Screening methodology to identify potential endocrine disruptors according to different options in the context of an impact assessment

Several pieces of EU legislation regulate the marketing and use of chemical substances. While several regulations, including the regulations on Plant Protection Products (PPPR), Biocidal Products (BPR) and Chemicals (REACH), include provisions for endocrine disrupting substances (EDs), objective scientific criteria are lacking. In order to evaluate the potential health, socio-economic and environmental impacts of applying four different options for criteria defining EDs across these pieces of legislation, the Commission initiated an Impact Assessment (IA). This IA has been supported by two studies, focusing on (a) selection of substances for the IA and the screening of their potential for identification as EDs according to different options for defining criteria for identification of endocrine disruptors and (b) the potential impacts of various policy options on health, environment, trade, agriculture and socio-economy. This report describes a screening methodology that has been developed by the JRC to support the first study which has assessed all pesticide and biocide active ingredients and a selection of substances falling under REACH, the Cosmetic Products Regulation and the Water Framework Directive. This screening methodology is not intended to replace an in-depth risk assessment process, and the results obtained are not intended to pre-empt regulatory conclusions that may eventually be made under different pieces of EU legislation.JRC.I.5-Systems Toxicolog

Alternative methods for regulatory toxicology – a state-of-the-art review

This state-of-the art review is based on the final report of a project carried out by the European Commission’s Joint Research Centre (JRC) for the European Chemicals Agency (ECHA). The aim of the project was to review the state of the science of non-standard methods that are available for assessing the toxicological and ecotoxicological properties of chemicals. Non-standard methods refer to alternatives to animal experiments, such as in vitro tests and computational models, as well as animal methods that are not covered by current regulatory guidelines. This report therefore reviews the current scientific status of non-standard methods for a range of human health and ecotoxicological endpoints, and provides a commentary on the mechanistic basis and regulatory applicability of these methods. For completeness, and to provide context, currently accepted (standard) methods are also summarised. In particular, the following human health endpoints are covered: a) skin irritation and corrosion; b) serious eye damage and eye irritation; c) skin sensitisation; d) acute systemic toxicity; e) repeat dose toxicity; f) genotoxicity and mutagenicity; g) carcinogenicity; h) reproductive toxicity (including effects on development and fertility); i) endocrine disruption relevant to human health; and j) toxicokinetics. In relation to ecotoxicological endpoints, the report focuses on non-standard methods for acute and chronic fish toxicity. While specific reference is made to the information needs of REACH, the Biocidal Products Regulation and the Classification, Labelling and Packaging Regulation, this review is also expected to be informative in relation to the possible use of alternative and non-standard methods in other sectors, such as cosmetics and plant protection products.JRC.I.5-Systems Toxicolog

Study of the expression, the regulation and the biological role of adhesion molecules on luteinized granulosa cells in the human ovary

