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

Ana Claudia Zenclussen, Valentina Belmar Erilkin, Linda Böhmert, Petra

Borilova Linhartova, Albert Braeuning, Georg Braun, Cécile Chevrier,

Liesbeth Duijts, Beate Isabella Escher, Janine Felix, et al.

► To cite this version:

Ana Claudia Zenclussen, Valentina Belmar Erilkin, Linda Böhmert, Petra Borilova Linhartova, Albert Braeuning, et al.. The ENDOMIX project: an interdisciplinary approach to understanding how real-life chemical mixtures target the immune system to trigger disease. Open Research Europe, 2025, 4, pp.271. 10.12688/openreseurope.19088.2. . hal-05425434

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OPEN LETTER

REVISED

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

[version 2; peer review: 1 approved, 3 approved with reservations]

Ana Claudia Zenclussen<sup>1,2</sup>, Valentina Belmar Erilkin<sup>3</sup>, Linda Böhmert<sup>4</sup>,
Petra Borilova Linhartova<sup>5</sup>, Albert Braeuning<sup>4</sup>, Georg Braun<sup>6</sup>,
Cécile Chevrier<sup>7</sup>, Liesbeth Duijts<sup>8-10</sup>, Beate Isabella Escher<sup>2,6</sup>, Janine Felix<sup>8,9</sup>,
Sergio Gómez-Olarte<sup>1</sup>, Mònica Guxens<sup>11</sup>, Gunda Herberth<sup>1</sup>,
Klara Hilscherova<sup>5</sup>, Jana Klanova<sup>5</sup>, Yvonne Kohl<sup>12</sup>, Katharina Krischak<sup>3</sup>,
Dominique Lagadic-Gossmann<sup>7</sup>, Sophie Langouët<sup>7</sup>, Sabrina Llop<sup>13,14</sup>,
Maria Jose Lopez-Espinosa<sup>13-15</sup>, Léa Maitre<sup>11</sup>, Corinne Martin-Chouly<sup>7</sup>,
Nicole Meyer<sup>1</sup>, Marion Ouidir<sup>16</sup>, Thi Anh Mai Pham<sup>1</sup>, Claire Philippat<sup>16</sup>,
Raymond Pieters<sup>17</sup>, Marie-Laure Pinel-Marie<sup>7</sup>, Normand Podechard<sup>7</sup>,
Tobias Polte<sup>1</sup>, Elliott Price<sup>5</sup>, Oliver Robinson<sup>18</sup>, Kristin Schubert<sup>19</sup>,
Anne Schumacher<sup>1</sup>, Violeta Stojanovska<sup>1</sup>, Tamara Tal<sup>20</sup>, Paolo Vineis<sup>18</sup>,
Robert van Vorstenbosch<sup>17</sup>, Roel Vermeulen<sup>17</sup>, Charline Warembourg<sup>7</sup>

<footnote n="1"><sup>1</sup>Department of Environmental Immunology, Helmholtz-Centre for Environmental Research - UFZ, Leipzig, Saxony, 04318, Germany</footnote>

<footnote n="2"><sup>2</sup>German Center for Child and Adolescent Health (DZKJ), partner site Leipzig/Dresden, Leipzig, Germany</footnote>

<footnote n="3"><sup>3</sup>European Institute for Biomedical Imaging Research (EIBIR), Vienna, Austria</footnote>

<footnote n="4"><sup>4</sup>German Federal Institute for Risk Assessment (BfR), Dept. Food Safety, Berlin, 10589, Germany</footnote>

<footnote n="5"><sup>5</sup>RECETOX, Faculty of Science, Masaryk University, Brno, South Moravian Region, Czech Republic</footnote>

<footnote n="6"><sup>6</sup>Department of Cell Toxicology, Helmholtz-Centre for Environmental Research - UFZ, Leipzig, Saxony, 04318, Germany</footnote>

<footnote n="7"><sup>7</sup>Univ Rennes, Inserm, EHESP, Institut de Recherche en Santé, Environnement et Travail - UMR_S 1085, Rennes, France</footnote>

<footnote n="8"><sup>8</sup>Department of Pediatrics, Erasmus MC, University Medical Center, Rotterdam, The Netherlands</footnote>

