Determination of in vitro hepatotoxic potencies of a series of perfluoroalkyl substances (PFASs) based on gene expression changes in HepaRG liver cells.

Per- and polyfluoroalkyl substances (PFASs) are omnipresent and have been shown to induce a wide range of adverse health effects, including hepatotoxicity, developmental toxicity, and immunotoxicity. The aim of the present work was to assess whether human HepaRG liver cells can be used to obtain insight into differences in hepatotoxic potencies of a series of PFASs. Therefore, the effects of 18 PFASs on cellular triglyceride accumulation (AdipoRed assay) and gene expression (DNA microarray for PFOS and RT-qPCR for all 18 PFASs) were studied in HepaRG cells. BMDExpress analysis of the PFOS microarray data indicated that various cellular processes were affected at the gene expression level. From these data, ten genes were selected to assess the concentration-effect relationship of all 18 PFASs using RT-qPCR analysis. The AdipoRed data and the RT-qPCR data were used for the derivation of in vitro relative potencies using PROAST analysis. In vitro relative potency factors (RPFs) could be obtained for 8 PFASs (including index chemical PFOA) based on the AdipoRed data, whereas for the selected genes, in vitro RPFs could be obtained for 11-18 PFASs (including index chemical PFOA). For the readout OAT5 expression, in vitro RPFs were obtained for all PFASs. In vitro RPFs were found to correlate in general well with each other (Spearman correlation) except for the PPAR target genes ANGPTL4 and PDK4. Comparison of in vitro RPFs with RPFs obtained from in vivo studies in rats indicate that best correlations (Spearman correlation) were obtained for in vitro RPFs based on OAT5 and CXCL10 expression changes and external in vivo RPFs. HFPO-TA was found to be the most potent PFAS tested, being around tenfold more potent than PFOA. Altogether, it may be concluded that the HepaRG model may provide relevant data to provide insight into which PFASs are relevant regarding their hepatotoxic effects and that it can be applied as a screening tool to prioritize other PFASs for further hazard and risk assessment.

Towards best use and regulatory acceptance of generic physiologically based kinetic (PBK) models for in vitro-to-in vivo extrapolation (IVIVE) in chemical risk assessment.

With an increasing need to incorporate new approach methodologies (NAMs) in chemical risk assessment and the concomitant need to phase out animal testing, the interpretation of in vitro assay readouts for quantitative hazard characterisation becomes more important. Physiologically based kinetic (PBK) models, which simulate the fate of chemicals in tissues of the body, play an essential role in extrapolating in vitro effect concentrations to in vivo bioequivalent exposures. As PBK-based testing approaches evolve, it will become essential to standardise PBK modelling approaches towards a consensus approach that can be used in quantitative in vitro-to-in vivo extrapolation (QIVIVE) studies for regulatory chemical risk assessment based on in vitro assays. Based on results of an ECETOC expert workshop, steps are recommended that can improve regulatory adoption: (1) define context and implementation, taking into consideration model complexity for building fit-for-purpose PBK models, (2) harmonise physiological input parameters and their distribution and define criteria for quality chemical-specific parameters, especially in the absence of in vivo data, (3) apply Good Modelling Practices (GMP) to achieve transparency and design a stepwise approach for PBK model development for risk assessors, (4) evaluate model predictions using alternatives to in vivo PK data including read-across approaches, (5) use case studies to facilitate discussions between modellers and regulators of chemical risk assessment. Proof-of-concepts of generic PBK modelling approaches are published in the scientific literature at an increasing rate. Working on the previously proposed steps is, therefore, needed to gain confidence in PBK modelling approaches for regulatory use.

Applicability of generic PBK modelling in chemical hazard assessment: A case study with IndusChemFate.