The formation of the corpus luteum (CL) is critical for the establishment of a successful pregnancy. Following ovulation, under the influence of luteogenic hormones, the CL develops from the remnants of the ovulated ovarian follicle. This process involves intense reorganization of constituent cells, phenomena that includes varying cell-matrix interactions. These events, however, are poorly characterized. Adhesion molecules, especially integrins, have crucial role in the physiological functions of many different systems. Many studies, in the past, have established for several cell types that the binding of cell surface integrins to their ligands in the extracellular matrix facilitates cell proliferation, migration and survival. Integrins have also been found to participate in many important functions of the reproductive system, such as fertilization, interactions between the preimplantation embryo and endometriun as well as trophoblast outgrowth during implantation. Moreover, it has been demonstrated that activity of integrins was regulated by human chorionic gonadotropin (hCG) and vascular endothelial growth factor (VEGF). In order to understand the role and potential regulation of cell-matrix interactions in the formation of the CL, we investigated the expression of the matrix protein fibronectin (FN) and selected FN-binding integrin receptors on luteinized granulosa cells (GCs). We further examined the possible regulation of that expression by the luteogenic hormone, hCG and the involvement of mitogenic VEGF. Lastly, we investigated the role of the aforementioned integrins in promoting the adhesion, migration and survival of GCs. The present data reported that FN is detected around GCs with luteinization, while several FN-binding integrins, along with the VEGF receptor (Flt-1), appeared on the surface of these cells during the early luteal phase. Expression of these proteins declined in the late luteal phase with regression of the CL. In vitro, GCs released FN and stimulation of these cells with hCG, increased the surface expression of α5β1 and αvβ3 integrins, as well as the amount of FN associated with the cell surface. These effects were reproduced by stimulation with VEGF and hCG-stimulated up-regulation of FN, α5β1 and αvβ3 on the surface of GCs was inhibited by anti-VEGF antibody. In addition, there has been shown that expression of α5β1 and αvβ3 integrins mediates adhesion to FN, an ability that promotes cell movement and prevents apoptosis. In conclusion, the present study reports new data on the role of integrins in migration, adhesion and apoptosis of human GCs, and the regulation of these processes XI by the hCG and VEGF, and thus indicate the potential contribution of these molecules in formation and maintenance of the CLΟ σχηματισμός του ωχρού σωματίου (CL) αποτελεί βασική λειτουργία για την επιτυχή έκβαση της εγκυμοσύνης. Την φάση της ωοθυλακιορρηξίας, ακολουθεί η ανάπτυξη του CL από τα υπολείμματα του ρηχθέντος θυλακίου, μία διαδικασία που ελέγχεται από τις ωχρινοποιητικές ορμόνες. Κατά την φάση αυτή, τα κύτταρα που συνιστούν το ωχρό σωμάτιο υφίστανται έντονες τροποποιήσεις, που απαιτούν πολύπλοκες αλληλεπιδράσεις μεταξύ του κυττάρου και του εξωκυττάριου χώρου, η πλειονότητα των οποίων παραμένει ασαφής. Τα μόρια συνάφειας, και ιδιαίτερα οι ιντεγκρίνες, διαδραματίζουν εξέχοντα ρόλο σε φυσιολογικές λειτουργίες ενός μεγάλου αριθμού διαφορετικών συστημάτων. Η πρόσδεση των ιντεγκρινικών υποδοχέων στους αντίστοιχους εξωκυττάριους προσδέτες τους έχει ήδη αποδειχτεί ότι προάγει την κυτταρική κίνηση, τον πολλαπλασιαμό και την απόπτωση, σε πολλούς διαφορετικούς κυτταρικούς τύπους. Επιπροσθέτως, οι ιντεγκρίνες συμμετέχουν σε αρκετές σημαντικές λειτουργίες του αναπαραγωγικού συστήματος, συμπεριλαμβανομένης της γονιμοποίησης, της εμφύτευσης του εμβρύου και της αναδιάταξης του πλακούντα. Πιο πρόσφατες μελέτες υποστηρίζουν ότι η ιντεγκρινική δραστηριότητα ελέγχεται, σε πολλές περιπτώσεις, από την ωχρινοποιητική ορμόνη, χοριακή γοναδοτροπίνη (hCG), καθώς και από τον μιτογενετικό παράγοντα (VEGF). Στα πλαίσια διερεύνησης του ρόλου των κυτταρικών αλληλεπιδράσεων με τα μόρια του εξωκυττάριου χώρου στην διαδικασία δημιουργίας του CL, μελετήσαμε την έκφραση μία εξωκυττάριας πρωτεΐνης, της φιμπρονεκτίνης (FN) και επιλεγμένων FN- προσδενόμενων ιντεγκρινών σε ωχρινοποιημένα κοκκώδη κύτταρα (GCs). Επιπλέον, ερευνήσαμε πιθανή ρύθμιση της έκφρασης αυτής από την hCG, καθώς και την ενδεχόμενη συμμετοχή του VEGF παράγοντα στην ίδια διαδικασία. Βασιζόμενοι στα προκύπτοντα αποτελέσματα, επεκτείναμε την έρευνά μας, μελετώντας τον ρόλο των προαναφερόμενων ιντεγκρινών στις διαδικασίες προσκόλλησης, μετανάστευσης και απόπτωσης των ωχρινοποιημένων GCs. Στην παρούσα εργασία αποδείχτηκε ότι η FN ανιχνεύεται στον εξωκυττάριο χώρο των ωχρινοποιημένων GCs, ενώ ταυτόχρονα, οι FN-προσδενόμενες ιντεγκρίνες και ο υποδοχέας του VEGF (Flt-1) εκφράζονται στην κυτταρική τους επιφάνεια, μόνο στα πρώτα στάδια της ωχρινικής φάσης. Η έκφραση αυτή μειώνεται σημαντικά στα τελευταία στάδια της ωχρινικής φάσης, ταυτόχρονα με την ωχρινολυτική διαδικασία. IX Επιπλέον, βρέθηκε ότι τα GCs εκκρίνουν FN, μία λειτουργία που επηρεάζεται από την hCG. Η ορμόνη αυτή αυξάνει και την έκφραση των α5β1 και αvβ3 ιντεγκρινών, με ταυτόχρονη αύξηση της συγκέντρωσης της FN, που δεσμεύεται στην κυτταρική επιφάνεια. Επανάληψη των αποτελεσμάτων παρατηρήθηκε και μετά από διέγερση των κυττάρων με VEGF, ενώ η προσθήκη αντι-VEGF αντισώματος οδηγούσε σε αναστολή της hCG-επαγόμενης αύξησης της έκφρασης. Εν συνεχεία, βρέθηκε ότι οι α5β1 και αvβ3 ιντεγκρίνες ελέγχουν την κυτταρική σύνδεση με την FN του εξωκυττάριου χώρου, μία δυνατότητα που προωθεί την κυτταρική μετανάστευση και αναστέλλει την απόπτωση. Συμπερασματικά, στην συγκεκριμένη εργασία παρουσιάζονται νέα δεδομένα για την συμμετοχή των ιντεγκρινών στις διαδικασίες προσκόλλησης, μετανάστευσης και απόπτωσης των ωχρινοποιημένων GCs ανθρώπινης ωοθήκης, καθώς και για την ρύθμιση των επιδράσεων αυτών από την hCG και τον VEGF. Τα αποτελέσματά μας επομένως, υποδεικνύουν τον σημαντικό ρόλο που διαδραματίζουν τα προαναφερόμενα μόρια στην διαδικασία της δημιουργίας και διατήρησης του CL

