The ENDOMIX project: an interdisciplinary approach to understanding how real-life chemical mixtures target the immune system to trigger disease

OPEN LETTER The ENDOMIX project: an interdisciplinary approach to understanding how real-life chemical mixtures target the immune system to trigger disease [version 1; peer review: 3 approved with reservations] Ana Claudia Zenclussen 1,2, Valentina Belmar Erilkin3, Linda Böhmert4, Petra Borilova Linhartova 5, Albert Braeuning4, Georg Braun 6, Cécile Chevrier7, Liesbeth Duijts8-10, Beate Isabella Escher2,6, Janine Felix8,9, Sergio Gómez-Olarte 1, Mònica Guxens 11, Gunda Herberth 1, Klara Hilscherova5, Jana Klanova5, Yvonne Kohl12, Katharina Krischak3, Dominique Lagadic-Gossmann7, Sophie Langouët 7, Sabrina Llop13,14, Maria Jose Lopez-Espinosa13-15, Léa Maitre11, Corinne Martin-Chouly 7, Nicole Meyer1, Marion Ouidir 16, Thi Anh Mai Pham 1, Claire Philippat16, Raymond Pieters17, Marie-Laure Pinel-Marie7, Normand Podechard7, Tobias Polte1, Elliott Price5, Oliver Robinson18, Kristin Schubert19, Anne Schumacher1, Violeta Stojanovska1, Tamara Tal20, Paolo Vineis18, Robert van Vorstenbosch17, Roel Vermeulen17, Charline Warembourg 7 1Department of Environmental Immunology, Helmholtz-Centre for Environmental Research - UFZ, Leipzig, Saxony, 04318, Germany 2German Center for Child and Adolescent Health (DZKJ), partner site Leipzig/Dresden, Leipzig, Germany 3European Institute for Biomedical Imaging Research (EIBIR), Vienna, Austria 4German Federal Institute for Risk Assessment (BfR), Dept. Food Safety, Berlin, 10589, Germany 5RECETOX, Faculty of Science, Masaryk University, Brno, South Moravian Region, Czech Republic 6Department of Cell Toxicology, Helmholtz-Centre for Environmental Research - UFZ, Leipzig, Saxony, 04318, Germany 7Univ Rennes, Inserm, EHESP, Institut de Recherche en Santé, Environnement et Travail - UMR_S 1085, Rennes, France 8Department of Pediatrics, Erasmus MC, University Medical Center, Rotterdam, The Netherlands 9Generation R Study Group, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands 10Department of Neonatal and Intensive Care, Division of Neonatology, Erasmus MC, University Medical Center, Rotterdam, Rotterdam, The Netherlands 11Institute for Global Health, ISGlobal, Barcelona, Spain 12Fraunhofer Institute for Biomedical Engineering IBMT, Sulzbach, Germany 13Epidemiology and Environmental Health Joint Research Unit, FISABIO–Universitat Jaume I–Universitat de València, Valencia, Spain 14Spanish Consortium for Research on Epidemiology and Public Health (CIBERESP), Madrid, Spain 15Department of Nursing, Faculty of Nursing and Chiropody, University of Valencia, Valencia, Spain 16University Grenoble Alpes, Inserm U-1209, CNRS-UMR-5309, Environmental Epidemiology Applied to Development and Respiratory Health Team, Institute for Advanced Biosciences, Grenoble, France 17Institute for Risk Assessment Sciences, Utrecht University, Utrecht, The Netherlands 18MRC Centre for Environment and Health, School of Public Health, Imperial College London, London, UK 19Department of Molecular Toxicology, Helmholtz-Centre for Environmental Research - UFZ, Leipzig, Saxony, 04318, Germany 20Department of Ecotoxicology, Helmholtz-Centre for Environmental Research - UFZ, Leipzig, Saxony, 04318, Germany Open Research Europe  Page 1 of 25 Open Research Europe 2024, 4:271 Last updated: 25 NOV 2025 First published: 19 Dec 2024, 4:271 https://doi.org/10.12688/openreseurope.19088.1 Latest published: 21 Nov 2025, 4:271 https://doi.org/10.12688/openreseurope.19088.2 v1 Abstract The true impact of endocrine disrupting chemicals (EDCs) on human health is far from being understood. Humans are exposed to mixtures of chemicals throughout their lives, yet regulations and most studies focus on individual chemicals. ENDOMIX takes a novel approach to identifying associations and causality between EDCs and adverse health outcomes by focusing on exposure to mixtures of EDCs over the life course, including windows of susceptibility, using human biomonitoring data from several European cohorts. We will model and measure how real-life EDC mixtures act together and target the immune system to initiate, trigger or maintain disease. Health effects will be investigated using pioneering methodologies ranging from high-throughput in vitro bioassays, sophisticated organoid and coculture systems, to in vivo models. In combination, they will provide valuable information on mechanistic pathways and transgenerational effects of EDC exposure. We aim to identify biomarkers and patterns of chemical exposures that are easy to measure, available for large cohorts and indicative for adverse health outcomes. We will use in vitro, in silico and in vivo data to strengthen causal inference using a weight-of-evidence approach. Moreover, using novel text mining methods, we will create knowledge graphs to capture and summarize the complexity of biomechanistic information, which aids rapid risk assessments and the creation of network models. The knowledge generated by ENDOMIX will provide an evidence base for policymaking and also reach people of all ages to raise awareness of the risks of EDC exposure and encourage health-promoting behaviors. Plain language summary Endocrine disrupting chemicals (EDCs) are natural or man-made substances that mess with the normal function of the body’s hormone system in both humans and animals. The true effect of EDCs on human health is far from being understood. People are exposed to a mixture of many different chemicals over their lifetime, yet regulations and most research studies often look at one chemical at a time. The ENDOMIX project uses a new approach to study the link between a variety of EDCs and harmful effects on health throughout the person’s lifetime. We will use data from human biological monitoring of several European study groups. We will further study how real-life mixtures of EDCs act together on the immune system to cause or perpetuate diseases. We will generate and use data from laboratory experiments (in vitro, in silico)), computational experiments and animal experiments (in vivo) to better understand the cause-andeffect relationship. The evidence produced by ENDOMIX will be a solid Open Peer Review Approval Status 123 version 2 (revision) 21 Nov 2025 version 1 19 Dec 2024 view view view Lyuba Varticovski , National Institutes of Health, Maryland, USA 1. Jacques Robert , University of Rochester Medical Center, Rochester, USA 2. Josef Köhrle , Charité-Universitätsmedizin Berlin, Berlin, Germany 3. Any reports and responses or comments on the article can be found at the end of the article. Open Research Europe  Page 2 of 25 Open Research Europe 2024, 4:271 Last updated: 25 NOV 2025 Corresponding author: Ana Claudia Zenclussen ([email protected]) Author roles: Zenclussen AC: Conceptualization, Funding Acquisition, Writing – Original Draft Preparation, Writing – Review & Editing; Belmar Erilkin V: Writing – Review & Editing; Böhmert L: Writing – Review & Editing; Borilova Linhartova P: Writing – Review & Editing; Braeuning A: Writing – Review & Editing; Braun G: Writing – Review & Editing; Chevrier C: Writing – Review & Editing; Duijts L: Writing – Review & Editing; Escher BI: Writing – Review & Editing; Felix J: Writing – Review & Editing; Gómez-Olarte S: Writing – Review & Editing; Guxens M: Writing – Review & Editing; Herberth G: Writing – Review & Editing; Hilscherova K: Writing – Review & Editing; Klanova J: Writing – Review & Editing; Kohl Y: Writing – Review & Editing; Krischak K: Writing – Review & Editing; Lagadic-Gossmann D: Writing – Review & Editing; Langouët S: Writing – Review & Editing; Llop S: Writing – Review & Editing; Lopez-Espinosa MJ: Writing – Review & Editing; Maitre L: Writing – Review & Editing; Martin-Chouly C: Writing – Review & Editing; Meyer N: Writing – Review & Editing; Ouidir M: Writing – Review & Editing; Pham TAM: Writing – Review & Editing; Philippat C: Writing – Review & Editing; Pieters R: Writing – Review & Editing; Pinel-Marie ML: Writing – Review & Editing; Podechard N: Writing – Review & Editing; Polte T: Writing – Review & Editing; Price E: Writing – Review & Editing; Robinson O: Writing – Review & Editing; Schubert K: Writing – Review & Editing; Schumacher A: Writing – Review & Editing; Stojanovska V: Writing – Review & Editing; Tal T: Writing – Review & Editing; Vineis P: Writing – Review & Editing; van Vorstenbosch R: Writing – Review & Editing; Vermeulen R: Writing – Review & Editing; Warembourg C: Writing – Review & Editing Competing interests: No competing interests were disclosed. Grant information: Funded by the European Union. Views and opinions expressed are however those of the author(s) only and do not necessarily reflect those of the European Union or the European Health and Digital Executive Agency (HADEA). Neither the European Union nor the granting authority can be held responsible for them. (Understanding how endocrine disruptors and chemical mixtures of concern target the immune system to trigger or perpetuate disease [ENDOMIX, GA No 101136566]). This project has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No 857560 (CETOCOEN Excellence). We acknowledge support from the grant CEX2023-0001290-S funded by MCIN/AEI/ 10.13039/501100011033, and support from the Generalitat de Catalunya through the CERCA Program. Authors thank RECETOX RI (No LM2023069) and BBMRI.cz (No LM2023033) financed by the Czech MEYS for supportive background. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Copyright: © 2024 Zenclussen AC et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. How to cite this article: Zenclussen AC, Belmar Erilkin V, Böhmert L et al. The ENDOMIX project: an interdisciplinary approach to understanding how real-life chemical mixtures target the immune system to trigger disease [version 1; peer review: 3 approved with reservations] Open Research Europe 2024, 4:271 https://doi.org/10.12688/openreseurope.19088.1 First published: 19 Dec 2024, 4:271 https://doi.org/10.12688/openreseurope.19088.1 foundation for policy-making and help to inform people of all ages about the risk of exposure to EDCs and encourage health-promoting behavior. Keywords endocrine disrupting chemicals, EDC exposure, chemical mixtures, immune system, long-term health effects, interdisciplinary approach, science to policy translation This article is included in the Horizon Europe gateway. This article is included in the Horizon 2020 gateway. Open Research Europe  Page 3 of 25 Open Research Europe 2024, 4:271 Last updated: 25 NOV 2025 Introduction Endocrine disrupting chemicals (EDCs) are natural or synthetic chemicals that interfere with the normal functioning of the endocrine system in humans and animals. However, the impact of EDCs in general and man-made chemicals in particular on human health is not so well-understood. They are ubiquitous and have been implicated in the increase in the prevalence of common non-communicable diseases (NCDs) over the last decade1. Man-made EDC chemicals, from here on referred to as EDCs, can be found not only in the environment (water, soil, air), but also in consumer products (plastics, food packaging, electronics and food packaging), in personal care products (e.g., sunscreen, cosmetics), as well as in food and drinking water2. Efforts to minimize exposure to harmful EDCs include regulations on their use in consumer products, bans on certain chemicals, and public education campaigns to raise awareness of their potential adverse health risks. Many of these approaches rely on results obtained from scenarios on exposure to singlechemicals or at best to a family of chemicals. In reality, however, we are exposed to multiple EDCs simultaneously throughout our lives. Not only do some EDCs accumulate in the environment and enrich in certain tissues3, but their mixtures will lead to additive, and in rare cases, synergistic or antagonistic effects4. Deviations from normal healthy state or development do cause individual suffering and impose enormous costs on health care systems5. Although ample evidence of the adverse effects of EDC exposure in wildlife and animals was already reported by the International Program on Chemical Safety in 2002 and has been complemented by a large number of epidemiological studies over the past decades, significant gaps still remain in our understanding of the health effects and mechanisms of EDC in the human population3,6–8 The so-called immunotoxicology lists among the less explored topics. Indeed, the effects of EDCs on the immune system have been poorly investigated. However, dysregulated immune pathways may underlie and are important drivers of common human diseases. The fact that EDCs may affect the immune system and cause abnormalities relevant to disease is poorly addressed5. Despite growing evidence feeding to his emergent field of research, more research is needed to fully understand the breadth of EDCs´ impact on the immune system, especially concerning long-term health effects and the combined impact of multiple disruptors to which we are exposed chronically. Additionally, understanding individual and group susceptibility needs to be considered. In this article we present the concept and objectives of the ENDOMIX project to address the above-mentioned challenges and needs. ENDOMIX conception The vision and aim of ENDOMIX is to thoroughly clarify and understand the overall immunotoxic or immunomodulatory impact of EDC mixtures and underlying mechanisms leading to adverse health outcomes. ENDOMIX is a cutting-edge research initiative that aims to uncover the true impact of EDCs on human health by bridging existing knowledge gaps between science and policy. It was born out of the need to synthesize available information from different approaches and sources, and the urgent demands of society to protect individual health by reducing exposure to and minimizing chemical risks. The project takes an interdisciplinary approach, covering the full chain from population-based studies to mechanistic understanding of EDC effects at the cellular and target organ level, and translating this into policy recommendations. ENDOMIX strongly focuses on EDCs and their possible combined effects with other stressors, including socioeconomic aspects, and addresses an area that is under-researched and where knowledge is grossly lacking. Exposure to EDCs is associated with important adverse health outcomes in humans across the life course, including impaired respiratory and cardiometabolic health, neurodevelopment autoimmunity, and altered reproductive health9. These constitute health outcomes with a large burden of disease worldwide10. The exact underlying biological pathways are mostly unclear. Many studies overlook the fact that there are age windows in which exposure to EDCs are more critical, making certain populations like pregnant women, neonates, infants and children more vulnerable to their effects. ENDOMIX takes a unique approach to this problem by focusing on a) chemical mixtures (real life EDCs), b) immunotoxicity as a critical central mechanism, c) strong study designs, of multiple European cohorts with data on chemical exposures and NCDs across the life course, d) modelling approaches complemented by bioassays that provide novel knowledge about real-life mixtures and their effects, e) mechanistic and causal molecular investigation including in vitro barrier and target organ models as well as different animal species considering the 3R rules, and f) knowledge synthesis through novel text mining approaches for biomechanistic insights, modelling and risk estimation. Existing gaps in scientific understanding, policy, and knowledge transfer hamper the effectiveness of European regulations in assessing, preventing, and reducing human and environmental exposure to EDCs. Addressing this uncertainty by integrating robust data sources, existing data from biobank samples, modelling strategies, and bioassays will be transformative in identifying real-life mixtures that are of concern and should be looked at more carefully. The effects of chemical mixtures may be more significant than their components, potentially causing irreversible adverse effects during vulnerable periods of life such as pregnancy, early childhood, and adolescence. This aligns with the Developmental Origins of Health and Disease (DOHaD) framework, which posits that many adult diseases originate in early life, with chemical exposure during these vulnerable periods potentially driving adverse health outcomes7,11. Adolescence is a key period of rapid and unique development, in which individuals may be very prone to major endocrine and metabolic changes, and thus potentially highly sensitive to EDCs, but this has rarely been examined in epidemiological studies. Further, it is not considered in the design of single in vivo studies nor experimental settings for risk assessment. ENDOMIX will therefore break new ground by focussing on puberty endpoints. Page 4 of 25 Open Research Europe 2024, 4:271 Last updated: 25 NOV 2025 A recent report of the Lancet Commission for Planetary Health identified immunotoxicity, the focus of ENDOMIX, as one of “three particularly worrisome, and inadequately charted consequences of chemical pollution”12, the other two being neurotoxicity and reproductive toxicity. Even though that it is documented that acute or chronic EDC exposure on the immune system results in autoimmune diseases13, or common diseases like diabetes, obesity, allergies and respiratory diseases, the impact of EDCs on health outcomes mediated by the