Predicting optimal treatment outcomes in phase-based treatment and direct trauma-focused treatment among patients with posttraumatic stress disorder stemming from childhood abuse.

Research over the last few decades has demonstrated the effectiveness of various treatments for posttraumatic stress disorder (PTSD). However, the question of which treatment works best remains, especially for patients with PTSD stemming from childhood abuse. Using the Personalized Advantage Index (PAI), we explored which patients benefit more from phase-based treatment and which benefit more from direct trauma-focused treatment. Data were obtained from a multicenter randomized controlled trial (RCT) comparing a phase-based treatment condition (i.e., eye-movement desensitization and reprocessing [EMDR] therapy preceded by Skills Training in Affect and Interpersonal Regulation [STAIR]; n = 57) and a direct trauma-focused treatment (EMDR only; n = 64) among individuals with PTSD related to childhood abuse. Machine learning techniques were used to examine all pretreatment variables included in the trial as potential predictors and moderators, with selected variables combined to build the PAI model. The utility of the PAI was tested by comparing actual posttreatment outcomes of individuals who received PAI-indicated treatment with those allocated to a non-PAI-indicated treatment. Although eight pretreatment variables between PTSD treatment outcome and treatment condition were selected as moderators, there was no significant difference between participants assigned to their PAI-indicated treatment and those randomized to a non-PAI-indicated treatment, d = 0.25, p = .213. Hence, the results of this study do not support the need for personalized medicine for patients with PTSD and a history of childhood abuse. Further research with larger sample sizes and external validation is warranted.

Identification of genomic biomarkers for anthracycline-induced cardiotoxicity in human iPSC-derived cardiomyocytes: an in vitro repeated exposure toxicity approach for safety assessment.

The currently available techniques for the safety evaluation of candidate drugs are usually cost-intensive and time-consuming and are often insufficient to predict human relevant cardiotoxicity. The purpose of this study was to develop an in vitro repeated exposure toxicity methodology allowing the identification of predictive genomics biomarkers of functional relevance for drug-induced cardiotoxicity in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). The hiPSC-CMs were incubated with 156 nM doxorubicin, which is a well-characterized cardiotoxicant, for 2 or 6 days followed by washout of the test compound and further incubation in compound-free culture medium until day 14 after the onset of exposure. An xCELLigence Real-Time Cell Analyser was used to monitor doxorubicin-induced cytotoxicity while also monitoring functional alterations of cardiomyocytes by counting of the beating frequency of cardiomyocytes. Unlike single exposure, repeated doxorubicin exposure resulted in long-term arrhythmic beating in hiPSC-CMs accompanied by significant cytotoxicity. Global gene expression changes were studied using microarrays and bioinformatics tools. Analysis of the transcriptomic data revealed early expression signatures of genes involved in formation of sarcomeric structures, regulation of ion homeostasis and induction of apoptosis. Eighty-four significantly deregulated genes related to cardiac functions, stress and apoptosis were validated using real-time PCR. The expression of the 84 genes was further studied by real-time PCR in hiPSC-CMs incubated with daunorubicin and mitoxantrone, further anthracycline family members that are also known to induce cardiotoxicity. A panel of 35 genes was deregulated by all three anthracycline family members and can therefore be expected to predict the cardiotoxicity of compounds acting by similar mechanisms as doxorubicin, daunorubicin or mitoxantrone. The identified gene panel can be applied in the safety assessment of novel drug candidates as well as available therapeutics to identify compounds that may cause cardiotoxicity.

Conventional over-the-top-aiming devices with short offset fail to hit the center of the human femoral ACL footprint in medial portal technique, whereas medial-portal-aiming devices with larger offset hit the center reliably.

