Periodic Reporting for period 3 - ENDpoiNTs (Novel Testing Strategies for Endocrine Disruptors in the Context of Developmental NeuroToxicity)
Periodo di rendicontazione: 2022-01-01 al 2023-06-30
ENDpoiNTs is developing new test methods to meet the regulatory, scientific and societal needs for improved assessment of endocrine disrupting chemicals (EDCs). A significant knowledge gap is how EDCs affect neurodevelopment, and endocrine disruption (ED)-induced developmental neurotoxicity (DNT) is hardly covered by the testing tools in regulatory use. The main objective of ENDpoiNTs is to generate new scientific knowledge on how ED is linked to DNT at the molecular, cellular, tissue, and organism level. Based on this new knowledge, the project will:
• Develop predictive computational tools for chemical screening
• Develop and validate cellular testing and screening tools
• Develop novel molecular endpoints for existing animal-based test guidelines
• Ensure human relevance by linking experimental and epidemiological evidence
• Engage with key stakeholders and develop novel strategies for EDC testing and assessment into European and international chemical regulatory frameworks
• Develop predictive computational tools for chemical screening
• Develop and validate cellular testing and screening tools
• Develop novel molecular endpoints for existing animal-based test guidelines
• Ensure human relevance by linking experimental and epidemiological evidence
• Engage with key stakeholders and develop novel strategies for EDC testing and assessment into European and international chemical regulatory frameworks
A number of models exist to study DNT endpoints, addressing cellular key events and neurodevelopmental outcomes. However, the ability of these models to detect ED-induced DNT is largely unknown. In ENDpoiNTs, we have assessed the responsiveness of well-established DNT key events to endocrine interference. Potentially relevant ED pathways were selected using in silico modelling to predict targets of EDCs associated with neurodevelopmental impairments in humans. Using receptor agonists and antagonists, their link to cellular key events was established in a number of in vitro models. This screening effort showed that all investigated key events are dependent on one or several of the selected hormonal receptors. Thereby, novel roles for hormonal receptors in neurodevelopmental key events were uncovered. Some results were in accordance between human and rodent models while others diverged, underscoring species differences. For the most sensitive and robust assays, positive and negative controls as well as standard operating procedures (SOPs) have been established. Additionally, brain organoids have been established for ED-DNT testing and a strategy developed to use such 3D cell models to unravel the contribution of genetic variability in response to ED-induced DNT. The response of selected assays have been tested with EDCs that are associated with DNT outcomes in humans. Depending on the assays between 5 and 25 chemicals have already been tested. Results show that some of these EDCs indeed affect cellular endpoints in low doses. One of the developed cellular assays is currently undergoing pre-validation including inter-laboratory transfer. Additionally, six in vitro assays addressing ED-induced DNT are prepared for a submission to the OECD test guideline program.
To link the molecular and cellular key events addressed in the in vitro models to adverse outcomes in whole organisms, several in vivo models are employed. Rats were developmentally exposed to six EDCs. Behavioural analyses in the adults showed sex-specific effects on learning and memory for some of these EDCs. Currently, molecular analyses, including transcriptomics, epigenomics, metabolomics, lipidomics, and steroidomics are in progress. First results show that a number of gene networks, steroids, neurotransmitters and lipids are dysregulated by the six EDCs, and compound and sex-specific effects were observed. These molecular effects will be correlated to the behavioural outcomes and transcriptional data sets from in vitro models, to provide a link between the cellular and the organismal outcomes. Also in Xenopus laevis, a systematic correlation between early transcriptional dysregulation and adult adverse outcomes is ongoing using the same EDCs as well as hormone agonists and antagonists.
For the management and handling of the experimental data, a standardised database platform was developed. It includes data capture, curation procedures, and statistical analysis approaches. All ENDpoiNTs data is contained in the database (currently 2.316.378 data records) and, at the end of the project, will be made available as an invaluable resource for the community. Furthermore, 12 first tier screening high confidence Quantitative Structure Activity Relationships (QSARs) were developed for predicting the agonistic and antagonistic modes of action of the selected receptors. This is the basis for in silico models identifying chemicals that can induce DNT via these interactions. To extrapolate in vitro to in vivo concentrations of EDCs, a physiology-based toxicokinetic (PBTK) approach has been developed, predicting fetal brain concentrations. Furthermore, data produced in ENDpoiNTs is integrated into existing Adverse Outcome Pathways (AOPs) and AOP networks are built.