Development of the Adverse Outcome Pathway (AOP): Chronic binding of antagonist to N-methyl-D-aspartate receptors (NMDARs) during brain development induces impairment of learning and memory abilities of children

The Adverse Outcome Pathways (AOPs) are designed to provide mechanistic understanding of complex biological systems and pathways of toxicity that result in adverse outcomes (AOs) relevant to regulatory endpoints. AOP concept captures in a structured way the causal relationships resulting from initial chemical interaction with biological target(s) (molecular initiating event) to an AO manifested in individual organisms and/or populations through a sequential series of key events (KEs), which are cellular, anatomical and/or functional changes in biological processes. An AOP provides the mechanistic detail required to support chemical safety assessment, the development of alternative methods and the implementation of an integrated testing strategy. An example of the AOP relevant to developmental neurotoxicity (DNT) is described here following the requirements of information defined by the OECD Users' Handbook Supplement to the Guidance Document for developing and assessing AOPs. In this AOP, the binding of an antagonist to glutamate receptor N-methyl-Daspartate (NMDAR) receptor is defined as MIE. This MIE triggers a cascade of cellular KEs including reduction of intracellular calcium levels, reduction of brain derived neurotrophic factor release, neuronal cell death, decreased glutamate presynaptic release and aberrant dendritic morphology. At organ level, the above mentioned KEs lead to decreased synaptogenesis and decreased neuronal network formation and function causing learning and memory deficit at organism level, which is defined as the AO. There are in vitro, in vivo and epidemiological data that support the described KEs and their causative relationships rendering this AOP relevant to DNT evaluation in the context of regulatory purposes.JRC.F.3-Chemicals Safety and Alternative Method

Development and application of the adverse outcome pathway framework for understanding and predicting chronic toxicity: II. A focus on growth impairment in fish

Adverse outcome pathways (AOPs) organize the knowledge on the progression of toxicity through levels of biological organization. By elucidating the linkages between toxicity events on different levels, the AOPs lay the foundation for mechanism-based alternative testing approaches to hazard assessment. We have previously suggested that development of chronic toxicity AOPs can improve understanding of chronic toxicity and facilitate development of alternative tests. Here, we illustrate this process by focusing on fish growth, which is an apical adverse outcome commonly assessed in chronic toxicity tests, for which the alternatives are actively sought-for. Based on four criteria, namely importance of a particular process for growth-related outcomes, pathway conservation across species, frequency of occurrence of a certain disruption and environmental relevance of chemical-induced effects, we selected to focus on reduction in food intake to initiate middle-out AOP development. To explore the link between reduction in food intake and growth impairment, as well as the mechanisms that cause reduction in food intake, we developed three AOP case studies for pyrethroids, selective serotonin reuptake inhibitors (SSRIs) and cadmium. Our analysis demonstrated that the reduction in food intake is strongly linked to growth impairment in case of pyrethroids and SSRIs, but not cadmium. For pyrethroids, impairment of locomotion is strongly linked to the effects on food intake and growth, but in case of SSRIs, their direct effects on appetite may play a role more important than their impacts on locomotion. For cadmium, the main cause of growth impairment is reallocation of energy resources due to increased maintenance costs. We further discuss which alternative tests can be developed and used to inform on the key events we identified to be predictive of effects on growth. In conclusion, our work demonstrated how the AOP concept can be used in practice to critically assess the knowledge available for specific chronic toxicity cases in order to identify existing knowledge gaps and potential alternative tests.JRC.H.1-Water Resource