INTRODUCTION: Aim of this study was to investigate the accuracy of a conventional over-the-top-guide (OTG) with a typically short offset to hit the center of the native femoral ACL footprint through the anteromedial portal in comparison to a specific medial-portal-aimer (MPA) with larger offset. MATERIALS AND METHODS: In 20 matched human cadaveric knees, insertion sites of the ACL were marked in medial arthrotomy. An OTG with an offset of 5.5 mm, respectively, the MPA with 9 mm offset was used in a medial portal approach to locate the center of a single bundle ACL reconstruction tunnel with k-wires. Distances from the footprint center, the OTG drilling and the MPA drilling to the roof of the intercondylar notch and to the deep cartilage margin were determined. After positioning of radiological markers, radiographic analysis was performed according to the quadrant technique as described by Bernard and Hertel. RESULTS: The distance from ACL origin to the roof of the notch was 10.3 (±2.1) mm, in the OTG group 6.7 (±1.5) mm and in the MPA group 9.6 (±1.9) mm. The distance to the deep cartilage margin was 9.5 (±1.7) mm from ACL origin, 4.8 (±1.3) mm with OTG and 8.7 (±1.4) mm with MPA. There were statistically significant differences between the distances of the footprint center and the OTG group after measuring and also after radiographic analysis (p < 0.0001). Using the MPA, no significant different distances in comparison to the anatomical ACL center were found (p > 0.0001). There was an increased risk for femoral blow (9/10 vs. 0/10) in the OTG group after overdrilling with a 9 mm drill. CONCLUSION: Short (5.5 mm) offset femoral aiming devices fail to locate the native ACL footprint center in medial portal approach with an increased risk for femoral blowout when overdrilling. The special medial-portal-aiming device with 9 mm offset hit the center reliably.

Guidance on classification for reproductive toxicity under the globally harmonized system of classification and labelling of chemicals (GHS).

The Globally Harmonised System of Classification (GHS) is a framework within which the intrinsic hazards of substances may be determined and communicated. It is not a legislative instrument per se, but is enacted into national legislation with the appropriate legislative instruments. GHS covers many aspects of effects upon health and the environment, including adverse effects upon sexual function and fertility or on development. Classification for these effects is based upon observations in humans or from properly designed experiments in animals, although only the latter is covered herein. The decision to classify a substance based upon experimental data, and the category of classification ascribed, is determined by the level of evidence that is available for an adverse effect on sexual function and fertility or on development that does not arise as a secondary non-specific consequence of other toxic effect. This document offers guidance on the determination of level of concern as a measure of adversity, and the level of evidence to ascribe classification based on data from tests in laboratory animals.

Human embryonic stem cell-derived test systems for developmental neurotoxicity: a transcriptomics approach.

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.

Embryonic stem cells in safety pharmacology and toxicology.

Embryonic stem (ES) cells undergo self-renewal and are pluripotent, i.e., they can give rise to all the types of specialised cells in the body. Scientific knowledge on ES cells is increasing rapidly, leading to opportunities for establishment of ES cell-based in vitro tests for drug discovery, preclinical safety pharmacology and toxicology. The main properties of ES cells making them useful in in vitro assays are that they have a normal diploid karyotype and can provide a large number of cells for high-throughput assays. Human ES cells additionally have the potential to provide solutions to problems related to interspecies differences and methods for screening for human polymorphisms, thus supporting robust human hazard identification and optimised drug discovery strategies. Importantly, ES cell based assays could be potential tools to reduce and perhaps replace, animal experiments. This chapter will describe ongoing research in the use of ES cells in toxicology and safety pharmacology, focusing on the major areas of progress, namely, embryotoxicology, cardiotoxicology and hepatoxicology.

ECVAM and new technologies for toxicity testing.

