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 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.

Neurotoxicity is one of the most challenging fields for the development of in vitro testing systems. In the last years, alternative in vitro testing strategies for chemical risk assessment have been designed, according to the current REACH legislation, to reduce the number of animal required for testing. However, to date in vitro assays for neurotoxicity have not been formally validated yet [1]. This is mostly due to the extreme complexity of the nervous system in which many different cell types are organized in a well orchestrated and functional network difficult to reproduce in vitro, but also to the lack of in vitro systems and methods capable to fully cover some of the endpoints of the in vivo tests such as the neurobehavioral and neurocognitive aspects as well as the motor functionality. Nowadays, many cellular models are available for the nervous system, including primary cultures of fetal and adult neurons and glial cells, tumor-derived cell lines, hippocampal brain slices and neural progenitor cells, but all of this models suffer from considerable drawbacks, e.g. non-human origin, limited access or non-physiological transformed cell types [2]. Human pluripotent stem cells (hPSCs) are considered as a powerful tool for drug screening and the development of new in vitro testing strategies. Indeed, these cells can be indefinitely expanded and efficiently differentiated into neuronal derivatives, including different regionalized neuronal subtypes, glial cells and peripheral neurons [3]. In this context, the European project ESNATS has taken the first step toward the design of developmental neurotoxicity tests based on the use of hPSCs, particularly developing battery of tests covering different aspects of neural teratogenicity [4-6]. Many of these hPSCs-based models are very well characterized on the molecular basis, but to date there are few data indicating how they reflect the functionality of the in vivo central nervous system (CNS)/ peripheral nervous system (PNS) and clearly none of these systems can completely resemble the complex physiology of the entire nervous system. Consequently, the main problem in the development of novel test strategies relies on the fact that the mechanisms underlying neurotoxicity are too extensive to be covered with a single model and a small set of endpoints. Therefore, in vitro neurotoxicity tests should include different cellular models and multiple levels of evaluation, ranging from cytotoxicity and cell physiology to neuronal specific cell function endpoints. Moreover, the obtained data, in order to be considered reliable and predictive, should be compared across diverse in vitro models, extrapolated and further aligned to in vivo available data sets, in order to bridge the gap between in vivo and in vitro neurotoxicity. URI: Authors: ROSAS Vania ZAGOURA Dimitra PISTOLLATO Francesca HAUPT Simone COLLEONI Silvia BREMER Susanne PRICE Anna PESCHANSKI Marc Publication Year: 2014 Type:

Introduction: Human stem cell lines and their derivatives, as alternatives to the use of animal cells or cancer cell lines, have been widely discussed as cellular models in predictive toxicology. However, the role of stem cells in the development of long-term toxicities and carcinogenesis has not received great attention so far, despite growing evidence indicating the relationship of stem cell damage to adverse effects later in life. However, testing this in vitro is a scientific/technical challenge in particular due to the complex interplay of factors existing under physiological conditions. Current major research programs in stem cell toxicity are not aiming to demonstrate that stem cells can be targeted by toxicants. Therefore, this knowledge gap needs to be addressed in additional research activities developing technical solutions and defining appropriate experimental designs.Areas covered: The current review describes selected examples of the role of stem cells in the development of long-term toxicities in the brain, heart or liver and in the development of cancer.Expert opinion: The presented examples illustrate the need to analyze the contribution of stem cells to chronic toxicity in order to make a final conclusion whether stem cell toxicities are an underestimated risk in mechanism-based safety assessments. This requires the development of predictive in vitro models allowing the assessment of adverse effects to stem cells on chronic toxicity and carcinogenicity.