immune system is poorly acknowledged or completely overlooked in most of current approaches. ENDOMIX will advance the field by studying immunotoxicity as a driver of deviations in human health development following exposure to EDCs, and a perpetuator of pathologies. Dysregulated immune pathways are known drivers of common diseases such as diabetes, obesity and respiratory diseases. In particular, dysregulated immune responses in pregnancy may have long-term effects on offspring, even if the pregnancy seemed normal. The fact that EDCs can affect immune responses upon binding to the hormone receptors expressed on the surface of or in immune cells14,15 and cause abnormalities relevant to pathology is poorly recognized or not recognized at all. The primary focus of ENDOMIX on the emerging field of immunotoxicity is not limited to immune-related diseases, but applies to a wide range of pathophysiological conditions. Acute or chronic exposure to EDC chemicals and mixtures can impact the immunome, a term used to comprise the genes and proteins that constitute the immune system16, and also affect the frequency, phenotype and functionality of immune cells, with different effects depending on individual predisposition, time and duration of exposure. However, whether and how exposure to chemicals, and in particular EDCs and chemical mixtures of concern, is associated with immune-mediated pathologies has not been studied in depth. The vision of ENDOMIX is to fully elucidate and understand the overall effects of complex EDC mixtures and the underlying mechanisms, Our strategy is to cover the entire knowledge chain to achieve this ambitious goal (Figure 1). In a first step candidate EDC mixtures relevant for endocrine disruption, immunotoxicity and immunomodulation will be identified using a previously developed prioritization workflow for neurotoxic chemicals based on simulated worst case exposure, high-throughput toxicokinetics models and high-throughput toxicity data with data-gap filling through toxicity prediction models17. The identified potential mixture effect contributors will be searched for in population-based blood exposure data from a large collection of 16 cohorts and cohort consortia European cohorts Figure 1. The ENDOMIX strategy from data generation to its translation into policy guidelines and recommendations. With a combination of life-course population-based cohort data, high-throughput screening and modelling to identify new mixtures of concern, in vitro cell, barrier and organoid models, in vivo models, omics, and computational approaches, ENDOMIX will deliver evidence-based guidelines and advice to citizens on improving human health. Page 5 of 25 Open Research Europe 2024, 4:271 Last updated: 25 NOV 2025 including target and non-target analysis using high-resolution mass spectrometry (HRMS) data. Based on this information, complex EDC mixtures will be reconstituted in the laboratory at the concentration ratios as they were detected in human blood. The reconstituted mixtures will be used to investigate their effects at the level of immune cells and target organs. The concentration ratios of the chemicals in the mixtures correspond to the concentrations found in the cohort samples, but the overall concentration is higher to be able to measured but the results will be extrapolated down to the real-life concentrations. Tissue barrier systems, organ-on-a-chip-approaches and zebrafish embryo models behavioral assays will be utilized to understand EDCs’ impact on defined endpoints. By combining the gained knowledge on defined associations between exposures and health outcomes and identified celland organdependent mechanisms, we will continue using cohort samples and model systems to understand causality between these exposures and defined health outcomes and identify underlying mechanisms. Finally, at the end of the project, we will be able to provide evidence-based recommendations to regulators and policy makers, inform the public and contribute to improved risk assessment. ENDOMIX objectives ENDOMIX aims at unravel the overall immunotoxic or immunomodulatory impact of EDC mixtures leading to adverse health outcomes. The overarching objective of ENDOMIX is to deliver new information and concepts about the immune-mediated health impact of EDC mixtures, and their underlying biological mechanisms, which will build the basis to elaborate on approaches that help minimize EDC exposures in the future. Mixtures identified as potentially concerning will be tested in in vitro and in vivo assays, including primary immune cells, organoids, alternative models such as Caenorhabditis elegans and zebrafish embryos, and classic mouse models to uncover mechanistic insights. Barrier models will be used to determine the transport and targets of EDC mixtures. Computational approaches will bolster associations between multiple exposures and health outcomes. Our objective is to provide clear scientific evidence on exposure scenarios, co-exposures’ effects (including lifestyle and socioeconomic factors), target cells and organs, and mechanistic pathways. ENDOMIX will integrate harmonised data on EDC exposures, immune markers, and health outcome endpoints from multiple European cohorts to accelerate this integrative approach. Additionally, we will identify novel bioactive EDCs through predictive exposure and toxicokinetic modelling, combined with data mining from high-throughput screening (HTS) bioassays, with an emphasis on immunotoxicity and immunomodulation, to prioritise drivers of mixture effects. Objective 1: Identification of complex EDC mixtures in European populations Existing knowledge about EDC impact on health mostly came from studies of single chemicals and previous experimental design is often insufficient as they focus on known chemicals only. People are exposed to a wide range of EDCs that can potentially interact and will certainly act together in mixtures. Therefore, it is vital to improve our understanding of the effects of exposure to real-life EDC scenarios at different life stages. Additionally, the problem is that there are potentially thousands of chemicals in our blood and there are hundreds of chemicals that potentially cause endocrine disruption and immunomodulation, but they were never systematically matched to identify chemicals that are mixture effect drivers. ENDOMIX cutting edge and novel approach is that we will simulate mixtures of potential concern by data mining of the toxicology literature, simulating mixture exposure with help of highthroughput toxicokinetic modelling and mixture effect models. This approach has so far led to >7000 candidate chemicals out of a starting list of 100,000 chemicals that could potentially be contributing to mixture effects. ENDOMIX will derive complex EDC mixtures and health outcomes in European populations using an exceptional wealth of data and biosamples analysis by systematic in silico prioritization leveraging toxicology literature, high-throughput toxicity & exposure methods and mixture effect models. In silico mixtures predictions will be augmented with existing blood biomonitoring data through searching for candidate chemicals of concern data from cohort samples within the consortium. Newly detected bioactive compounds will be confirmed in a small subset of blood samples from the cohorts before complex chemical mixtures are designed for testing in the HTS assays. ENDOMIX will design in vitro studies to analyse endpoints that are relevant for immunotoxicants and associated mediators of health outcomes as defined in the cohort studies. Based on key characteristics (KC) of immunotoxicity, we will select adequate HTS assays addressing relevant key events of endocrine disruption and immunomodulation as a comprehensive apical endpoint of immunotoxicity in innate and adaptive immune cells. These assays are suitable for screening large numbers of chemicals, mixtures and ultimately even extracts from blood samples of the cohorts. The large number of screened chemicals and mixtures in the HTS assays will then be used to develop a smaller number of representative mixtures of immunotoxicants that will be tested in more specialised and more complex bioassays, primary cell cultures and in vivo model systems. Our strategy starts with population-based studies that include many cohorts covering the life course (prenatal, perinatal, infancy, childhood, adolescence, adulthood) and comprising regions across Europe and beyond with very detailed data (Participating cohorts, Figure 2). ENDOMIX not only brings together large multiple existing cohorts with already measured EDCs in biosamples, but its novelty lies in the identification and study of EDC as mixtures and the focus on the immunemediated effects of EDCs. In addition to the identification