[Show abstract][Hide abstract] ABSTRACT: Numerous environmental contaminants have been linked to adverse reproductive health outcomes. However, the complex correlation structure of exposures and multiple testing issues limit the interpretation of existing evidence. Our objective was to identify, from a large set of contaminant exposures, exposure profiles associated with biomarkers of male reproductive function.
[Show abstract][Hide abstract] ABSTRACT: Based on a proposal made at the ICH Workshop in Tallinn, Estonia (2010), the value of the rabbit embryo-fetal development (EFD) versus the rodent EFD was examined by the HESI DART group. A cross-industry data survey provided anonymised EFD and toxicokinetic data from EFD studies on over 400 marketed and unmarketed drugs (over 800 studies) that were entered by experts at RIVM into US EPA's ToxRefDB style database. The nature and severity of findings at the lowest observed adverse effect level (LOAEL) are being reviewed to quantitate the frequency with which lesser signs of embryo-fetal effects (e.g., delays in ossification, minor changes in frequency of variants) are driving the LOAELs. Interpretation was based on exposure rather than administered dose. This paper provides an update of this ongoing project as discussed during a workshop of the European Teratology Society in Ispra, Italy (2013). This was the first presentation of the initial data set, allowing debate on future directions, to provide a better understanding of the implications of either delaying a rabbit EFD or waiving the need in particular circumstances.
[Show abstract][Hide abstract] ABSTRACT: To improve the predictability of the zebrafish embryotoxicity test (ZET) for developmental (neuro) toxicity screening, we used a multiple-endpoints strategy, including morphology, motor activity (MA), histopathology and kinetics. The model compounds used were antiepileptic drugs (AEDs): valproic acid (VPA), carbamazepine (CBZ), ethosuximide (ETH) and levetiracetam (LEV). For VPA, histopathology was the most sensitive parameter, showing effects already at 60μM. For CBZ, morphology and MA were the most sensitive parameters, showing effects at 180μM. For ETH, all endpoints showed similar sensitivity (6.6mM), whereas MA was the most sensitive parameter for LEV (40mM). Inclusion of kinetics did not alter the absolute ranking of the compounds, but the relative potency was changed considerably. Taking all together, this demo-case study showed that inclusion of multiple-endpoints in ZET may increase the sensitivity of the assay, contribute to the elucidation of the mode of toxic action and to a better definition of the applicability domain of ZET.
[Show abstract][Hide abstract] ABSTRACT: Many brominated flame retardants (BFRs)-including polybrominated diphenyl ethers (PBDEs)-have been shown to persist in the environment, and some have been associated with adverse health effects. The aim of the present study was to quantify serum concentrations of common brominated flame retardants in Inuit men from across Greenland, and in men from Warsaw, Poland and Kharkiv, Ukraine. Serum was sampled between 2002 and 2004 from men 19 to 50years of age. 299 samples were analyzed for BDE-28, 47, 99, 100, 153, 154 and 183 and the brominated biphenyl BB-153 using gas chromatography-high resolution mass spectrometry. BDE-47 and BDE-153 were detected in more than 95% of samples from all three populations. All other congeners, except BDE-154, were detected in more than 70% of samples from Greenland; lower detection frequencies were observed in Polish and Ukrainian samples. Concentrations of individual congeners were 2.7 to 15 fold higher in Greenlandic relative to Polish and Ukrainian men. Geometric mean concentrations of the sum of the most abundant PBDEs of the Penta-BDE commercial mixture (BDE-47, 99, 100, 153 and 154) were 6.1, 1.7 and 0.87ng/g lipids in the Greenlandic, Polish and Ukrainian men, respectively. Furthermore, significant geographical differences in BFR concentrations were observed within Greenland. Principal component analysis revealed distinct clustering of samples by country of origin. The associations between ΣPBDEs and age were inconsistent, varying from no association in Greenlandic and Polish study populations to a U-shaped relationship in Ukrainians. We report BFR levels for three populations for which sparse biomonitoring data exists.