Toxicology is moving away from animal testing towards in vitro tools to assess chemical safety. This new testing framework requires a quantitative method, i.e. kinetic modelling, which extrapolates effective concentrations in vitro to a bioequivalent human dose in vivo and which can be applied on "high throughput screening" of a wide variety of chemicals. Generic physiologically based kinetic (PBK) models help account for the role of toxicokinetics in setting human toxic exposure levels. Furthermore these models may be parameterized only on in silico QSARs and in vitro metabolism assays, thereby circumventing the use of in vivo toxicokinetics for this purpose. Though several such models exist their applicability domains have yet to be comprehensively assessed. This study extends previous evaluations of the PBK model IndusChemFate and compares it with its more complex biological complement ("TNO Model"). Both models were evaluated with a broad span of chemicals, varying regarding physicochemical properties. The results reveal that the "simpler" performed best, illustrating that IndusChemFate can be a useful first-tier for simulating toxicokinetics based on QSARs and in vitro parameters. Finally, proper quantitative in vitro to in vivo extrapolation conditions were illustrated starting with acetaminophen induced in vitro cytotoxicity in human HepaRG cells.

Applicability of organ-on-chip systems in toxicology and pharmacology.

Organ-on-chip (OoC) systems are microfabricated cell culture devices designed to model functional units of human organs by harboring an in vitro generated organ surrogate. In the present study, we reviewed issues and opportunities related to the application of OoC in the safety and efficacy assessment of chemicals and pharmaceuticals, as well as the steps needed to achieve this goal. The relative complexity of OoC over simple in vitro assays provides advantages and disadvantages in the context of compound testing. The broader biological domain of OoC potentially enhances their predictive value, whereas their complexity present issues with throughput, standardization and transferability. Using OoCs for regulatory purposes requires detailed and standardized protocols, providing reproducible results in an interlaboratory setting. The extent to which interlaboratory standardization of OoC is feasible and necessary for regulatory application is a matter of debate. The focus of applying OoCs in safety assessment is currently directed to characterization (the biology represented in the test) and qualification (the performance of the test). To this aim, OoCs are evaluated on a limited scale, especially in the pharmaceutical industry, with restricted sets of reference substances. Given the low throughput of OoC, it is questionable whether formal validation, in which many reference substances are extensively tested in different laboratories, is feasible for OoCs. Rather, initiatives such as open technology platforms, and collaboration between OoC developers and risk assessors may prove an expedient strategy to build confidence in OoCs for application in safety and efficacy assessment.

Systemic PFOS and PFOA exposure and disturbed lipid homeostasis in humans: what do we know and what not?

Associations between per- and polyfluoroalkyl substances (PFASs) and increased blood lipids have been repeatedly observed in humans, but a causal relation has been debated. Rodent studies show reverse effects, i.e. decreased blood cholesterol and triglycerides, occurring however at PFAS serum levels at least 100-fold higher than those in humans. This paper aims to present the main issues regarding the modulation of lipid homeostasis by the two most common PFASs, PFOS and PFOA, with emphasis on the underlying mechanisms relevant for humans. Overall, the apparent contrast between human and animal data may be an artifact of dose, with different molecular pathways coming into play upon exposure to PFASs at very low versus high levels. Altogether, the interpretation of existing rodent data on PFOS/PFOA-induced lipid perturbations with respect to the human situation is complex. From a mechanistic perspective, research on human liver cells shows that PFOS/PFOA activate the PPARα pathway, whereas studies on the involvement of other nuclear receptors, like PXR, are less conclusive. Other data indicate that suppression of the nuclear receptor HNF4α signaling pathway, as well as perturbations of bile acid metabolism and transport might be important cellular events that require further investigation. Future studies with human-relevant test systems would help to obtain more insight into the mechanistic pathways pertinent for humans. These studies shall be designed with a careful consideration of appropriate dosing and toxicokinetics, so as to enable biologically plausible quantitative extrapolations. Such research will increase the understanding of possible perturbed lipid homeostasis related to PFOS/ PFOA exposure and the potential implications for human health.

Occupational exposure to hexavalent chromium. Part II. Hazard assessment of carcinogenic effects.