A number of in vitro toxicity assays based on human embryonic stem cells (hESCs) are under development in order to provide alternative methods for the screening of chemicals and drugs and to reduce the number of animals needed for developmental toxicity assessment. The major challenge is to demonstrate the reliability of these in vitro methods by correlating the in vitro produced results to the available in vivo data. In this context transcriptomic approaches associated to toxicogenomic database analysis give the possibility to screen, annotate and cluster high numbers of genes and to identify the molecular changes that univocally mark the toxicity induced processes or are indicative of the early initiating events that lead to cellular toxicity. In this retrospective study we compare microarray transcriptomic data derived from two different hESCs lines (HUES1 and H9) exposed to valproic acid (VA) while applying the same differentiation protocol. We present the results of this comparative analysis in light of the known teratogenic effects of VA. The results show molecular changes in the processes of neural development, neural crest migration, apoptosis and regulation of transcription, indicating a good correspondence with the available in vivo data. We also describe common toxicological signatures and provide an interpretation of the observed qualitative differences referring to known biological features of the two hESCs lines.

tests for developmental toxicity follow OECD guidelines 414 (2-generation study), 426 (developmental neurotoxic-ity) or others. These tests analyze, for example, the num-bers of embryo-fetal deaths, altered total and organ weight and anatomical and behavioral abnormalities. They require exposure and analysis of animals over long periods. For example, according to OECD 426, exposure is performed during gestation and lactation and the offspring has to be analyzed for neurological, histological, neurochemical and behavioral alterations. These complex in vivo tests are too laborious and expensive to allow the required testing for thousands of chemicals (Krug et2013 al. ), and might also not well reflect the human situation because of inter-species variation. Therefore, there is a general agreement that relia-ble, faster and more accurate in vitro tests of developmental toxicity are urgently needed (Krause et al. 2013; Leist et al. 2012; van Thriel et al. 2012).

The National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM) coordinated an international interlaboratory validation study of the BG1Luc estrogen receptor (ER) transactivation (TA) test method (BG1Luc ER TA) developed by Xenobiotic Detection Systems, Inc. In 2010, the validation study finished evaluating the usefulness and limitations of the BG1Luc ER TA test method to screen for substances with in vitro ER agonist or antagonist activity. The international validation study was sponsored by NICEATM, with participation from the European Centre for the Validation of Alternative Methods and the Japanese Center for the Validation of Alternative Methods. In 2011, NICEATM and the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) released a test method evaluation report on the usefulness and limitations of the BG1Luc ER TA test method. ICCVAM recommended the use of the BG1Luc ER TA as a screening test to identify substances with in vitro ER agonist and antagonist activity and recommended that the BG1Luc ER TA test method could be considered as an alternative to the existing ER TA test guideline (EPA OPPTS 890.1300/OECD TG 455). All 15 ICCVAM member agencies, including the U.S. Environmental Protection Agency, concurred with the ICCVAM recommendations. NICEATM sponsored the new method for evaluation by the Organisation for Economic Cooperation and Development (OECD), which approved the BG1Luc ER TA test method and added the BG1 agonist protocol to the existing Test Guideline 455. The BG1 antagonist method has been adopted as OECD Test Guideline 457. Acceptance of the BG1Luc ER TA test method by U.S. and international agencies is an example of increased cooperation and collaboration to support the international adoption of scientifically valid test methods that will protect people, animals, and the environment while reducing, refining, and replacing animal use. (ILS staff supported by NIEHS contract N01-ES 35504.)

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 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.

In 2011, the European Commission has restricted the use of Bisphenol A in plastic infant feeding bottles. In a response to this restriction, Bisphenol S is now often used as a component of plastic substitutes for the production of babybottles. One of the major concerns leading to the restriction of Bisphenol A was its weak estrogenic activity. By using two highly standardised transactivation assays, we could demonstrate that the estrogenic activity of Bisphenol A and Bisphenol S is of a comparable potency. Furthermore, some insights about the structure-activity relationships of these two chemicals and their metabolites could be gained from in silico predictions of their relative estrogen receptor-binding affinities and their liver phase-I biotransformation.

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.

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.

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 (1). 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.

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.