Environment international 09/2013; 61C:8-16. · 6.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Some recent EU Regulations have focused on the potential risks posed by the presence of Endocrine Disrupters (ED) into the environment. However there are conflicting opinions on how to assess the risk from exposure to these molecules that can reversibly modulate hormonal activity, Endocrine Active Substances (EAS) rather than causing irreversible damage (ED).The present paper attempts to discuss that perturbation of normal endocrine homeostasis in itself may not be an adverse effect, since the endocrine system is naturally dynamic and responsive to various stimuli as part of its normal function and it is modulated according to the characteristic trend of the dose response curve.EDs should be evaluated using a weight-of-evidence (WoE) approach. If a chemical meets the criteria to be defined as an ED in experimental animals, the relevance of observed effects to the human then needs to be addressed.Hazard-based risk management is therefore not justified since does not meet the criteria for a sound scientifically-based assessment.
[Show abstract][Hide abstract] ABSTRACT: From 15-17 June 2011, a dedicated workhop was held on the subject of in vitro models for mammalian spermatogenesis and their applications in toxicological hazard and risk assessment. The workshop was sponsored by the Dutch ASAT initiative (Assuring Safety without Animal Testing), which aims at promoting innovative approaches towards toxicological hazard and risk assessment on the basis of human and in vitro data, and replacement of animal studies. Participants addressed the state of the art regarding human and animal evidence for compound mediated testicular toxicity, reviewed existing alternative assay models, and brainstormed about future approaches, specifically considering tissue engineering. The workshop recognized the specific complexity of testicular function exemplified by dedicated cell types with distinct functionalities, as well as different cell compartments in terms of microenvironment and extracellular matrix components. This complexity hampers quick results in the realm of alternative models. Nevertheless, progress has been achieved in recent years, and innovative approaches in tissue engineering may open new avenues for mimicking testicular function in vitro. Although feasible, significant investment is deemed essential to be able to bring new ideas into practice in the laboratory. For the advancement of in vitro testicular toxicity testing, one of the most sensitive end points in regulatory reproductive toxicity testing, such an investment is highly desirable.
[Show abstract][Hide abstract] ABSTRACT: The application of alternative methods in developmental and reproductive toxicology is challenging in view of the complexity of mechanisms involved. A battery of complementary test systems may provide a better prediction of developmental and reproductive toxicity than single assays. We tested twelve compounds with varying mechanisms of toxic action in an assay battery including 24 CALUX transcriptional activation assays, mouse cardiac embryonic stem cell test, ReProGlo assay, zebrafish embryotoxicity assay, and two CYP17 and two CYP19 activity assays. The battery correctly detected 11/12 compounds tested, with one false negative occurring, which could be explained by the absence of the specific mechanism of action of this compound in the battery. Toxicokinetic modeling revealed that toxic concentrations were in the range expected from in vivo reproductive toxicity data. This study illustrates added value of combining assays that contain complementary biological processes and mechanisms, increasing predictive value of the battery over individual assays.
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: Availability of chemical response-specific lists of genes (gene sets) for pharmacological and/or toxic effect prediction for compounds is limited. We hypothesize that more gene sets can be created by next-generation text mining (next-gen TM), and that these can be used with gene set analysis (GSA) methods for chemical treatment identification, for pharmacological mechanism elucidation, and for comparing compound toxicity profiles. METHODS: We created 30,211 chemical response-specific gene sets for human and mouse by next-gen TM, and derived 1,189 (human) and 588 (mouse) gene sets from the Comparative Toxicogenomics Database (CTD). We tested for significant differential expression (SDE) (false discovery rate -corrected p-values < 0.05) of the next-gen TM-derived gene sets and the CTD-derived gene sets in gene expression (GE) data sets of five chemicals (from experimental models). We tested for SDE of gene sets for six fibrates in a peroxisome proliferator-activated receptor alpha (PPARA) knock-out GE dataset and compared to results from the Connectivity Map. We tested for SDE of 319 next-gen TM-derived gene sets for environmental toxicants in three GE data sets of triazoles, and tested for SDE of 442 gene sets associated with embryonic structures. We compared the gene sets to triazole effects seen in the Whole Embryo Culture (WEC), and used principal component analysis (PCA) to discriminate triazoles from other chemicals. RESULTS: Next-gen TM-derived gene sets matching the chemical treatment were significantly altered in three GE data sets, and the corresponding CTD-derived gene sets were significantly altered in five GE data sets. Six next-gen TM-derived and four CTD-derived fibrate gene sets were significantly altered in the PPARA knock-out GE dataset. None of the fibrate signatures in cMap scored significant against the PPARA GE signature. 33 environmental toxicant gene sets were significantly altered in the triazole GE data sets. 21 of these toxicants had a similar toxicity pattern as the triazoles. We confirmed embryotoxic effects, and discriminated triazoles from other chemicals. CONCLUSIONS: Gene set analysis with next-gen TM-derived chemical response-specific gene sets is a scalable method for identifying similarities in gene responses to other chemicals, from which one may infer potential mode of action and/or toxic effect.