Hexavalent chromium (Cr(VI)) compounds have been studied extensively and several agencies have described their toxicological profile. In the past, personnel of the Dutch Ministry of Defence may have been exposed to Cr(VI) during maintenance activities on NATO equipment. To investigate if this exposure may have caused irreversible adverse health effects, the Dutch National Institute for Public Health and the Environment (RIVM) summarized all available knowledge from previous evaluations. This information was complemented with a scoping review to retrieve new scientific literature. All scientific evidence was evaluated in workshops with external experts to come to an overview of irreversible adverse health effects that could be caused by occupational exposure to Cr(VI) compounds. This review provides the hazard assessment for occupational exposure to Cr(VI) and carcinogenic effects by integrating and weighting evidence provided by international agencies complemented with newly published studies. It was concluded that occupational exposure to Cr(VI) can cause lung cancer, nose and nasal sinus cancer in humans. Cr(VI) is suspected to cause stomach cancer and laryngeal cancer in humans. It is currently insufficiently clear if Cr(VI) can cause cancer of the small intestine, oral cavity, pancreas, prostate or bladder in humans.

Occupational exposure to hexavalent chromium. Part I. Hazard assessment of non-cancer health effects.

Hexavalent chromium (Cr(VI)) compounds have been studied extensively and several agencies have described their toxicological profile. In the past, personnel of the Dutch Ministry of Defence may have been exposed to Cr(VI) during maintenance activities. To investigate if this exposure may have caused irreversible adverse health effects, the Dutch National Institute for Public Health and the Environment (RIVM) summarized all available knowledge from previous evaluations. This information was complemented with a scoping review to retrieve new scientific literature. All scientific evidence was evaluated in workshops with external experts to come to an overview of irreversible adverse health effects that could be caused by occupational exposure to Cr(VI) compounds. This review focuses on non-cancer health effects. It was concluded that occupational exposure to Cr(VI) can cause perforation of the nasal septum by chromium ulcers, chronic lung diseases, including asthma, rhinitis, pulmonary fibrosis and COPD, skin ulcers and allergic contact dermatitis in humans. It is currently insufficiently clear if Cr(VI) can cause irreversible diseases due to disturbances of the immune system (other than allergic contact eczema, allergic asthma and rhinitis and chronic lung diseases) or adverse effects on fertility or prenatal development in humans.

Oxygen tension influences embryonic stem cell maintenance and has lineage specific effects on neural and cardiac differentiation.

The importance of oxygen tension in in vitro cultures and its effect on embryonic stem cell (ESC) differentiation has been widely acknowledged. Research has mainly focussed on ESC maintenance or on one line of differentiation and only few studies have examined the potential relation between oxygen tension during ESC maintenance and differentiation. In this study we investigated the influence of atmospheric (20%) versus physiologic (5%) oxygen tension in ESC cultures and their differentiation within the cardiac and neural embryonic stem cell tests (ESTc, ESTn). Oxygen tension was set at 5% or 20% and cells were kept in these conditions from starting up cell culture until use for differentiation. Under these oxygen tensions, ESC culture showed no differences in proliferation and gene and protein expression levels. Differentiation was either performed in the same or in the alternative oxygen tension compared to ESC culture creating four different experimental conditions. Cardiac differentiation in 5% instead of 20% oxygen resulted in reduced development of spontaneously beating cardiomyocytes and lower expression of cardiac markers Nkx2.5, Myh6 and MF20 (myosin), regardless whether ESC had been cultured in 5% or 20% oxygen tension. As compared to the control (20% oxygen during stem cell maintenance and differentiation), neural differentiation in 5% oxygen with ESC cultured in 20% oxygen led to more cardiac and neural crest cell differentiation. The opposite experimental condition of neural differentiation in 20% oxygen with ESC cultured in 5% oxygen resulted in more glial differentiation. ESC that were maintained and differentiated in 5% oxygen showed an increase in neural crest and oligodendrocytes as compared to 20% oxygen during stem cell maintenance and differentiation. This study showed major effects on ESC differentiation in ESTc and ESTn of oxygen tension, which is an important variable to consider when designing and developing a stem cell-based in vitro system.

An update on the hazard of and exposure to diethyl hexyl phthalate (DEHP) alternatives used in medical devices.

