Periodic Reporting for period 2 - ENDpoiNTs (Novel Testing Strategies for Endocrine Disruptors in the Context of Developmental NeuroToxicity) Reporting period: 2020-07-01 to 2021-12-31 Summary of the context and overall objectives of the project ENDpoiNTs is developing a new testing strategy to meet the regulatory, scientific and societal needs for improved hazard and risk 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 brain is among the most vulnerable organs with respect to toxic insults, particularly during development, and EDCs have indeed been shown to target the developing brain.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• Develop an integrated approach to identify endocrine disrupting chemicals inducing DNT• Engage with key stakeholders and develop novel strategies for EDC testing and assessment into European and international chemical regulatory frameworks Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far A number of cellular and animal-based models exist to study DNT endpoints, addressing cellular key events and neurodevelopmental outcomes. However, the predictive value of these models to detect ED-induced DNT is largely unknown. In ENDpoiNTs we are assessing the responsiveness of these DNT endpoints to endocrine interference. ED pathways potentially relevant for DNT cellular key events were selected using in silico modelling to predict targets of EDCs that are known to affect human neurodevelopment. 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 almost all key events investigated are dependent on one or several of the selected hormonal receptors. Thereby, novel roles for hitherto unstudied hormonal receptors in the context of EDCs in neurodevelopmental key events were uncovered. On the other hand, the estrogen and androgen systems, that are beside thyroid hormone the most studied in the context of EDCs, were not prominently involved in DNT-related cellular key events. Some results were in accordance between human and rodent models while others diverged, underscoring species differences. Moreover, when comparing genetic sex in the cell models, some pathways were only affected in one but not the other sex. First results with model chemicals show that some of the cellular endpoints are also affected by low doses of EDCs. For most of the hormone-depended endpoints, standard operating procedures (SOPs) were established, and one of the newly developed cellular assays is currently undergoing pre-validation, including assessment of inter-laboratory transferability. 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. The rat model entails a developmental exposure scheme to six model compounds (Bisphenol F (BPF), Permethrin (PMT), PFOS, Butylbenzyl phthalate, Triphenyl phosphate, and 1,2-Cyclohexane dicarboxylic acid diisononyl ester (DINCH) and subsequent molecular as well as behavioural and cognitive measurements. All six studies are finalized, behavioural data is being analysed, and molecular studies are in progress. The molecular analyses include transcriptomics, epigenomics, metabolomics, lipidomics, and steroidomics. For the latter, a new sensitive platform has been developed for plasma and tissue samples to be able to perform targeted investigations using low amounts of samples. Similar analyses with more model EDCs are conducted in zebrafish embryos and tadpoles of Xenopus laevis. For the management and handling of the experimental data, a standardised database platform was developed. It includes data capture, curation procedures and data treatment and statistical analysis approaches relevant for ENDpoiNTs. Currently, all data of the in vitro models are fed into, and analysed by, this platform in order to compare the sensitivity and specificity between the models and, based on this, a final selection of assays for pre-validation will be made. 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. Using data from the public domain and produced in ENDpoiNTs, the molecular interactions, cellular effects and adverse outcomes are now being integrated using the Adverse Outcome Pathway (AOP) framework. To enable this, the relevant literature has been reviewed to identify brain regions, neurotransmitter systems, and endocrine systems of interest. For establishing human relevance of the test methods, doses producing an adverse effect in test systems will be compared with human exposure data from the ENDpoiNTs cohorts and other international data sets 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 has been initiated, 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 ensure the uptake of the developed assays and strategies into the regulatory context, ENDpoiNTs is actively engaging with key stakeholders. Within the EURION cluster, a stakeholder workshop was organised, discussing the views, needs and expectations related to EDC test method development and international strategies and guidelines. Furthermore, communication channels with relevant working groups of the OECD have been established or strengthened to enable continuous discussion of the readiness of the developed assays and endpoints for validation and regulatory implementation. Finally, to disseminate the project’s results to the scientific community, key stakeholders, and the public at large, ENDpoiNTs has produced project flyers and animated short films about the aims and first results of the project. It is actively maintaining its webpage (https://endpoints.eu/) as well as continuously publishing its results in scientific journals with open access. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) ENDpoiNTs will advance the current foundations of regulatory science in this area clearly beyond the state of the art by i) enhancing human-relevance of screening methods, and ii) developing an integrated platform of testing strategies and methods to test and assess ED-induced DNT. The development of a strategy for integrating the battery of testing tools in the Integrated Approach to Testing and Assessment (IATA) framework and integrating epidemiological research and data in risk assessment of EDCs will also advance the scientific basis for risk management. Ultimately, this will have impact on policymaking and regulations and enable better protection of vulnerable populations and particularly, the developing brain. endpoints-picture-for-summary.jpg