BMC Medical Genomics 01/2013; 6(1):2. · 3.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Toxicological hazard assessment currently finds itself at a crossroads where the existing classical test paradigm is challenged by a host of innovative approaches. Animal study protocols are being enhanced for additional parameters and improved for more efficient effect assessment with reduced animal numbers. Whilst existing testing paradigms have generally proven conservative for chemical safety assessment, novel alternative in silico and in vitro approaches and assays are being introduced that begin to elucidate molecular mechanisms of toxicity. Issues such as animal welfare, alternative assay validation, endocrine disruption, and the US-NAS report on toxicity testing in the twenty-first century have provided directionality to these developments. The reductionistic nature of individual alternative assays requires that they be combined in a testing strategy in order to provide a complete picture of the toxicological profile of a compound. One of the challenges of this innovative approach is the combined interpretation of assay results in terms of toxicologically relevant effects. Computational toxicology aims at providing that integration. In order to progress, we need to follow three steps: (1) Learn from past experience in animal studies and human diseases about critical end points and pathways of toxicity. (2) Design alternative assays for essential mechanisms of toxicity. (3) Build an integrative testing strategy tailored to human hazard assessment using a battery of available alternative tests for critical end points that provides optimal in silico and in vitro filters to upgrade toxicological hazard assessment to the mechanistic level.
[Show abstract][Hide abstract] ABSTRACT: In developmental toxicity testing, in vitro screening assays are highly needed to increase efficiency and to reduce animal use. A promising in vitro assay is the cardiac mouse embryonic stem cell test (ESTc), in which the effect of developmental toxicants on cardiomyocyte differentiation is assessed. Recently, we developed a neural differentiation variant of the stem cell test (ESTn). In both of these models, we have previously performed a series of transcriptomic studies to characterize gene expression changes 1) across time during normal differentiation and 2) in response to a series of developmental toxicants in the ESTn and ESTc. Here, , using the cumulative of these studies, we compared gene expression profiles of ESTn and ESTc over time as well as model-specific changes induced by seven compounds, comprising of six known in vivo developmental toxicants and one negative control. Time-related gene expression profiles showed similarities between the two EST systems. However, specific genes could be identified changing over time differently in each model related to the two different lineages of differentiation. . Interestingly, compound-induced gene-expression changes were generally model-specific, especially for methylmercury and flusilazole, which were predicted better in ESTn and in ESTc, respectively. Valproic acid-induced gene expression changes were most comparable out of the six developmental toxicants between the ESTn and ESTc. Direct transcriptomic comparisons between the ESTn and ESTc indicate that combined transcriptomic analyses support and compliment each other. . Therefore, a combined approach incorporating ESTc and ESTn may improve developmental toxicant detection over individual assays.
[Show abstract][Hide abstract] ABSTRACT: Developmental toxicity testing according to the globally standardized OECD 414 protocol is an important basis for decisions on classification and labeling of developmental toxicants in the European Union (EU). This test requires relatively large animal numbers, given that parental and offspring generations are involved. In vitro assay designs and systems biology paradigms are being developed to reduce animal use and to improve prediction of human hazard. Such approaches could benefit from the long-term experience with animal protocols and more specifically from information on the relevance of effects observed in these tests for developmental toxicity. Therefore, we have analyzed relative parameter sensitivity in 22 publicly available developmental toxicity studies, representing about one third of all classified developmental toxicants under European legislation. Maternal and fetal weight effects and fetal survival were most often affected parameters at the developmental Lowest Observed Adverse Effect Level (dLOAEL), followed by skeletal malformations. Specific end points such as cleft palate were observed in fewer studies at dLOAEL, but if observed may have been crucial in classification and labeling decisions. These results are similar to earlier studies using different selections of chemicals, indicating that in general classified developmental toxicants have a similar pattern of effects at the dLOAEL as chemicals in general. These findings are discussed within the perspective of the development of innovative alternative approaches to developmental hazard assessment.