The development of alternative empirical (testing) and non-empirical (non-testing) methods to traditional toxicological tests for complex human health effects is a tremendous task. Toxicants may potentially interfere with a vast number of physiological mechanisms thereby causing disturbances on various levels of complexity of human physiology. Only a limited number of mechanisms relevant for toxicity ('pathways' of toxicity) have been identified with certainty so far and, presumably, many more mechanisms by which toxicants cause adverse effects remain to be identified. Recapitulating in empirical model systems (i.e., in vitro test systems) all those relevant physiological mechanisms prone to be disturbed by toxicants and relevant for causing the toxicity effect in question poses an enormous challenge. First, the mechanism(s) of action of toxicants in relation to the most relevant adverse effects of a specific human health endpoint need to be identified. Subsequently, these mechanisms need to be modeled in reductionist test systems that allow assessing whether an unknown substance may operate via a specific (array of) mechanism(s). Ideally, such test systems should be relevant for the species of interest, i.e., based on human cells or modeling mechanisms present in humans. Since much of our understanding about toxicity mechanisms is based on studies using animal model systems (i.e., experimental animals or animal-derived cells), designing test systems that model mechanisms relevant for the human situation may be limited by the lack of relevant information from basic research. New technologies from molecular biology and cell biology, as well as progress in tissue engineering, imaging techniques and automated testing platforms hold the promise to alleviate some of the traditional difficulties associated with improving toxicity testing for complex endpoints. Such new technologies are expected (1) to accelerate the identification of toxicity pathways with human relevance that need to be modeled in test methods for toxicity testing (2) to enable the reconstruction of reductionist test systems modeling at a reduced level of complexity the target system/organ of interest (e.g., through tissue engineering, use of human-derived cell lines and stem cells etc.), (3) to allow the measurement of specific mechanisms relevant for a given health endpoint in such test methods (e.g., through gene and protein expression, changes in metabolites, receptor activation, changes in neural activity etc.), (4) to allow to measure toxicity mechanisms at higher throughput rates through the use of automated testing. In this chapter, we discuss the potential impact of new technologies on the development, optimization and use of empirical testing methods, grouped according to important toxicological endpoints. We highlight, from an ECVAM perspective, the areas of topical toxicity, skin absorption, reproductive and developmental toxicity, carcinogenicity/genotoxicity, sensitization, hematopoeisis and toxicokinetics and discuss strategic developments including ECVAM's database service on alternative methods. Neither the areas of toxicity discussed nor the highlighted new technologies represent comprehensive listings which would be an impossible endeavor in the context of a book chapter. However, we feel that these areas are of utmost importance and we predict that new technologies are likely to contribute significantly to test development in these fields. We summarize which new technologies are expected to contribute to the development of new alternative testing methods over the next few years and point out current and planned ECVAM projects for each of these areas.

Report of the EPAA-ECVAM workshop on the validation of Integrated Testing Strategies (ITS).

The use of Integrated Testing Strategies (ITS) permits the combination of diverse types of chemical and toxicological data for the purposes of hazard identification and characterisation. In November 2008, the European Partnership for Alternative Approaches to Animal Testing (EPAA), together with the European Centre for the Validation of Alternative Methods (ECVAM), held a workshop on Overcoming Barriers to Validation of Non-animal Partial Replacement Methods/Integrated Testing Strategies, in Ispra, Italy, to discuss the extent to which current ECVAM approaches to validation can be used to evaluate partial replacement in vitro test methods (i.e. as potential ITS components) and ITS themselves. The main conclusions of these discussions were that formal validation was only considered necessary for regulatory purposes (e.g. the replacement of a test guideline), and that current ECVAM approaches to validation should be adapted to accommodate such test methods. With these conclusions in mind, a follow-up EPAA-ECVAM workshop was held in October 2009, to discuss the extent to which existing validation principles are applicable to the validation of ITS test methods, and to develop a draft approach for the validation of such test methods and/or overall ITS for regulatory purposes. This report summarises the workshop discussions that started with a review of the current validation methodologies and the presentation of two case studies (skin sensitisation and acute toxicity), before covering the definition of ITS and their components, including their validation and regulatory acceptance. The following main conclusions/recommendations were made: that the validation of a partial replacement test method (for application as part of a testing strategy) should be differentiated from the validation of an in vitro test method for application as a stand-alone replacement, especially with regard to its predictive capacity; that, in the former case, the predictive capacity of the whole testing strategy (rather than of the individual test methods) would be more important, especially if the individual test methods had a high biological relevance; that ITS allowing for flexible and ad hoc approaches cannot be validated, whereas the validation of clearly defined ITS would be feasible, although practically quite difficult; and that test method developers should be encouraged to develop and submit to ECVAM not only full replacement test methods, but also partial replacement methods to be placed as parts of testing strategies. The added value from the formal validation of testing strategies, and the requirements needed in view of regulatory acceptance of the data, require further informed discussion within the EPAA forum on the basis of case studies provided by industry.