The use of the plasticizer diethyl hexyl phthalate (DEHP) in PVC medical devices is being questioned due to its potential reprotoxic effects in patients exposed as a result from migration from the device. This article reviews new information on migration and toxicity data of eleven alternative plasticizers that have previously been evaluated by the Danish EPA and the EU SCENIHR (Scientific Committee on Emerging and Newly Identified Health Risks). The new toxicity data did not justify the reconsideration of the critical NOAELs as established by SCENIHR and Danish EPA. The dataset on oral toxicity studies is rather complete for most substances; however, in particular for reproductive toxicity and endocrine disruption, data gaps still exist for many alternatives. Toxicity data on intravenous exposure are lacking and these are essential to conclude on hazard characteristics of alternatives that are poorly absorbed via the oral exposure route. Migration data are emerging for a few alternatives but still sparse for the majority of the alternatives. Taking all data on migration and toxicity in consideration, 1,2-cyclohexanedicarboxylic acid, diisononylester (DINCH), and tris(2-ethylhexyl)benzene-1,2,4-tricarboxylate display a more favorable profile compared to DEHP. For these promising alternatives, a risk assessment for use in medical devices should be conducted. As a next step, we recommend the (further) generation of relevant migration data and, where needed, relevant toxicity data for the alternative substances, in order to be able to conduct a benefit-risk analysis of DEHP and the alternatives as obligatory in the new European Union Medical Device Regulation.

Culture Conditions Affect Chemical-Induced Developmental Toxicity In Vitro: The Case of Folic Acid, Methionine and Methotrexate in the Neural Embryonic Stem Cell Test.

In vitro tests are increasingly applied in chemical hazard assessment. Basic culture conditions may affect the outcome of in vitro tests and should be optimised to reduce false predictions. The neural embryonic stem cell test (ESTn) can predict early neurodevelopmental effects of chemicals, as it mimics the differentiation of stem cells towards the neuroectodermal lineage. Normal early neural differentiation depends crucially on folic acid (FA) and methionine (MET), both elements of the one-carbon (1C) cycle. The aim of this study was to assess the concentration-dependent influence of FA and MET on neural differentiation in the ESTn, and its consequences for assay sensitivity to methotrexate (MTX), a compound that interferes with the 1C cycle. Neural differentiation was inhibited below 0.007 mM and above 0.22 mM FA, while both stem cell viability (< 0.097 mM, > 1.52 mM) and neural differentiation (< 0.181 mM, > 1.35 mM) were affected when changing MET concentrations. A 10-day exposure to 13 nM MTX inhibited neural differentiation, especially in FA- and MET-deficient conditions. However, a 24-hour exposure to 39 nM MTX decreased neural cell and neural crest cell differentiation markers only when the concentration of FA in the medium was three times the standard concentration, which was expected to have a protective effect against MTX. These results show the importance of nutrient concentrations, exposure scenarios and timing of read-outs for cell differentiation and compound sensitivity in the ESTn. Caution should be taken when interpreting results from a single in vitro test, especially when extrapolating to effects on complex morphogenetic processes, like neural tube development.

Testing developmental toxicity in a second species: are the differences due to species or replication error?

Developmental toxicity studies for chemical and pharmaceutical safety are primarily performed in rats. Regulatory frameworks may require testing in a second, non-rodent species, for which the rabbit is usually chosen. This study shows that differences in NOAELs or LOAELs (N(L)OAELs) observed between rat and rabbit developmental toxicity studies performed according to OECD guidelines could just as well be caused by study replication errors, and not necessarily by differences in species sensitivity. This conclusion follows from an analysis of a database with rat and rabbit developmental toxicity studies for over 1000 industrial chemicals, pesticides, veterinary drugs and human pharmaceuticals, which included 143 compounds with multiple oral rat studies and 124 compounds with multiple oral rabbit studies. Our analysis confirms earlier findings that, on average over all compounds, rat and rabbit do not differ in sensitivity to developmental effects. There is substantial scatter in the correlation plots comparing rat and rabbit developmental N(L)OAELs, which is easily interpreted as species differences for individual compounds. However, for compounds tested twice in the same species, these N(L)OAELs may differ up to a factor of 25. Thus, potential interspecies differences in developmental N(L)OAEL will be overwhelmed by the reproducibility error, rendering the added value of a second species study questionable. As N(L)OAELs serve as point of departure (POD) for setting health-based guidance values in risk assessment, the large reproducibility error of N(L)OAELs should be taken into account by the introduction of an additional uncertainty factor. It is recommended to aim for reducing the reproducibility error by applying dose-response (BMD) analysis, optimize study designs and harmonize study protocols.

Why Do Countries Regulate Environmental Health Risks Differently? A Theoretical Perspective.