of new mixture contributors, ENDOMIX will use existing targeted screening data of a number of EDCs: polychlorinated biphenyls, organochlorine pesticides, perand polyfluorinated compounds, metals, parabens, phthalates, phenols, and organophosphate pesticides. Further, for cohorts with limited availability of Page 6 of 25 Open Research Europe 2024, 4:271 Last updated: 25 NOV 2025 EDC data, such as for relatively rare outcomes like autoimmune diseases, we will proceed to quantify chemicals suspected to contribute to immunotoxic mixture effects, such as polychlorinated biphenyls, organochlorine pesticides, and perand polyfluorinated compounds. Another strength is the covering of various life stages allowing the study of different windows of susceptibility to EDCs including early-life and adolescence, two major sensitive periods subject to important endocrine and physiological changes. We expect that EDC mixtures may vary over time due to changes in EDC use, exposure sources, and policies, and across different populations depending on determinants such as age, sex, socioeconomic status, and lifestyle habits. In addition to most of the identification of new mixture contributors, involved cohorts benefit from exposure and health outcome data already harmonised in the context of previous or ongoing EU-funded projects including the LifeCycle project18 and projects that are part of the European Human Exposome Network (EHEN, including ATHLETE19, LongITools20 and Expanse21. ENDOMIX will use existing targeted screening data of a number of EDCs: polychlorinated biphenyls, organochlorine pesticides, perand polyfluorinated compounds, metals, parabens, phthalates, phenols, and organophosphate pesticides. Objective 2: Associations between exposure to EDC mixtures, occurrence of allergies and respiratory, cardiometabolic and reproductive health as well as autoimmunity Mixtures that occur in real life will be tested for immunotoxic effects from HTS using in vitro bioassays and predictive exposure models. After identifying mixtures of concern and testing their effects in simple bioassays, we will examine their potential determinants including socioeconomic, environmental and lifestyle-related factors and their link with several important health outcomes, of which; the choice was based on the potential involvement of the immune system as a mediator of disease development. Immune cells can be targeted by EDCs, are ubiquitous in the human body, can traffic to target organs and if dysfunctional, they are able to initiate, trigger or perpetuate disease. Health outcomes to be studied within ENDOMIX therefore are (1) allergies & respiratory health (eczema, atopic dermatitis, allergic rhinitis, wheezing, asthma, lung function, COPD), (2) cardiometabolic & cardiovascular health (body mass index, waist circumference, adiposity, dyslipidemia, insulin resistance, diabetes, stroke, acute myocardial infarction) (3) reproductive health (age at menarche, age at voice break, Tanner stages, menstrual cycle characteristics, Figure 2. Overview of the observational studies involved in ENDOMIX. Cohort study name, life course timepoints (pregnancy, infancy, childhood, adolescence, and adulthood) and illustration with coloured signs representing the availability of chemical exposure data measured with targeted analysis throughout the life course is shown. Further, available microbiome, epigenome, transcriptomics, and metabolomics data are indicated. Page 7 of 25 Open Research Europe 2024, 4:271 Last updated: 25 NOV 2025 sex hormone levels), and (4) autoimmune diseases (rheumatoid arthritis, ulcerative colitis, Crohn´s disease). The health outcomes will be studied at different age periods, from childhood to adulthood (e.g., from early changes in blood pressure to the onset of cardiovascular diseases) in cohorts and disease models. We will additionally explore how EDCs may be associated with overall health by analysing their potential effects on more than one health outcome, an approach particularly relevant for public health to identify the EDC mixtures that contribute to multimorbidity. The association of EDCs with the health outcomes may be linked to central immunotoxicity processes with a focus on inflammation. ENDOMIX will identify immune markers focused on inflammation using a proteomics approach and will examine their relation with EDC exposure. Immune markers focused on inflammation will comprise cytokines, chemokines, their respective receptors and a wide range of other immune proteins that will be identified through novel proteomic analyses using Olink® inflammation panels. Many biological pathways. metabolic perturbations may serve as biological pathways involved in immune-related effects of EDCs on human health. ENDOMIX will make use of available epigenetic, microbiomic, and endogenous metabolomic data of participants of the human cohorts. By doing so, we will offer insights into the relationships of exposure to EDCs, immune markers and DNA methylation, microbiome, and metabolome at multiple life stages and their association with human health outcomes. Studying EDC mixtures in epidemiology raises methodological challenges due to the high correlations between different EDCs and the high dimension of data to be considered (tens to hundreds of chemicals). ENDOMIX will overcome these challenges by using advanced statistical methods suitable for chemical mixtures. These include unsupervised dimensionality reduction and clustering methods that will allow the identification of specific profiles of exposure independently of their health effects, and supervised techniques (e.g., Bayesian Kernel Machine Regression, Quantile G-computation) to estimate both the overall effect of the EDC mixture on disease risk and the contribution of each individual compound to the mixture effect. Another statistical challenge is the integration of immunological markers as potential intermediate endpoints between EDC exposure and health. We will perform mediation analysis in a high dimensional context to disentangle the direct and indirect effect of EDCs through changes in immunological markers. To synthesize information across different biological layers, we will use a multi-omics framework using graphical networks and multi-block approaches, such as two-way orthogonal PLS (O2PLS) and multi-omics factor analysis (MOFA), to integrate the information from EDC exposures, omics layers, possible sex differences and disease outcome. Objective 3: Impact of prioritized EDC mixtures on the immunome, innate and adaptive immune cell functionality and target organs To investigate the direct effects of priority EDC mixtures on the immunome, innate and adaptive immune cells’ frequency, phenotype and functionality, we will first characterise the imunome and which cell subtypes are the most affected by EDC mixtures by mass cytometry, based on CyTOF® (cytometry by time of flight) technology. The unique metal labelling of antibodies allows the simultaneous identification of up to 50 protein markers in one cell with the further advantage of no background signal compared to other methods. After identifying the cell subtypes affected the most, more specific immunoassays involving innate and adaptive immune cell populations will be employed to test the impact of chemicals. Moreover, cytokine-bead arrays will be applied to determine cytokines in monocyte, dendritic cell, T and B cell culture supernatants to gain further insights into cytokine secretion patterns22. For T cells, maturation and differentiation assays will be conducted under chemical treatment. For B cells, alterations in antibody production (Ab) capacity (e.g. IgG, IgM) will be determined under the impact of EDC mixtures. Together, these assays will provide detailed information about EDC effects on immune cell differentiation, maturation, plasticity and functionality and will thus increase our knowledge on chemical drivers of acute and chronic diseases. Finally, gene expression analyses using semi-high-throughput qPCR and omics approaches will be employed in chemical-exposed immune cell populations to gain further insights in altered intracellular pathways. Further, barrier and organ in vitro models including organoids will unravel how EDCs behave within tissues, and how EDC mixtures cross barriers and if immune cells work as messengers or mediators of organ toxicity. The models were carefully chosen to be meaningful regarding the health endpoints studied in cohorts. Besides using human in vitro models, as described above, we will employ innovative models that faithfully mimic the organs of interest and human response (Figure 3). Here, barrier models, co-culture and 3D models, including organoids and organ-on-a-chip models that are already published and running in the laboratories of