Alternative (non-animal) methods for cosmetics testing: current status and future prospects-2010.

The 7th amendment to the EU Cosmetics Directive prohibits to put animal-tested cosmetics on the market in Europe after 2013. In that context, the European Commission invited stakeholder bodies (industry, non-governmental organisations, EU Member States, and the Commission's Scientific Committee on Consumer Safety) to identify scientific experts in five toxicological areas, i.e. toxicokinetics, repeated dose toxicity, carcinogenicity, skin sensitisation, and reproductive toxicity for which the Directive foresees that the 2013 deadline could be further extended in case alternative and validated methods would not be available in time. The selected experts were asked to analyse the status and prospects of alternative methods and to provide a scientifically sound estimate of the time necessary to achieve full replacement of animal testing. In summary, the experts confirmed that it will take at least another 7-9 years for the replacement of the current in vivo animal tests used for the safety assessment of cosmetic ingredients for skin sensitisation. However, the experts were also of the opinion that alternative methods may be able to give hazard information, i.e. to differentiate between sensitisers and non-sensitisers, ahead of 2017. This would, however, not provide the complete picture of what is a safe exposure because the relative potency of a sensitiser would not be known. For toxicokinetics, the timeframe was 5-7 years to develop the models still lacking to predict lung absorption and renal/biliary excretion, and even longer to integrate the methods to fully replace the animal toxicokinetic models. For the systemic toxicological endpoints of repeated dose toxicity, carcinogenicity and reproductive toxicity, the time horizon for full replacement could not be estimated.

Cardiotoxicity testing using pluripotent stem cell-derived human cardiomyocytes and state-of-the-art bioanalytics: a review.

In this article, recent progress in cardiotoxicity testing based on the use of immortalized cell lines or human embryonic stem cell (hESC) derived cardiomyocytes in combination with state-of-the-art bioanalytical methods and sensors is reviewed. The focus is on hESC-derived cells and their refinement into competent testing cells, but the access and utility of other relevant cell types are also discussed. Recent developments in sensor techniques and bioanalytical approaches for measuring critical cardiotoxicity parameters are highlighted, together with aspects of data evaluation and validation. Finally, recommendations for further research are given.

PREDICtors for health-related quality of life after elective sigmoidectomy for DIVerticular disease: the PREDIC-DIV study protocol of a prospective multicentric transnational observational study.

INTRODUCTION: Diverticulitis is among the most common abdominal disorders. The best treatment strategy for this complicated disease as well as for recurrent stages is still under debate. Moreover, little knowledge exists regarding the effect of different therapeutic strategies on the health-related quality of life (HrQoL). Therefore, the PREDIC-DIV (PREDICtors for health-related quality of life after elective sigmoidectomy for DIVerticular disease) study aims to assess predictors of a change in HrQoL in patients after elective sigmoidectomy for diverticular disease. METHODS AND ANALYSIS: A prospective multicentre transnational observational study was started in November 2017. Patients undergoing elective sigmoid resection for diverticular disease were included. Primary outcome includes HrQoL 6 months postoperatively, staged by the Gastrointestinal Quality of Life Index (GIQLI). Secondary outcomes include HrQoL 6 months after sigmoidectomy, assessed using the Short Form 36 Questionnaire and a custom-made Visual Analogue Scale-based inventory; HrQoL after 12 and 24 months; postoperative morbidity; mortality; influence of surgical technique (conventional laparoscopic multiport operation vs robotic approach); histological grading of inflammation and morphological characteristics of the bowel wall in the resected specimen; postoperative functional changes (faecal incontinence, faecal urge, completeness of emptying, urinary incontinence, sexual function); disease-specific healthcare costs; and changes in economic productivity, measured by the iMTA Productivity Cost Questionnaire. The total follow-up will be 2 years. ETHICS AND DISSEMINATION: The protocol was approved by the medical ethical committee of the Bavarian Medical Council (report identification number: 2017-177). The study was conducted in accordance with the Declaration of Helsinki. The findings of this study will be submitted to a peer-reviewed journal (BMJ Open, Annals of Surgery, British Journal of Surgery, Diseases of the Colon and the Rectum). Abstracts will be submitted to relevant national and international conferences. TRIAL REGISTRATION NUMBER: The study is registered with the ClinicalTrials.gov register as NCT03527706; Pre-results.