Why do countries regulate, or prefer to regulate, environmental health risks such as radiofrequency electromagnetic fields and endocrine disruptors differently? A wide variety of theories, models, and frameworks can be used to help answer this question, though the resulting answer will strongly depend on the theoretical perspective that is applied. In this theoretical review, we will explore eight conceptual frameworks, from different areas of science, which will offer eight different potential explanations as to why international differences occur in environmental health risk management. We are particularly interested in frameworks that could shed light on the role of scientific expertise within risk management processes. The frameworks included in this review are the Risk Assessment Paradigm, research into the roles of experts as policy advisors, the Psychometric Paradigm, the Cultural Theory of Risk, participatory approaches to risk assessment and risk management, the Advocacy Coalition Framework, the Social Amplification of Risk Framework, and Hofstede's Model of National Cultures. We drew from our knowledge and experiences regarding a diverse set of academic disciplines to pragmatically assemble a multidisciplinary set of frameworks. From the ideas and concepts offered by the eight frameworks, we derive pertinent questions to be used in further empirical work and we present an overarching framework to depict the various links that could be drawn between the frameworks.

Design and validation of an ontology-driven animal-free testing strategy for developmental neurotoxicity testing.

Developmental neurotoxicity entails one of the most complex areas in toxicology. Animal studies provide only limited information as to human relevance. A multitude of alternative models have been developed over the years, providing insights into mechanisms of action. We give an overview of fundamental processes in neural tube formation, brain development and neural specification, aiming at illustrating complexity rather than comprehensiveness. We also give a flavor of the wealth of alternative methods in this area. Given the impressive progress in mechanistic knowledge of human biology and toxicology, the time is right for a conceptual approach for designing testing strategies that cover the integral mechanistic landscape of developmental neurotoxicity. The ontology approach provides a framework for defining this landscape, upon which an integral in silico model for predicting toxicity can be built. It subsequently directs the selection of in vitro assays for rate-limiting events in the biological network, to feed parameter tuning in the model, leading to prediction of the toxicological outcome. Validation of such models requires primary attention to coverage of the biological domain, rather than classical predictive value of individual tests. Proofs of concept for such an approach are already available. The challenge is in mining modern biology, toxicology and chemical information to feed intelligent designs, which will define testing strategies for neurodevelopmental toxicity testing.

Consensus statement on the need for innovation, transition and implementation of developmental neurotoxicity (DNT) testing for regulatory purposes.

This consensus statement voices the agreement of scientific stakeholders from regulatory agencies, academia and industry that a new framework needs adopting for assessment of chemicals with the potential to disrupt brain development. An increased prevalence of neurodevelopmental disorders in children has been observed that cannot solely be explained by genetics and recently pre- and postnatal exposure to environmental chemicals has been suspected as a causal factor. There is only very limited information on neurodevelopmental toxicity, leaving thousands of chemicals, that are present in the environment, with high uncertainty concerning their developmental neurotoxicity (DNT) potential. Closing this data gap with the current test guideline approach is not feasible, because the in vivo bioassays are far too resource-intensive concerning time, money and number of animals. A variety of in vitro methods are now available, that have the potential to close this data gap by permitting mode-of-action-based DNT testing employing human stem cells-derived neuronal/glial models. In vitro DNT data together with in silico approaches will in the future allow development of predictive models for DNT effects. The ultimate application goals of these new approach methods for DNT testing are their usage for different regulatory purposes.

In vitro to in vivo extrapolation of effective dosimetry in developmental toxicity testing: Application of a generic PBK modelling approach.