To establish human relevance of the test methods, doses producing an adverse effect in test systems are currently being compared with human exposure data for both single reference EDCs and for their mixture. For comparison, the metrics have been evaluated using established regulatory values translated to biomonitoring equivalent concentrations. Furthermore, a mixture study of EDCs is being finalised, with the aim to compare effects of a real-life mixture, established based on human data, to single compounds in the developed in vitro models. To further align experimental with human evidence, molecular data on the level of metabolomics and epigenomics will be compared. To this end, analyses in the epidemiological data have been conducted to provide links between molecular patterns and both prenatal exposures and behavioural outcomes.
To ensure the uptake of the developed assays and strategies into the regulatory context, ENDpoiNTs is actively engaging with key stakeholders, e.g. in workshops. Furthermore, communication channels with relevant working groups of the OECD have been strengthened to enable continuous discussion of the readiness of the developed assays for validation and regulatory implementation. Finally, to disseminate the project’s results to different target groups, ENDpoiNTs has produced project flyers and animated short films, and is actively maintaining its webpage (https://endpoints.eu/) as well as continuously publishing its results in scientific journals with open access.
To link the molecular and cellular key events addressed in the in vitro models to adverse outcomes in whole organisms, several in vivo models are employed. Rats were developmentally exposed to six EDCs. Behavioural analyses in the adults showed sex-specific effects on learning and memory for some of these EDCs. Currently, molecular analyses, including transcriptomics, epigenomics, metabolomics, lipidomics, and steroidomics are in progress. First results show that a number of gene networks, steroids, neurotransmitters and lipids are dysregulated by the six EDCs, and compound and sex-specific effects were observed. These molecular effects will be correlated to the behavioural outcomes and transcriptional data sets from in vitro models, to provide a link between the cellular and the organismal outcomes. Also in Xenopus laevis, a systematic correlation between early transcriptional dysregulation and adult adverse outcomes is ongoing using the same EDCs as well as hormone agonists and antagonists.
For the management and handling of the experimental data, a standardised database platform was developed. It includes data capture, curation procedures, and statistical analysis approaches. All ENDpoiNTs data is contained in the database (currently 2.316.378 data records) and, at the end of the project, will be made available as an invaluable resource for the community. Furthermore, 12 first tier screening high confidence Quantitative Structure Activity Relationships (QSARs) were developed for predicting the agonistic and antagonistic modes of action of the selected receptors. This is the basis for in silico models identifying chemicals that can induce DNT via these interactions. To extrapolate in vitro to in vivo concentrations of EDCs, a physiology-based toxicokinetic (PBTK) approach has been developed, predicting fetal brain concentrations. Furthermore, data produced in ENDpoiNTs is integrated into existing Adverse Outcome Pathways (AOPs) and AOP networks are built.
To establish human relevance of the test methods, doses producing an adverse effect in test systems are currently being compared with human exposure data for both single reference EDCs and for their mixture. For comparison, the metrics have been evaluated using established regulatory values translated to biomonitoring equivalent concentrations. Furthermore, a mixture study of EDCs is being finalised, with the aim to compare effects of a real-life mixture, established based on human data, to single compounds in the developed in vitro models. To further align experimental with human evidence, molecular data on the level of metabolomics and epigenomics will be compared. To this end, analyses in the epidemiological data have been conducted to provide links between molecular patterns and both prenatal exposures and behavioural outcomes.
To ensure the uptake of the developed assays and strategies into the regulatory context, ENDpoiNTs is actively engaging with key stakeholders, e.g. in workshops. Furthermore, communication channels with relevant working groups of the OECD have been strengthened to enable continuous discussion of the readiness of the developed assays for validation and regulatory implementation. Finally, to disseminate the project’s results to different target groups, ENDpoiNTs has produced project flyers and animated short films, and is actively maintaining its webpage (https://endpoints.eu/) as well as continuously publishing its results in scientific journals with open access.
Scientifically, ENDpoiNTs has contributed with significant new knowledge on hormonal involvement in neurodevelopmental key events, which is the basis for understanding ED-induced DNT and targeted methods for this kind of toxicity. First tools advancing chemical testing beyond the state of the art have been developed, including 12 QSARS interrogating modes of actions identified as relevant for ED-induced DNT, and 6 well-characterised novel in vitro assays. The OMICs analyses will reveal novel early molecular endpoints that can be used in whole organism models to predict later adversity, as well as human biomarkers of exposure and adverse outcomes. Other tools are PBTK models to predict fetal brain concentrations from maternal serum concentrations and for in vitro-in vivo extrapolations, and the ENDpoiNTs database including statistical analyses tools that will be an invaluable tool to make all the produced data FAIR. All of this will contribute to novel testing strategies for ED-induced DNT, and impact on policy making and regulations to enable better protection of vulnerable populations.