Hazard assessment of methylmercury toxicity to neuronal induction in embryogenesis using human embryonic stem cells.

Pluripotent human embryonic stem cell (hESC) lines can to some extent mimic in vitro the development of the embryo, providing the scientific rationale for the use of these cells to establish tests for toxicity to embryogenesis. Such humanised in vitro tests have potential to improve human hazard prediction by avoiding interspecies differences. We explored the potential of a hESC-based assay for detection of toxicity to neuronal induction in embryonic development. Neuronal precursor differentiation was performed according to a previous publication, while we established a new protocol for maturation of the precursors into neuron-like cells. Appearance of neuronal derivatives was demonstrated by real-time PCR, showing up-regulation of several neuronal marker genes, and immunohistochemistry, demonstrating the appearance of neurofilament medium polypeptide, beta-tubulin III and microtubule-associated protein 2 positive cells. In order to assess whether the hESC model could detect chemically induced developmental toxicity, we exposed the differentiating cells to methylmercury (MeHg) causing structural developmental abnormalities in the brain. Two separate exposure intervals were used to determine the effects of MeHg on neuronal precursor formation and their further maturation, respectively. The formation of precursors was sensitive to MeHg in non-cytotoxic concentrations, as the expression of several neuronal mRNA markers changed. In contrast, non-cytotoxic MeHg concentrations did not effect the mRNA marker expression in matured cells, indicating that neuronal precursor formation is more sensitive to MeHg than later stages of neuronal differentiation. Overall, our experiments demonstrate that the hESC assay can provide alerts for the adverse effects of MeHg on neuronal induction.

Overview of the blood compatibility of nanomedicines: A trend analysis of in vitro and in vivo studies.

As nanomedicines have the potential to address many currently unmet medical needs, the early identification of regulatory requirements that could hamper a smooth translation of nanomedicines from the laboratory environment to clinical applications is of utmost importance. The blood system is especially relevant as many nanomedicinal products that are currently under development are designed for intravenous administration and cells of the blood system will be among the first biological systems exposed to the injected nanomedicine. This review collects and summarizes the current knowledge related to the blood compatibility of nanomedicines and nanomaterials with a potential use in biomedical applications. Different types of nanomedicines were analyzed for their toxicity to the blood system, and the role of their physicochemical properties was further elucidated. Trends were identified related to: (a) the nature of the most frequently occurring blood incompatibilities such as thrombogenicity and complement activation, (b) the contribution of physicochemical properties to these blood incompatibilities, and (c) the similarities between data retrieved from in vivo and in vitro studies. Finally, we provide an overview of available standards that allow evaluating the compatibility of a material with the blood system. This article is categorized under: Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials Therapeutic Approaches and Drug Discovery > Emerging Technologies Toxicology and Regulatory Issues in Nanomedicine > Regulatory and Policy Issues in Nanomedicine.

Digital movie analysis for quantification of beating frequencies, chronotropic effects, and beating areas in cardiomyocyte cultures.