Incorporation of kinetics to quantitative in vitro to in vivo extrapolations (QIVIVE) is a key step for the realization of a non-animal testing paradigm, in the sphere of regulatory toxicology. The use of Physiologically-Based Kinetic (PBK) modelling for determining systemic doses of chemicals at the target site is accepted to be an indispensable element for such purposes. Nonetheless, PBK models are usually designed for a single or a group of compounds and are considered demanding, with respect to experimental data needed for model parameterization. Alternatively, we evaluate here the use of a more generic approach, i.e. the so-called IndusChemFate model, which is based on incorporated QSAR model parametrization. The model was used to simulate the in vivo kinetics of three diverse classes of developmental toxicants: triazoles, glycol ethers' alkoxyacetic acid metabolites and phthalate primary metabolites. The model required specific input per each class of compounds. These compounds were previously tested in three alternative assays: the whole-embryo culture (WEC), the zebrafish embryo test (ZET), and the mouse embryonic stem cell test (EST). Thereafter, the PBK-simulated blood levels at toxic in vivo doses were compared to the respective in vitro effective concentrations. Comparisons pertaining to relative potency and potency ranking with integration of kinetics were similar to previously obtained comparisons. Additionally, all three in vitro systems produced quite comparable results, and hence, a combination of alternative tests is still preferable for predicting the endpoint of developmental toxicity in vivo. This approach is put forward as biologically more plausible since plasma concentrations, rather than external administered doses, constitute the most direct in vivo dose metric.

Embryotoxic and pharmacologic potency ranking of six azoles in the rat whole embryo culture by morphological and transcriptomic analysis.

Differential gene expression analysis in the rat whole embryo culture (WEC) assay provides mechanistic insight into the embryotoxicity of test compounds. In our study, we hypothesized that comparative analysis of the transcriptomes of rat embryos exposed to six azoles (flusilazole, triadimefon, ketoconazole, miconazole, difenoconazole and prothioconazole) could lead to a better mechanism-based understanding of their embryotoxicity and pharmacological action. For evaluating embryotoxicity, we applied the total morphological scoring system (TMS) in embryos exposed for 48h. The compounds tested showed embryotoxicity in a dose-response fashion. Functional analysis of differential gene expression after 4h exposure at the ID10 (effective dose for 10% decreased TMS), revealed the sterol biosynthesis pathway and embryonic development genes, dominated by genes in the retinoic acid (RA) pathway, albeit in a differential way. Flusilazole, ketoconazole and triadimefon were the most potent compounds affecting the RA pathway, while in terms of regulation of sterol function, difenoconazole and ketoconazole showed the most pronounced effects. Dose-dependent analysis of the effects of flusilazole revealed that the RA pathway related genes were already differentially expressed at low dose levels while the sterol pathway showed strong regulation at higher embryotoxic doses, suggesting that this pathway is less predictive for the observed embryotoxicity. A similar analysis at the 24-hour time point indicated an additional time-dependent difference in the aforementioned pathways regulated by flusilazole. In summary, the rat WEC assay in combination with transcriptomics could add a mechanistic insight into the embryotoxic potency ranking and pharmacological mode of action of the tested compounds.

Comparing rat and rabbit embryo-fetal developmental toxicity data for 379 pharmaceuticals: on systemic dose and developmental effects.

A database of embryo-fetal developmental toxicity (EFDT) studies of 379 pharmaceutical compounds in rat and rabbit was analyzed for species differences based on toxicokinetic parameters of area under the curve (AUC) and maximum concentration (Cmax) at the developmental lowest adverse effect level (dLOAEL). For the vast majority of cases (83% based on AUC of n = 283), dLOAELs in rats and rabbits were within the same order of magnitude (less than 10-fold different) when compared based on available data on AUC and Cmax exposures. For 13.5% of the compounds the rabbit was more sensitive and for 3.5% of compounds the rat was more sensitive when compared based on AUC exposures. For 12% of the compounds the rabbit was more sensitive and for 1.3% of compounds the rat was more sensitive based on Cmax exposures. When evaluated based on human equivalent dose (HED) conversion using standard factors, the rat and rabbit were equally sensitive. The relative extent of embryo-fetal toxicity in the presence of maternal toxicity was not different between species. Overall effect severity incidences were distributed similarly in rat and rabbit studies. Individual rat and rabbit strains did not show a different general distribution of systemic exposure LOAELs as compared to all strains combined for each species. There were no apparent species differences in the occurrence of embryo-fetal variations. Based on power of detection and given differences in the nature of developmental effects between rat and rabbit study outcomes for individual compounds, EFDT studies in two species have added value over single studies.

Scientific principles for the identification of endocrine-disrupting chemicals: a consensus statement.