the ENDOMIX partners, will be used. When adequate or possible, immune cells will be combined with the cell systems. These systems include: in vitro lung barrier23,24, in vitro blood-brain-barrier25,26, in vitro placenta barrier27 and in vitro intestinal barrier28,29. Further, for target organ models, we use lung organoids30, in vitro heart spheroids31,32, intestinal organoids33,34 and liver spheroids35,36. Immune cells will be co-cultured to understand EDC effect on their interaction. At the molecular level, we will identify gene and protein expressions of nutrient transporters, regulation of genes related to lipid and glucose metabolism gene and associated transcriptional factors, and perform nuclear receptor transactivation assays. Established gene and protein signatures indicative of triglyceride accumulation37 will be transferred to further models to derive characteristic and predictive gene expression patterns in vitro, indicative of a certain endocrine effect. Organoids will be generated from primary samples, like placenta organoids or will be stem-cell derived Multi-organ Page 8 of 25 Open Research Europe 2024, 4:271 Last updated: 25 NOV 2025 in vitro models will be used to study interactions between different organs (e.g. intestinal-hepatic interaction or liveradipocyte interaction) and the effect on chemical biotransformation. Because of its defined short life cycle, C. elegans can be exploited to test the effect of chemicals on the whole life cycle of an organism (from egg production to development to mature organism). Our innate immune models with C. elegans include bacterial infection models and detection of antimicrobial proteins and peptides. EDC mixtures and individual chemicals will be tested on development and innate immune responses by C. elegans38,39. Moreover, a zebrafish embryo model will be used to complement in vitro approaches as C. elegans to evaluate the impact of EDC on the outcome of metabolic liver diseases and on related intestine-liver axis disruptions including alterations of microbiota and immune cell functions. The zebrafish embryo model represents a 3R-compliant40, alternative model that, up to 5 days post-fertilization (dpf), is considered to be a non-protected life stage, according to EU regulations41. There is a gap in the availability of complex, higher-throughput immunotoxicity screening systems. We propose that the zebrafish embryo model can fill this gap, particularly for identifying innate immunotoxicants, as the innate immune system develops in isolation in 5-day-old zebrafish embryos. A new alternative assay to detect innate system immunomodulation will be developed and applied in embryonic zebrafish that quantitates, in parallel, a concentration-response relationship on a diverse suite of ~25 immunomodulatory molecules including markers for phagocytes (neutrophils, monocytes, macrophages), inflammation-related serum proteins (complement, C-reactive protein), antimicrobial peptides (defensins), cell receptors that signal a defensive response (TLRs), cells that release inflammatory mediators (macrophages, mast cells, NK cells), and markers of physical barriers (tight junctions). In addition, automated morphometrics will be applied to quantitate gastrointestinal area which, when increased in the absence of other morphological effects, may indicate intestinal inflammation. Individual chemicals and mixtures developed in this project will be tested in a concentration response format. Transgenic lines labelling neutrophils or macrophages will be used to quantify immune cell migration into the gastrointestinal (GI) tract. Critical periods of exposure and possible sex differences are also considered in ENDOMIX´s design as for example placenta organoids and immune cells from both sexes are employed to represent exposure that affects not only mother and offspring in a transgenerational approach but also sex-related disease development. The use of alternative models including transgenic zebrafish with labelled macrophages and C. elegans as a model of dysbiosis will provide valuable data on EDC impact and immune-mediated mechanisms and thereby greatly reduce the use of vertebrate animal experiments. Objective 4: Establishment of causality of EDC exposure on health outcomes The direct causality between exposure to EDC mixtures and deviations from normal healthy development or disease outcomes needs to be confirmed to deliver evidence for improved chemical regulations. ENDOMIX will utilize the information obtained in Objectives 1–3 to design studies addressing causality of EDC exposures on health outcomes. The models will further consider the EDC association with health outcomes through their impact on immune cells and target organs. These approaches include deep characterisation in cohort samples using omics-based biomarkers and the effect and perinatal exposure using transgenerational in vivo models. Experimental mouse models will be used whenever in vitro or alternative models cannot answer the scientific question. Mixtures identified previously will be tested in well-defined transgenerational 3R-conform mouse models to verify adverse effects observed in human cohorts and in in vitro models. Control treatment will depend on the nature of the defined mixtures. These models may include: asthma in the offspring of exposed mothers, rheumatoid arthritis in offspring of exposed mothers and will include mechanistic studies42–44. As for reproductive health, we will study potential EDC effects on Figure 3. Models used in ENDOMIX. Different models are used in ENDOMIX to study the impact of endocrine disrupting chemical (EDC) mixtures in tissue barriers, cell-cell interaction and whole organisms. Page 9 of 25 Open Research Europe 2024, 4:271 Last updated: 25 NOV 2025 The last and 5th objective focusses on future guidance for consumers and the society to avoid and reduce EDC exposure. Are there any strategies, benchmarks, criteria already available how potential endpoints of preventive measures can be established, validated and tested for their applicability and success? This project will provide novel insight into the role of immunotoxic effects of EDC exposure during various life phases. It will contribute to a better understanding of the impact, role and interactions of the immune system for developmental processes relevant for the endocrine, reproduction and neurological systems. It can be expected that data accumulated by the EU-funded ENDOMIX consortium can contribute to better guidance for reduction of individual, environmental and societal EDC exposure which can only complement EU regulatory measures to stop the production, further distribution and application of identified and suspected endocrine disrupting chemicals (EDCs), which are substance of very high concern (SVHC) like mutagens, carcinogens and reprotoxicants. Probably, several of these chemicals and their mixtures not only act as EDCs but are also neurotoxic and/or immunotoxic, adversely impacting the health of living organisms including humans, especially during development in an irreversible manner. Is the rationale for the Open Letter provided in sufficient detail? (Please consider whether existing challenges in the field are outlined clearly and whether the purpose of the letter is explained) Yes Does the article adequately reference differing views and opinions? Partly Are all factual statements correct, and are statements and arguments made adequately supported by citations? Yes Is the Open Letter written in accessible language? (Please consider whether all subjectspecific terms, concepts and abbreviations are explained) Yes Where applicable, are recommendations and next steps explained clearly for others to follow? (Please consider whether others in the research community would be able to implement guidelines or recommendations and/or constructively engage in the debate) Partly Competing Interests: No competing interests were disclosed. Reviewer Expertise: molecular aspects of the thyroid hormone system (biosynthesis, distribution, metabolism, action, regulation); effects of endocrine active compounds (endocrine disruptors) upon the thyroid hormone system; interaction between essential trace elements (iodine, selenium, iron) and the endocrine system; development of analytical methods for endocrine-relevant ligands. Open Research Europe  Page 16 of 25 Open Research Europe 2024, 4:271 Last updated: 25 NOV 2025 I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Reviewer Report22 July 2025 https://doi.org/10.21956/openreseurope.20658.r55598 © 2025 Robert J. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Jacques Robert University of Rochester Medical Center, Rochester, USA This an interesting manuscript that presents the ENDOMIX project, which is an ambitious integrative approach to model and measure how real-life endocrine disrupting chemicals (EDCs) act in mixtures to target the immune system and initiate, trigger or maintain disease. ENDOMIX is also anticipated to allow the identification of biomarkers and patterns of chemical exposures that would be convenient to measure, available for large cohorts and indicative for adverse health outcomes. The manuscript is mainly a description of the ENDOMIX approach and anticipate outcomes. As such, the plan is sufficiently detailed and convincing, although it would be great to see some real data to determine how successful this approach will be. The manuscript would benefit from a short preliminary set of real data using ENDOMIX. Overall, the manuscript is well written. There are a few points listed below that the authors may consider that would improve the manuscript.  1) It is unclear how robust the prediction of the potential additive or synergistic effects of different EDCs can be based on their structure. Some clarification would be helpful. In addition, the possibility of interaction between degradation products of the different EDCs as well as the possible interaction of EDCs and their degradation products with metabolites in human tissues should be considered. Some discussion of this complex issue and potential limitation of ENDOMIX would be welcomed. 2) An important immune function targeted by EDCs that is unfortunately completely omitted in this manuscript is the defects in immune responses to pathogens leading to increase susceptibility to infectious diseases such as influenza and other bacterial pathogens. There are multiple published data from mouse and the amphibian Xenopus models as well as epidemiologic data. Including EDC impacts on this key function of the immune system is crucial and would markedly strengthen the manuscript. 3) While zebrafish is mentioned as an alternative model, the Xenopus model is left out, which is unfortunate owing its potential for assessing the direct impacts of EDCs on the development of adaptive immunity as well as the indirect EDC effect on immune system development through the thyroid axis. In addition, the Amphibian Metamorphosis Assay (AMA) is a well-established test to determine if a given toxicant has potential to be a thyroid-disrupting chemical (TDC), and Open Research Europe  Page 17 of 25 Open Research Europe 2024, 4:271 Last updated: 25 NOV 2025 guidelines for the AMA have been endorsed by the US Environmental Protection Agency and Organization for Economic Co-operation and Development (OECD). This makes Xenopus an important element in the regulation of industrial chemicals by government entities. 4) There is little discussion regarding the validation of the data obtained through ENDOMIX. For such a large approach a systematic validation system should be delineated. Is the rationale for the Open Letter provided in sufficient detail? (Please consider whether existing challenges in the field are outlined clearly and whether the purpose of the letter is explained) Yes Does the article adequately reference differing views and opinions? Partly Are all factual statements correct, and are statements and arguments made adequately supported by citations? Yes Is the Open Letter written in accessible language? (Please consider whether all subjectspecific terms, concepts and abbreviations are explained) Yes Where applicable, are recommendations and next steps explained clearly for others to follow? (Please consider whether others in the research community would be able to implement guidelines or recommendations and/or constructively engage in the debate) Partly Competing Interests: No competing interests were disclosed. Reviewer Expertise: Comparative immunology, immunotoxicology. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Author Response 14 Nov 2025 Thi Anh Mai Pham Thank you for your valuable and constructive feedback on our manuscript. We have carefully considered the points raised and prepared a detailed, point-by-point response to each comment, indicating the corresponding changes made in the manuscript and where they can be found in the revised version: This an interesting manuscript that presents the ENDOMIX project, which is an ambitious integrative approach to model and measure how real-life endocrine Open Research Europe  Page 18 of 25 Open Research Europe 2024, 4:271 Last updated: 25 NOV 2025 disrupting chemicals (EDCs) act in mixtures to target the immune system and initiate, trigger or maintain disease. ENDOMIX is also anticipated to allow the identification of biomarkers and patterns of chemical exposures that would be convenient to measure, available for large cohorts and indicative for adverse health outcomes. The manuscript is mainly a description of the ENDOMIX approach and anticipate outcomes. As such, the plan is sufficiently detailed and convincing, although it would be great to see some real data to determine how successful this approach will be. The manuscript would benefit from a short preliminary set of real data using ENDOMIX. Overall, the manuscript is well written. There are a few points listed below that the authors may consider that would improve the manuscript.  Author response: Thanks very much for this positive feedback. As for real data sets, we prefer at this stage to refrain of including data as this is not a classic manuscript but an open letter and this kind of format does not allow for primary data. Besides, given the complexity of a consortium project, it would be difficult to pick one data set to represent to whole consortium. 1) It is unclear how robust the prediction of the potential additive or synergistic effects of different EDCs can be based on their structure. Some clarification would be helpful. In addition, the possibility of interaction between degradation products of the different EDCs as well as the possible interaction of EDCs and their degradation products with metabolites in human tissues should be considered. Some discussion of this complex issue and potential limitation of ENDOMIX would be welcomed. Author response: From a public health and regulatory perspective, the central question is whether an endocrine-disrupting chemical (EDC) can exert detrimental effects on human health—regardless of whether the toxicity originates from the parent compound or its degradation products/metabolites. In epidemiological studies, the decision to measure either the parent compound or its metabolites is guided by the goal of accurately characterizing participants’ exposure levels. For EDCs that are rapidly metabolized upon entering the human body (e.g., phthalates, organophosphate pesticides), exposure is typically assessed via the quantification of their metabolites. In contrast, for more persistent EDCs (e.g., organochlorines, PFAS), exposure is generally evaluated by measuring the parent compounds directly. In whole-organism studies (both epidemiological and in vivo), the organism is exposed to the parent compound, which is subsequently metabolized by endogenous pathways. Consequently, the subject is effectively exposed to both the parent compound and its metabolites, and any observed toxicity may result from either or both forms. It is important to acknowledge, however, that humans may also be directly exposed to certain metabolites, and that interindividual variability in metabolic capacity can lead to exposure misclassification in population studies. Greater concern arises in in vitro (and potentially in silico) systems, where the metabolic capacity of the test model may be limited or absent. In such cases, exposing cells solely to the parent compound could yield misleading results—particularly if the toxic effect is mediated by metabolites rather than the parent chemical. To address this limitation, it is advisable to test both the parent compound and relevant metabolites when feasible, ensuring a more comprehensive assessment of potential toxic effects. However, our focus is on mixtures. In complex mixtures, the concentration-additive nature of effects becomes largely independent of chemical structure and synergistic effects less important. This Open Research Europe  Page 19 of 25 Open Research Europe 2024, 4:271 Last updated: 25 NOV 2025 additive hypothesis can be tested by comparing responses from single compounds with those from their mixtures—a strategy that has been shown to hold true within one order of magnitude in previous studies. In the ENDOMIX project, our goal is to analyze real-life mixtures. Therefore, as long as robust exposure data are available for both parent compounds and their transformation products, we will design mixtures that include both and systematically assess their effects. 