Software, named Cardio Analyser, was developed for digital movie analysis of beating frequencies, drug-induced chronotropic effects, and quantification of beating areas of contracting cardiomyocyte cultures. A major novelty of the software is the introduction of automated noise filtering and automated movie analysis of beating frequencies and areas of contracting cardiomyocyte cultures. The software was based on the observation that the intensity of light transmitted through a contractive tissue changes periodically in a way that correlates with the contractions. We provided proof of principle for the method by derivation of relevant data from movies of multicellular cardiomyocyte cultures derived from embryonic stem cells. Moreover, we compared the data to equivalent results obtained by extracellular electric field potential recordings. The comparison demonstrated higher sensitivity to chronotropic effects of the beta-adrenoceptor agonist isoprenaline, and hence implied that more embryonic stem cells underwent differentiation into beta-adrenoceptor-responding cardiomyocytes, in the experimental setup applied for movie analysis than in the setup used for extracellular electric field potential recordings. Our study indicates that the movie analysis method may have potential to be optimized for screening in early drug discovery, aiming to identify cardiac drug candidates or to alert for adverse effects on heart functionality or embryonic heart development.

Development of an in vitro test battery for assessing chemical effects on bovine germ cells under the ReProTect umbrella.

Current European legislation for the registration and authorization of chemicals (REACH) will require a dramatic increase in the use of animals for reproductive toxicity testing. Since one objective of REACH is to use vertebrates only as last resort, the development and validation of alternative methods is urgently needed. For this purpose ReProTect, an integrated research project funded by the European Union, joining together 33 partners with complementary expertise in reproductive toxicology, was designed. The study presented here describes a battery of two tests developed within ReProTect. The objective of these tests is the detection of chemical effects during the processes of oocyte maturation and fertilisation in a bovine model. The corresponding toxicological endpoints are the reaching of metaphase II and the formation of the pronuclei respectively. Fifteen chemicals have been tested (Benzo[a]pyrene, Busulfan, Butylparaben, Cadmium Chloride, Carbendazim, Cycloheximide, Diethylstilbestrol, Genistein, Ionomycin, Ketoconazole, Lindane, Methylacetoacetate, Mifepristone, Nocodazole and DMSO as solvent) demonstrating high intra-laboratory reproducibility of the tests. Furthermore, the responses obtained in both tests, for several substances, had a good correlation with the available in vivo and in vitro data. These tests therefore, could predictably become part of an integrated testing strategy that combines the bovine models with additional in vitro tests, in order to predict chemical hazards on mammalian fertility.

First steps in establishing a developmental toxicity test method based on human embryonic stem cells.

The use of embryonic stem cells is currently the most promising approach to assess developmental toxicity in vitro. In addition, the possibility of using human embryonic stem (hES) cells will increase safety of consumers and patients as false classification of substances due to inter-species variations can be avoided. One validated test based on murine embryonic stem cells, the embryonic stem cell test (EST), consists of following endpoints: IC(50) values of fibroblasts and embryonic stem cells as well as the inhibition of differentiation of mES cells into cardiomyocytes. As a follow up of its successful validation study we established a cytotoxicity assay based on hES cells and human fibroblasts employing two developmental toxicants: 5-fluorouracil (5-FU) and all-trans retinoic acid (RA). The results were compared to historical data from the EST. For 5-FU, no significant differences were obtained between the different cell lines. However, for RA, both test systems produced higher IC(50) values for the fibroblasts than for the stem cells, which is a well-known effect of developmental toxicants. Moreover, the reliability and relevance of several marker genes as possible toxicological endpoints were tested. During early differentiation Oct-4, hTert and Dusp6 showed the most reliable results. Brachyury and GATA-4 were found to be best suited to monitor cardiac differentiation. The late cardiac marker gene TNNT2 demonstrated significant results until day 18. Therefore, these marker genes have the highest potential to serve as endpoints for a developmental toxicity test.

Assessment of acute and chronic toxicity of doxorubicin in human induced pluripotent stem cell-derived cardiomyocytes.