Development and application of the adverse outcome pathway framework for understanding and predicting chronic toxicity: I. Challenges and research needs in ecotoxicology

To elucidate the effects of chemicals on populations of different species in the environment, efficient testing and modeling approaches are needed that consider multiple stressors and allow reliable extrapolation of responses across species. An adverse outcome pathway (AOP) is a concept that provides a framework for organizing knowledge about the progression of events resulting in toxicity across scales of biological organization. In this paper, we focus on exploring how the AOP concept can be used to guide research aimed at improving both our understanding of chronic toxicity, including delayed toxicity as well as epigenetic and transgenerational effects of chemicals, and our ability to predict adverse outcomes. A better understanding of the influence of subtle toxicity effects on individual and population fitness would support a broader integration of sublethal endpoints into risk assessment frameworks. Detailed mechanistic knowledge would facilitate the development of alternative testing methods as well as help prioritize higher tier toxicity testing. We argue that targeted development of AOPs supports both of these aspects by promoting the elucidation of molecular mechanisms and their contribution to relevant toxicity outcomes across biological scales. We further discuss information requirements and challenges in application of AOPs for chemical- and site-specific risk assessment and for extrapolation across species. We provide recommendations for potential extension of the AOP framework to incorporate information on exposure, toxicokinetics and situation-specific ecological contexts, and discuss common interfaces that can be employed to couple AOPs with computational modeling approaches and with evolutionary life history theory. The extended AOP framework can serve as a venue for integration of knowledge derived from various sources, including empirical data as well as molecular, quantitative and evolutionary-based models describing species responses to toxicants. In the future this will allow a more efficient application of AOP knowledge for quantitative chemical- and site-specific risk assessment as well as for extrapolation across species.JRC.H.1-Water Resource

Development and application of the adverse outcome pathway framework for understanding and predicting chronic toxicity. I. Challenges and research needs in ecotoxicology

To elucidate the effects of chemicals on populations of different species in the environment, efficient testing and modeling approaches are needed that consider multiple stressors and allow reliable extrapolation of responses across species. An adverse outcome pathway (AOP) is a concept that provides a framework for organizing knowledge about the progression of toxicity events across scales of biological organization that lead to adverse outcomes relevant for risk assessment. In this paper, we focus on exploring how the AOP concept can be used to guide research aimed at improving both our understanding of chronic toxicity, including delayed toxicity as well as epigenetic and transgenerational effects of chemicals, and our ability to predict adverse outcomes. A better understanding of the influence of subtle toxicity on individual and population fitness would support a broader integration of sublethal endpoints into risk assessment frameworks. Detailed mechanistic knowledge would facilitate the development of alternative testing methods as well as help prioritize higher tier toxicity testing. We argue that targeted development of AOPs supports both of these aspects by promoting the elucidation of molecular mechanisms and their contribution to relevant toxicity outcomes across biological scales. We further discuss information requirements and challenges in application of AOPs for chemical- and site-specific risk assessment and for extrapolation across species. We provide recommendations for potential extension of the AOP framework to incorporate information on exposure, toxicokinetics and situation-specific ecological contexts, and discuss common interfaces that can be employed to couple AOPs with computational modeling approaches and with evolutionary life history theory. The extended AOP framework can serve as a venue for integration of knowledge derived from various sources, including empirical data as well as molecular, quantitative and evolutionary-based models describing species responses to toxicants. This will allow a more efficient application of AOP knowledge for quantitative chemical- and site-specific risk assessment as well as for extrapolation across species in the future.ISSN:0045-6535ISSN:1879-129

Development and application of the adverse outcome pathway framework for understanding and predicting chronic toxicity. II. A focus on growth impairment in fish