2) An important immune function targeted by EDCs that is unfortunately completely omitted in this manuscript is the defects in immune responses to pathogens leading to increase susceptibility to infectious diseases such as influenza and other bacterial pathogens. There are multiple published data from mouse and the amphibian Xenopus models as well as epidemiologic data. Including EDC impacts on this key function of the immune system is crucial and would markedly strengthen the manuscript. Author response: We agree with the reviewer that this is highly relevant topic and very much related to the immune system. While infectious were not the focus of the project, basically because the health outcomes of the included cohorts do not specifically deal with infectious diseases, we still have included some aspects in our design. For example, in some in vitro approaches, effects of EDC mixtures will be tested under parallel stimulation with bacterial and viral motifs, namely pathogen-associated molecular patterns (PAMPs) and intracellular molecules associated with cellular damage, called damage-associated molecular patterns (DAMPs). Together, these assays will provide detailed information about EDC effects on immune cell differentiation, maturation, plasticity and functionality and will thus increase our knowledge on chemical drivers of acute and chronic diseases. Moreover, our findings will provide information on how EDCs may alter immune responses against pathogens. For clarification, we have revised the first paragraph under “Objective 3: Impact of prioritized EDC mixtures on the immunome, innate and adaptive immune cell functionality and target organs” providing the explanation mentioned and included three additional references (PMIDs: 33049673, 17355946, 7174118).We agree with the reviewer that the Xenopus model represents a strong system to investigate adaptive immunity and thyroid axis disruption. While the embryonic/larval zebrafish model is able to capture thyroid disrupting chemicals (PMID: 26765085), adaptive immunity can only be assessed in older fish, necessitating lower throughput testing that requires an approved animal use protocol. Here, we opted to complement mammalian testing with rapid, early life stage zebrafish-based screening to identify human-relevant chemicals and mixtures that trigger intestinal inflammation. Future work should consider using a Xenopus or juvenile-based zebrafish systems to study the effects of ENDOMIX mixtures on adaptive immunity development and function. 3) While zebrafish is mentioned as an alternative model, the Xenopus model is left out, which is unfortunate owing its potential for assessing the direct impacts of EDCs on the development of adaptive immunity as well as the indirect EDC effect on immune system development through the thyroid axis. In addition, the Amphibian Metamorphosis Assay (AMA) is a well-established test to determine if a given toxicant has potential to be a thyroid-disrupting chemical (TDC), and guidelines for the AMA have been endorsed by the US Environmental Protection Agency and Organization for Economic Co-operation and Development (OECD). This makes Xenopus an important Open Research Europe  Page 20 of 25 Open Research Europe 2024, 4:271 Last updated: 25 NOV 2025 element in the regulation of industrial chemicals by government entities. Author response:We appreciate the reviewer’s suggestion and acknowledge that Xenopus model offers advantages for thyroid hormone disruption-related investigations, nevertheless it is not a common model for studying human immunotoxicity, which is the primary focus of ENDOMIX project. In contrast, zebrafish have been widely adopted as a relevant and suitable model for immunotoxicity research, namely for targets within the innate immunity, as shown in recent literature (e.g. reviews by Zhao et al., 2024, doi: 10.1016/j.ecoenv.2024.116023; Franza et al., 2024, doi: 10.3390/ijms252212008). Moreover, the Amphibian Metamorphosis Assay (AMA) based on Xenopus laevis falls outside the general scope of our project, which focuses namely on the development and implementation of 3R (Replacement, Reduction, Refinement) -compliant approaches complemented by limited in vivo testing using specific mammalian models with direct relevance for human immune health. Unlike the involved in vitro methods and the Fish Embryo Toxicity (FET) assay, the AMA does not align with the 3Rs strategy due to its longer exposure duration of 21 days. Furthermore, AMA primarily focuses on metamorphosis as an endpoint, which is not directly relevant to human health outcomes. Therefore, we chose to include immune disruption-related endpoints in zebrafish embryos as a suitable model within the context of our study. 4) There is little discussion regarding the validation of the data obtained through ENDOMIX. For such a large approach a systematic validation system should be delineated. Author response:As for internal data validation, we envision different levels. Firstly, data analytical validation of results, internally but also comparing cross-cohort results takes place. We further have experimental validation, for example, the assessment of certain mixtures not only in populations, but also in vitro (cell models, organ models) and animal studies. Competing Interests: No competing interests were disclosed. Reviewer Report19 February 2025 https://doi.org/10.21956/openreseurope.20658.r49291 © 2025 Varticovski L. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The author(s) is/are employees of the US Government and therefore domestic copyright protection in USA does not apply to this work. The work may be protected under the copyright laws of other jurisdictions when used in those jurisdictions. Lyuba Varticovski National Institutes of Health, Maryland, USA The ENDOMIX project addresses a critical gap in research regarding the impact of EDCs on human health, with a particular emphasis on the immune system—an area that has been insufficiently Open Research Europe  Page 21 of 25 Open Research Europe 2024, 4:271 Last updated: 25 NOV 2025 explored in prior studies. This work is essential for gaining a comprehensive understanding of the extent of EDCs' effects on all living organisms, including both human and animal health. The main weaknesses of the present manuscript lie in the details of the design and execution of these studies. For instance, the authors claim that “ENDOMIX will design in vitro studies to analyse endpoints relevant for immunotoxicants and associated mediators of health outcomes, as defined in the cohort studies.” These are two major undertakings that will require significant time and research investment. Firstly, there are no established in vitro methods for assessing the immune system effects of lowlevel EDCs typically found in the environment, which are usually present at nano and picomolar concentrations. The authors do not appear to consider the labour-intensive and costly techniques currently used in studies on mammals and other species. Existing methodologies should be adapted to study biologically active relevant EDC concentrations in living cells, such as tracking the nuclear translocation of activated hormonal receptors. These are performed by analysing the nuclear/cytoplasmic ratio as the direct biological effect (PMID:32018941). While such studies are pertinent to the immune system, assessing the nuclear/cytoplasmic ratio may present challenges when applied to immune cells. Further references to existing methodologies (PMID 34339825), along with the authors' commentary on their potential applications in immune system research, are necessary to support the continuation of the ENDOMIX studies. Secondly, to our knowledge, no established methodologies currently exist for linking in vitro studies to the associated mediators of health outcomes. All previous information has been based on correlations rather than direct evidence. If the authors intend to propose a novel approach for identifying the direct effect of EDCs on the immune system, it would be a significant advancement and would require a thorough, detailed explanation. References 1. Varticovski L, Stavreva DA, McGowan A, Raziuddin R, et al.: Endocrine disruptors of sex hormone activities.Mol Cell Endocrinol. 2022; 539: 111415 PubMed Abstract | Publisher Full Text 2. Jones RR, Stavreva DA, Weyer PJ, Varticovski L, et al.: Pilot study of global endocrine disrupting activity in Iowa public drinking water utilities using cell-based assays.Sci Total Environ. 