The present study assesses acute and chronic toxicity of doxorubicin in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), with the aim to obtain in vitro biomarkers that can be used as readouts to predict in vivo cardiotoxicity. Possible acute toxicity was investigated by assessing effects on the beating rate and the field potential duration (FPD) of doxorubicin-exposed cardiomyocytes by measuring electrical activity using multi-electrode array (MEA) analyses. No effects on the beating rate and FPD were found at concentrations up to 6µM, whereas at 12µM no electrical activity was recorded, indicating that the cardiomyocytes stopped beating. Acute and chronic effects of doxorubicin on mitochondria, which have been reported to be affected in doxorubicin-induced cardiotoxicity, were assessed using high content imaging techniques. To this end hiPSC-CMs were exposed to 150 or 300nM doxorubicin using both single dosing (3h and 2days) and repetitive dosing (3 times, of 2days each), including washout studies to assess delayed effects (assessment at day 14) and effects on cell number, mitochondrial density, mitochondrial membrane potential, mitochondrial superoxide levels and mitochondrial calcium levels were assessed. No effects of doxorubicin were found on mitochondrial density and mitochondrial superoxide levels, whereas doxorubicin reduced cell survival and slightly altered mitochondrial membrane potential and mitochondrial calcium levels, which was most profound in the washout studies. Altogether, the results of the present study show that concentrations of doxorubicin in the micromolar range were required to affect electrical activity of hiPSC-CMs, whereas nanomolar concentrations already affected cell viability and caused mitochondrial disturbances. Integration of these data with other in vitro data may enable the selection of a series of in vitro biomarkers that can be used as readouts to screen chemicals for possible cardiotoxicity.

The value of alternative testing for neurotoxicity in the context of regulatory needs.

Detection and characterisation of chemical-induced toxic effects in the central and peripheral nervous system represent a major challenge for employing newly developed technologies in the field of neurotoxicology. Precise cellular predictive test batteries for chemical-induced neurotoxicity are increasingly important for regulatory decision making, but also the most efficient way to keep costs and time of testing within a reasonable margin. Current in vivo test methods are based on behavioural and sensory perturbations coupled with routine histopathological investigations. In spite of the empirical usefulness of these tests, they are not always sensitive enough and often, they do not provide information that facilitates a detailed understanding of potential mechanisms of toxicity, thus enabling predictions. In general, such in vivo tests are unsuitable for screening large number of agents. One way to meet the need for more powerful and comprehensive tests via an extended scientific basis is to study neurotoxicity in specific cell types of the brain and to derive generalised mechanisms of action of the toxicants from such series of experiments. Additionally, toxicokinetic models are to be developed in order to give a rough account for the whole absorption, distribution, metabolism, excretion (ADME) process including the blood-brain barrier (BBB). Therefore, an intensive search for the development of alternative methods using animal and human-based in vitro and in silico models for neurotoxic hazard assessment is appropriate. In particular, neurotoxicology represents one of the major challenges to the development of in vitro systems, as it has to account also for heterogeneous cell interactions of the brain which require new biochemical, biotechnological and electrophysiological profiling methods for reliable alternative ways with a high throughput.

The integrated project ReProTect: a novel approach in reproductive toxicity hazard assessment.

Validated alternative test methods are urgently needed for safety testing of drugs, chemicals and cosmetics. Whereas some animal tests for topical toxicity have been successfully replaced by alternative methods, systemic toxicity testing requires new test strategies in order to achieve an adequate safety level for the consumer. Substantial numbers of animals are required for the current in vivo assays for drugs, chemicals and cosmetics and a broad range of pioneering alternative methods were already developed. These prerequisites motivate the development of a tiered testing strategy based on alternative tests for reproductive toxicity hazard. In the Integrated Project ReProTect, a consortium set up by the European Centre for the Validation of Alternative Methods (ECVAM) takes the lead to manage the development of a testing strategy in the area of reproductive toxicity. The reproductive cycle can be broken down into well-defined sub-elements, namely male and female fertility, implantation and pre/postnatal development. In this project, in vitro, in silico and sensor technologies will be developed, leading to testing strategies, that shall be implemented and disseminated.