[Show abstract][Hide abstract] ABSTRACT: Alternative assays for developmental toxicity testing are needed to reduce animal use in regulatory toxicology. The in vitro murine neural embryonic stem cell test (ESTn) was designed as an alternative for neurodevelopmental toxicity testing. The integration of toxicogenomic-based approaches may further increase predictivity as well as provide insight into underlying mechanisms of developmental toxicity. In the present study, we investigated concentration-dependent effects of six mechanistically diverse compounds, acetaldehyde (ACE), carbamazepine (CBZ), flusilazole (FLU), monoethylhexyl phthalate (MEHP), penicillin G (PENG) and phenytoin (PHE), on the transcriptome and neural differentiation in the ESTn. All compounds with the exception of PENG altered ESTn morphology (cytotoxicity and neural differentiation) in a concentration-dependent manner. Compound induced gene expression changes and corresponding enriched gene ontology biological processes (GO-BP) were identified after 24h exposure at equipotent differentiation-inhibiting concentrations of the compounds. Both compound-specific and common gene expression changes were observed between subsets of tested compounds, in terms of significance, magnitude of regulation and functionality. For example, ACE, CBZ and FLU induced robust changes in number of significantly altered genes (≥ 687 genes) as well as a variety of GO-BP, as compared to MEHP, PHE and PENG (≤ 55 genes with no significant changes in GO-BP observed). Genes associated with developmentally related processes (embryonic morphogenesis, neuron differentiation, and Wnt signaling) showed diverse regulation after exposure to ACE, CBZ and FLU. In addition, gene expression and GO-BP enrichment showed concentration dependence, allowing discrimination of non-toxic versus toxic concentrations on the basis of transcriptomics. This information may be used to define adaptive versus toxic responses at the transcriptome level.
Toxicology and Applied Pharmacology 05/2012; 262(3):330-40. · 3.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: There are a large number of chemicals in current use for which adequate toxicity data are not available. Whilst there are clear ethical and legal obligations to obtain data from sources other than in vivo experiments wherever possible, in certain cases in vivo assays may be deemed necessary. In such circumstances, it is essential to ensure that the maximum amount of high quality data is obtained from the minimum number of animals, using the most humane procedures, in accordance with the philosophy of reduction, refinement and replacement (3Rs). The aim of this report is to provide a strategy for anyone involved in animal experimentation, for either toxicological or pharmacological purposes, as to how in vivo experiments may be optimised. The impact of generic and endpoint specific sources of variability has been highlighted in a proof-of-principle analysis considering the variation in protocols for assays for four human health endpoints (skin sensitisation, reproductive/developmental toxicity, mutagenicity and carcinogenicity). Other factors such as operator training, experimental/statistical design, use of lower species and use of combined assays are also discussed. Recommendations for optimisation of in vivo assays, in terms of the 3Rs philosophy, applied to performing tests, harvesting data and appropriate reporting are summarised as a checklist of issues to be addressed prior to undertaking such assays.
Regulatory Toxicology and Pharmacology 03/2012; 63(1):140-54. · 2.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Reproductive toxicity testing is characterized by high animal use. For registration of pharmaceutical compounds, developmental toxicity studies are usually conducted in both rat and rabbits. Efforts have been underway for a long time to design alternatives to animal use. Implementation has lagged, partly because of uncertainties about the applicability domain of the alternatives. The reproductive cycle is complex and not all mechanisms of development can be mimicked in vitro. Therefore, efforts are underway to characterize the available alternative tests with regard to the mechanism of action they include. One alternative test is the mouse embryonic stem cell test (EST), which has been studied since the late 1990s. It is a genuine 3R "alternative" assay as it is essentially animal-free. A meeting was held to review the state-of-the-art of various in vitro models for prediction of developmental toxicity. Although the predictivity of individual assays is improving, a battery of several assays is likely to have even higher predictivity, which is necessary for regulatory acceptance. The workshop concluded that an important first step is a thorough survey of the existing rat and rabbit studies, to fully characterize the frequency of responses and the types of effects seen. At the same time, it is important to continue the optimization of in vitro assays. As more experience accumulates, the optimal conditions, assay structure, and applicability of the alternative assays are expected to emerge.