2020; 714: 136317 PubMed Abstract | Publisher Full Text Is the rationale for the Open Letter provided in sufficient detail? (Please consider whether existing challenges in the field are outlined clearly and whether the purpose of the letter is explained) Yes Does the article adequately reference differing views and opinions? No Are all factual statements correct, and are statements and arguments made adequately supported by citations? No Open Research Europe  Page 22 of 25 Open Research Europe 2024, 4:271 Last updated: 25 NOV 2025 Is the Open Letter written in accessible language? (Please consider whether all subjectspecific terms, concepts and abbreviations are explained) Yes Where applicable, are recommendations and next steps explained clearly for others to follow? (Please consider whether others in the research community would be able to implement guidelines or recommendations and/or constructively engage in the debate) Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: Endocrine disruptors, Hormonal actions I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Author Response 14 Nov 2025 Thi Anh Mai Pham We appreciate the reviewers’ valuable comments and suggestions on our manuscript and would like to thank you for classifying the project as relevant and timely. We have carefully considered the points raised and prepared a detailed, point-by-point response to each comment, indicating the corresponding changes made in the manuscript and where they can be found in the revised version: The main weaknesses of the present manuscript lie in the details of the design and execution of these studies. For instance, the authors claim that “ENDOMIX will design in vitro studies to analyse endpoints relevant for immunotoxicants and associated mediators of health outcomes, as defined in the cohort studies.” These are two major undertakings that will require significant time and research investment. Firstly, there are no established in vitro methods for assessing the immune system effects of lowlevel EDCs typically found in the environment, which are usually present at nano and picomolar concentrations. The authors do not appear to consider the labour-intensive and costly techniques currently used in studies on mammals and other species. Existing methodologies should be adapted to study biologically active relevant EDC concentrations in living cells, such as tracking the nuclear translocation of activated hormonal receptors. These are performed by analysing the nuclear/cytoplasmic ratio as the direct biological effect (PMID: 32018941). While such studies are pertinent to the immune system, assessing the nuclear/cytoplasmic ratio may present challenges when applied to immune cells. Further references to existing methodologies (PMID 34339825), along with the authors' commentary on their potential applications in immune system research, are necessary to support the continuation of the ENDOMIX studies. Author response: Thank you for this valuable comment. We acknowledge that the methodological options for assessing the impact of chemicals at real-life concentrations remain limited, and addressing this gap is precisely where the ENDOMIX project aims to Open Research Europe  Page 23 of 25 Open Research Europe 2024, 4:271 Last updated: 25 NOV 2025 make a difference. It is important to distinguish between screening methods that can evaluate multiple chemicals based on general endpoints with regulatory potential, and more sophisticated methods that provide a detailed mechanistic understanding of how chemicals affect the immune system. In ENDOMIX, we employ a tiered approach to investigate the effects of chemicals and their mixtures across models of increasing physiological complexity, ranging from cell-based systems to in vivo models. The first tier comprises high-throughput assays designed to measure molecular initiating events and key biological responses, such as the quantitative activation of nuclear receptors and transcription factors in reporter gene assays (e.g., estrogen receptor, NF-κB, thyroid receptors). For the high-throughput systems, we test mixtures at concentration ratios corresponding to those detected in environmental samples, but at slightly higher absolute concentrations to ensure measurable effects. Provided that these effects fall within the linear range of the concentration–response curves and linearity at low doses has been established, it is then possible to extrapolate the results to environmentally relevant concentrations. Regarding the papers mentioned by the reviewer, we would like to note that the highthroughput in vitro assays used in ENDOMIX follow similar principles to those described in the first cited paper, which in fact references our earlier work on the application of reporter gene assays for water quality assessment. The main difference is that their setup is not high-throughput, as it relies on 96-well plates. The review article cited is indeed an excellent summary of endocrine disruptors acting on sex hormone pathways and describes many of the assays typically used in this field. We have extensive experience with these types of assays and have carefully considered them in our design. However, since our focus in ENDOMIX is on immunomodulatory endpoints, which are novel in this context, we prioritized selecting and optimizing assays relevant to immune function. The second tier involves more advanced methods that go beyond single endpoints by employing primary immune cells (PBMCs) obtained from donor blood samples. We have already established flow cytometry protocols to assess the impact of chemicals on multiple immune cell subsets (doi: 10.3389/fimmu.2023.1327960). Within ENDOMIX, we will further refine these methodologies to characterize how chemical mixtures influence immune cell subsets, functional parameters such as cytokine secretion, and activation status. This approach also allows us to identify immune cell subpopulations warranting more detailed investigation. Subsequently, we are developing specific assays for selected immune cell types to gain a deeper mechanistic understanding of chemical effects at the single-cell level. For clarification, we have added a new paragraph under the section “ENDOMIX conception” providing the explanation mentioned above. Secondly, to our knowledge, no established methodologies currently exist for linking in vitro studies to the associated mediators of health outcomes. All previous information has been based on correlations rather than direct evidence. If the authors intend to propose a novel approach for identifying the direct effect of EDCs on the immune system, it would be a significant advancement and would require a thorough, detailed explanation. Author response: We fully agree that establishing direct links between in vitro findings and health outcomes is inherently challenging. To address this complexity, ENDOMIX employs an innovative triangulation strategy that integrates evidence from cohort studies, in vitro, and in vivo models focusing on the same chemical mixtures. This multi-layered approach is Open Research Europe  Page 24 of 25 Open Research Europe 2024, 4:271 Last updated: 25 NOV 2025 designed to move beyond associative findings and towards the identification of causal immune targets of endocrine-disrupting chemicals (EDCs). In the cohort studies, chemical exposure data are linked to OLINK-derived proteomic profiles that capture subtle changes in immune parameters. This enables us to determine whether specific mixtures are associated with inflammatory signatures, immune suppression, or intracellular signaling alterations, and how these relate to defined health outcomes. By combining exposure data with molecular and clinical endpoints, we can generate biologically meaningful hypotheses that guide mechanistic testing. In the in vitro component, the same mixtures are systematically tested in human immune cell models to dissect their effects on inflammatory pathways, cytokine secretion, and immune functionality across defined cell subtypes. These mechanistic data provide insight into the cellular and molecular processes underlying the associations observed in human populations. In parallel, the in vivo studies allow us to assess whether the same mixtures can elicit or exacerbate specific health outcomes under controlled conditions, while concurrently evaluating their immunomodulatory effects. This offers a critical bridge between molecular mechanisms and organism-level responses. By integrating and cross-validating findings across these complementary tiers, ENDOMIX will generate a coherent, causality-oriented framework that connects environmental exposure to immune dysregulation and disease risk. This triangulated design represents a key strength of the project, enhancing both the robustness and translational relevance of the results. To address the reviewers’ comment, we have revised the paragraph describing the triangulated design under the section “ENDOMIX conception” according to the explanation mentioned above. Competing Interests: No competing interests were disclosed. Open Research Europe  Page 25 of 25 Open Research Europe 2024, 4:271 Last updated: 25 NOV 2025