Adverse outcome pathways (AOPs) organize knowledge on the progression of toxicity through levels of biological organization. By determining the linkages between toxicity events at different levels, AOPs lay the foundation for mechanism-based alternative testing approaches to hazard assessment. Here, we focus on growth impairment in fish to illustrate the initial stages in the process of AOP development for chronic toxicity outcomes. Growth is an apical endpoint commonly assessed in chronic toxicity tests for which a replacement is desirable. Based on several criteria, we identified reduction in food intake to be a suitable key event for initiation of middle-out AOP development. To start exploring the upstream and downstream links of this key event, we developed three AOP case studies, for pyrethroids, selective serotonin reuptake inhibitors (SSRIs) and cadmium. Our analysis showed that the effect of pyrethroids and SSRIs on food intake is strongly linked to growth impairment, while cadmium causes a reduction in growth due to increased metabolic demands rather than changes in food intake. Locomotion impairment by pyrethroids is strongly linked to their effects on food intake and growth, while for SSRIs their direct influence on appetite may play a more important role. We further discuss which alternative tests could be used to inform on the predictive key events identified in the case studies. In conclusion, our work demonstrates how the AOP concept can be used in practice to assess critically the knowledge available for specific chronic toxicity cases and to identify existing knowledge gaps and potential alternative tests.ISSN:0045-6535ISSN:1879-129

Luteogenic Hormones Act through a Vascular Endothelial Growth Factor-Dependent Mechanism to Up-Regulate α(5)β(1) and α(v)β(3) Integrins, Promoting the Migration and Survival of Human Luteinized Granulosa Cells

The formation of the corpus luteum (CL) is critical for the establishment of a successful pregnancy. After ovulation, the CL develops from the remnants of the ovulated ovarian follicle. This process, which involves varying cell-matrix interactions, is poorly characterized. To understand the role and potential regulation of cell-matrix interactions in the formation of the CL, we investigated the expression and activity of the matrix protein fibronectin (FN) and several of its integrin receptors on luteinized granulosa cells (GCs). In situ, FN and several FN-binding integrins were detected around luteinizing GCs during the early luteal phase, although expression declined in the late luteal phase. In vitro, GCs released FN, and stimulation of these cells with human chorionic gonadotropin increased the surface expression of FN, α(5)β(1), and α(v)β(3). Up-regulation of these proteins on GCs was reproduced by stimulation with vascular endothelial growth factor (VEGF) and was inhibited by anti-VEGF antibody. Lastly, expression of α(5)β(1) and α(v)β(3) mediated adhesion to FN, facilitated migration, and prevented apoptosis. These data suggest that in vivo luteogenic hormones, in part through a VEGF-dependent mechanism, stimulate selected integrin-matrix adhesive interactions that promote the motility and survival of GCs and thus contribute to the formation and preservation of the CL

Putative adverse outcome pathways relevant to neurotoxicity

The limitations of current in vivo testing coupled with the paucity of data for thousands of chemicals in the human chemosphere has significantly stymied hazard identification and risk assessment needed to protect the developing and mature human nervous system. To address this problem, new biochemical and cellular assay systems as well as computational predictive methods are being developed to more efficiently generate, interpret and use new and existing data for improved chemical safety evaluation. The Adverse Outcome Pathway (AOP) framework provides a template that facilitates understanding of complex biological systems and the pathways of toxicity that result in adverse outcomes (AOs). It has been developed to be used in human health risk assessment as it provides a tool for a knowledge-based safety assessment that relies on understanding toxicity, rather than simply observing its effects. The AOP approach describes a sequence of measurable key events (KEs) that link chemical exposure to an AO at the organism or population level. The AOP starts with an molecular initiating event (MIE) in which a chemical interacts with a biological target(s), followed by a sequential series of KEs, which are cellular, anatomical and functional changes in biological processes, that ultimate result in an AO manifest in individual organisms and populations. A large number of cellular and molecular processes are known to be crucial to proper development and function of the central and peripheral nervous systems (CNS and PNS, respectively). However, there are relatively few examples of well-documented pathways that include causally linked MIEs and KEs that result in adverse outcomes in the CNS or PNS. This lack of AOPs, and in particular the lack of MIEs, has made it difficult to evaluate the predictive ability of high-throughput chemical testing for neurotoxicity, which in turn has hampered discussion of how to use in vitro testing data for regulatory use. As a first step in applying the AOP framework to adverse health outcomes associated with exposure to exogenous neurotoxic substances, the EU Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) organized a workshop (March 2013, Ispra, Italy) to identify potential AOPs relevant to neurotoxic and developmental neurotoxic outcomes. Although the AOPs outlined during the workshop are not complete, they serve as a basis for further research and identification of MIEs and KEs that could help prioritise alternative assay development needed to advance human health protection.JRC.I.5-Systems Toxicolog