Regulatory Toxicology and Pharmacology 03/2012; 63(1):115-23. · 2.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The possible impact on classification and labelling decisions of effects observed in second generation parental (P1) and offspring (F2) parameters in multi-generation studies was investigated. This was done for 50 substances classified as reproductive toxicants in Europe, for which a multi-generation study was available. The P1 and F2 effects were compared to parental (P0) and first generation offspring (F1) effects with regard to type of effect as well as incidence, magnitude and severity (IMS), at any dose level. For every study with unique P1/F2 effects, or differences in IMS, the influence of the P1/F2 findings on the classification decision was investigated. Unique P1/F2 generation findings did not play a crucial role in the classification decision of any of the 50 classified substances, except for fenarimol. This substance however provided abundant alerts on the basis of its endocrine activity and developmental neurotoxicity and would therefore also be expected to be identified as a developmental neurotoxicant in an Extended One Generation Reproductive Toxicity Study (EOGRTS). These findings, in addition to the increased number of parameters analysed, increased statistical power and reduced animal use, provide strong further support for replacement of the classical two-generation reproductive toxicity study by the EOGRTS in regulatory reproductive toxicity assessment.
Regulatory Toxicology and Pharmacology 08/2011; 61(2):251-60. · 2.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This paper surveys the scientific basis for the current threshold approach for reproductive hazard and risk assessment. In some regulatory areas it was recently suggested to consider reproductive toxicants under the stringent linear extrapolation risk assessment paradigm that was developed for genotoxic carcinogens. First, the current risk assessment paradigm for genotoxic carcinogens is addressed, followed by an overview of reproductive toxicology and its threshold dose approach for hazard and risk assessment, the testing procedures for assessing the reproductive toxicity of chemicals, and the derivation of conclusions on their risk assessment and Classification, Labelling and Packaging (CLP). Relevant details of testing methodologies are discussed, such as exposure time windows, parameters determined, and the coverage of the entire reproductive cycle. In addition, the dose-response relationship is considered, illustrated with several examples. It is concluded that the current risk assessment methodology for genotoxic carcinogens is a debatable worst-case scenario and that for risk assessment of reproductive toxicants the threshold dose approach remains valid.
Critical Reviews in Toxicology 07/2011; 41(6):545-54. · 6.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Current globally harmonized Organisation for Economic Co-operation and Development (OECD) animal test guidelines for developmental toxicity require high numbers of experimental animals. To reduce animal use in this field, alternative developmental toxicity assays are highly desirable. We previously developed a dynamic in vitro model for screening effects of possible neurodevelopmental toxicants, using neural cell differentiation of pluripotent murine embryonic stem cells. To further mechanistically characterize the mouse neural embryonic stem cell test (ESTn) and to improve detection of possible neurodevelopmental toxicants, gene expression patterns were studied describing neural cell differentiation over time, as well as the impact on gene expression of exposure to the well-known neurotoxicant methylmercury (MeHg). A transcriptomics study was performed to examine whole-genome expression changes during the first 7 days of the cell differentiation protocol. Specific gene clusters were identified and enrichment analysis of Gene Ontology (GO) terms and gene sets derived from literature was performed using DAVID and T-profiler. Over time, a decrease of blastocyst and trophectoderm GO terms was observed, which included well-characterized pluripotency genes. Furthermore, an increase in the range of neural development-related GO terms, such as neuron differentiation and the wnt pathway, was observed. Analysis of gene expression using principle component analysis showed a time-dependent track in untreated cells, describing the process of neural differentiation. Furthermore, MeHg was shown to induce deviation from the predefined differentiation track. The compound inhibited general development GO terms and induced neural GO terms over time. This system appears promising for studying compound effects on neural differentiation in a mechanistic approach.