Endocrine disruption is a specific form of toxicity, where natural and/or anthropogenic chemicals, known as "endocrine disruptors" (EDs), trigger adverse health effects by disrupting the endogenous hormone system. There is need to harmonize guidance on the regulation of EDs, but this has been hampered by what appeared as a lack of consensus among scientists. This publication provides summary information about a consensus reached by a group of world-leading scientists that can serve as the basis for the development of ED criteria in relevant EU legislation. Twenty-three international scientists from different disciplines discussed principles and open questions on ED identification as outlined in a draft consensus paper at an expert meeting hosted by the German Federal Institute for Risk Assessment (BfR) in Berlin, Germany on 11-12 April 2016. Participants reached a consensus regarding scientific principles for the identification of EDs. The paper discusses the consensus reached on background, definition of an ED and related concepts, sources of uncertainty, scientific principles important for ED identification, and research needs. It highlights the difficulty in retrospectively reconstructing ED exposure, insufficient range of validated test systems for EDs, and some issues impacting on the evaluation of the risk from EDs, such as non-monotonic dose-response and thresholds, modes of action, and exposure assessment. This report provides the consensus statement on EDs agreed among all participating scientists. The meeting facilitated a productive debate and reduced a number of differences in views. It is expected that the consensus reached will serve as an important basis for the development of regulatory ED criteria.

Applying 'omics technologies in chemicals risk assessment: Report of an ECETOC workshop.

Prevailing knowledge gaps in linking specific molecular changes to apical outcomes and methodological uncertainties in the generation, storage, processing, and interpretation of 'omics data limit the application of 'omics technologies in regulatory toxicology. Against this background, the European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) convened a workshop Applying 'omics technologies in chemicals risk assessment that is reported herein. Ahead of the workshop, multi-expert teams drafted frameworks on best practices for (i) a Good-Laboratory Practice-like context for collecting, storing and curating 'omics data; (ii) the processing of 'omics data; and (iii) weight-of-evidence approaches for integrating 'omics data. The workshop participants confirmed the relevance of these Frameworks to facilitate the regulatory applicability and use of 'omics data, and the workshop discussions provided input for their further elaboration. Additionally, the key objective (iv) to establish approaches to connect 'omics perturbations to phenotypic alterations was addressed. Generally, it was considered promising to strive to link gene expression changes and pathway perturbations to the phenotype by mapping them to specific adverse outcome pathways. While further work is necessary before gene expression changes can be used to establish safe levels of substance exposure, the ECETOC workshop provided important incentives towards achieving this goal.

Comparison of rat and rabbit embryo-fetal developmental toxicity data for 379 pharmaceuticals: on the nature and severity of developmental effects.

Regulatory non-clinical safety testing of human pharmaceuticals typically requires embryo-fetal developmental toxicity (EFDT) testing in two species (one rodent and one non-rodent). The question has been raised whether under some conditions EFDT testing could be limited to one species, or whether the testing in a second species could be decided on a case-by-case basis. As part of a consortium initiative, we built and queried a database of 379 compounds with EFDT studies (in both rat and rabbit animal models) conducted for marketed and non-marketed pharmaceuticals for their potential for adverse developmental and maternal outcomes, including EFDT incidence and the nature and severity of adverse findings. Manifestation of EFDT in either one or both species was demonstrated for 282 compounds (74%). EFDT was detected in only one species (rat or rabbit) in almost a third (31%, 118 compounds), with 58% (68 compounds) of rat studies and 42% (50 compounds) of rabbit studies identifying an EFDT signal. For 24 compounds (6%), fetal malformations were observed in one species (rat or rabbit) in the absence of any EFDT in the second species. In general, growth retardation, fetal variations, and malformations were more prominent in the rat, whereas embryo-fetal death was observed more often in the rabbit. Discordance across species may be attributed to factors such as maternal toxicity, study design differences, pharmacokinetic differences, and pharmacologic relevance of species. The current analysis suggests that in general both species are equally sensitive on the basis of an overall EFDT LOAEL comparison, but selective EFDT toxicity in one species is not uncommon. Also, there appear to be species differences in the prevalence of various EFDT manifestations (i.e. embryo-fetal death, growth retardation, and dysmorphogenesis) between rat and rabbit, suggesting that the use of both species has a higher probability of detecting developmental toxicants than either one alone.