxicity data in rats reported in the literature. The results show that the BMDL10 values from predicted dose-response data differ about sixfold from the BMDL10 values obtained from in vivo data, pointing at the feasibility of using a combined in vitro-in silico approach for defining a point of departure for toxicological risk assessment.

Previously we showed a battery consisting of CALUX transcriptional activation assays, the ReProGlo assay, and the embryonic stem cell test, and zebrafish embryotoxicity assay as 'apical' tests to correctly predict developmental toxicity for 11 out of 12 compounds, and to explain the one false negative [7]. Here we report on applying this battery within the context of grouping and read across, put forward as a potential tool to fill data gaps and avoid animal testing, to distinguish in vivo non-or weak developmental toxicants from potent developmental toxicants within groups of structural analogs. The battery correctly distinguished 2-methylhexanoic acid, monomethyl phthalate, and monobutyltin trichloride as non-or weak developmental toxicants from structurally related developmental toxicants valproic acid, mono-ethylhexyl phthalate, and tributyltin chloride, respectively, and, therefore, holds promise as a biological verification model in grouping and read across approaches. The relevance of toxicokinetic information is indicated. (C) 2014 Elsevier Inc. All rights reserved.

Developmental neurotoxicity (DNT) and many forms of reproductive toxicity (RT) often manifest themselves in functional deficits that are not necessarily based on cell death, but rather on minor changes relating to cell differentiation or communication. The fields of DNT/RT would greatly benefit from in vitro tests that allow the identification of toxicant-induced changes of the cellular proteostasis, or of its underlying transcriptome network. Therefore, the 'human embryonic stem cell (hESC)- derived novel alternative test systems (ESNATS)' European commission research project established RT tests based on defined differentiation protocols of hESC and their progeny. Valproic acid (VPA) and methylmercury (MeHg) were used as positive control compounds to address the following fundamental questions: (1) Does transcriptome analysis allow discrimination of the two compounds? (2) How does analysis of enriched transcription factor binding sites (TFBS) and of individual probe sets (PS) distinguish between test systems? (3) Can batch effects be controlled? (4) How many DNA microarrays are needed? (5) Is the highest non-cytotoxic concentration optimal and relevant for the study of transcriptome changes? VPA triggered vast transcriptional changes, whereas MeHg altered fewer transcripts. To attenuate batch effects, analysis has been focused on the 500 PS with highest variability. The test systems differed significantly in their responses (\20 % overlap). Moreover, within one test system, little overlap between the PS changed by the two compounds has been observed. However, using TFBS enrichment, a relatively large 'common response' to VPA and MeHg could be distinguished from 'compound-specific' responses. In conclusion, the ESNATS assay battery allows classification of human DNT/RT toxicants on the basis of their transcriptome profiles.

The European cosmetics legislation foresees a review in 2011 and possible postponement of the 2013 marketing ban to enforce the testing ban for systemic and repeated-dose animal tests. For this purpose, a 119-page report commissioned by the European Commission was published recently. Here, a group of 17 independent experts from the US, Europe, and Japan was brought together to evaluate the report. The expert panel strongly endorsed the report and its conclusions. A number of important options not considered were identified; these do not, however, affect the overall conclusions regarding the current lack of availability of a full replacement, especially for the areas of repeated dose toxicity, carcinogenicity testing, and reproductive toxicity, though a roadmap for change is emerging. However, some of these options may provide adequate data for replacement of some animal studies in the near future pending validation. Various recommendations expand the original report. The reviewers agree with the report that there is greater promise in the short term for the areas of sensitization and toxicokinetics. Additional opportunities lie in more global collaborations and the inclusion of other industry sectors.

The European cosmetics legislation foresees a review in 2011 and possible postponement of the 2013 marketing ban to enforce the testing ban for systemic and repeated-dose animal tests. For this purpose, a 119-page report commissioned by the European Commission was published recently. Here, a group of 17 independent experts from the US, Europe, and Japan was brought together to evaluate the report. The expert panel strongly endorsed the report and its conclusions. A number of important options not considered were identified; these do not, however, affect the overall conclusions regarding the current lack of availability of a full replacement, especially for the areas of repeated dose toxicity, carcinogenicity testing, and reproductive toxicity, though a roadmap for change is emerging. However, some of these options may provide adequate data for replacement of some animal studies in the near future pending validation. Various recommendations expand the original report. The reviewers agree with the report that there is greater promise in the short term for the areas of sensitization and toxicokinetics. Additional opportunities lie in more global collaborations and the inclusion of other industry sectors.

The European cosmetics legislation foresees a review in 2011 and possible postponement of the 2013 marketing ban to enforce the testing ban for systemic and repeated-dose animal tests. For this purpose, a 119-page report commissioned by the European Commission was published recently. Here, a group of 17 independent experts from the US, Europe, and Japan was brought together to evaluate the report. The expert panel strongly endorsed the report and its conclusions. A number of important options not considered were identified; these do not, however, affect the overall conclusions regarding the current lack of availability of a full replacement, especially for the areas of repeated dose toxicity, carcinogenicity testing, and reproductive toxicity, though a roadmap for change is emerging. However, some of these options may provide adequate data for replacement of some animal studies in the near future pending validation. Various recommendations expand the original report. The reviewers agree with the report that there is greater promise in the short term for the areas of sensitization and toxicokinetics. Additional opportunities lie in more global collaborations and the inclusion of other industry sectors.