<footnote n="9"><sup>9</sup>Generation R Study Group, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands</footnote>

<footnote n="10"><sup>10</sup>Department of Neonatal and Intensive Care, Division of Neonatology, Erasmus MC, University Medical Center, Rotterdam, Rotterdam, The Netherlands</footnote>

<footnote n="11"><sup>11</sup>Institute for Global Health, ISGlobal, Barcelona, Spain</footnote>

<footnote n="12"><sup>12</sup>Fraunhofer Institute for Biomedical Engineering IBMT, Sulzbach, Germany</footnote>

<footnote n="13"><sup>13</sup>Epidemiology and Environmental Health Joint Research Unit, FISABIO-Universitat Jaume I-Universitat de València, Valencia, Spain</footnote>

<footnote n="14"><sup>14</sup>Spanish Consortium for Research on Epidemiology and Public Health (CIBERESP), Madrid, Spain</footnote>

<footnote n="15"><sup>15</sup>Department of Nursing, Faculty of Nursing and Chiropody, University of Valencia, Valencia, Spain</footnote>

<footnote n="16"><sup>16</sup>University Grenoble Alpes, Inserm U-1209, CNRS-UMR-5309, Environmental Epidemiology Applied to Development and Respiratory Health Team, Institute for Advanced Biosciences, Grenoble, France</footnote>

<footnote n="17"><sup>17</sup>Institute for Risk Assessment Sciences, Utrecht University, Utrecht, The Netherlands</footnote>

<footnote n="18"><sup>18</sup>MRC Centre for Environment and Health, School of Public Health, Imperial College London, London, UK</footnote>

<footnote n="19"><sup>19</sup>Department of Molecular Toxicology, Helmholtz-Centre for Environmental Research - UFZ, Leipzig, Saxony, 04318, Germany</footnote>

<footnote n="20"><sup>20</sup>Department of Ecotoxicology, Helmholtz-Centre for Environmental Research - UFZ, Leipzig, Saxony, 04318, Germany</footnote>
**v2** 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

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 co-
culture 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 policy-
making and also reach people of all ages to raise awareness of the
risks of EDC exposure and encourage health-promoting behaviors.

Keywords

endocrine disrupting chemicals, EDC exposure, chemical mixtures,
immune system, long-term health effects, interdisciplinary approach,
science to policy translation

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This article is included in the Horizon Europe
gateway.

Open Peer Review

Approval Status

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1. Lyuba Varticovski ID, National Institutes of Health, Maryland, USA

2. Jacques Robert ID, University of Rochester Medical Center, Rochester, USA

3. Josef Köhrle ID, Charité-Universitätsmedizin Berlin, Berlin, Germany

4. David Lopez-Rodriguez ID, F. Hoffmann-La Roche Ltd, Basel, Switzerland

Any reports and responses or comments on the
article can be found at the end of the article.
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This article is included in the Horizon 2020
gateway.

**Corresponding author:** Ana Claudia Zenclussen (ana.zenclussen@ufz.de)

**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:** © 2025 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 2; peer review: 1 approved, 3 approved with reservations] Open Research Europe 2025, 4:271 https://doi.org/10.12688/openreseurope.19088.2

**First published:** 19 Dec 2024, 4:271 https://doi.org/10.12688/openreseurope.19088.1
**REVISED** Amendments from Version 1

In response to the reviewers' comments, we have revised the manuscript to clarify and improve several aspects related to the ENDOMIX conception and objectives.

We have added a paragraph to the ENDOMIX conception section that provides a more detailed methodological explanation of the tiered approach used to investigate the effects of chemicals and their mixtures across models of increasing physiological complexity, ranging from cell-based systems to *in vivo* models. We have also elaborated further on the triangulation strategy employed within ENDOMIX to generate a coherent, causality-oriented framework that connects environmental exposure to immune dysregulation and disease risk.

In the Objective 3 section, we have also expanded the description of the approaches applied within ENDOMIX to illustrate how EDCs may alter immune responses against pathogens.

Any further responses from the reviewers can be found at the end of the article

**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 single chemicals 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 this 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 vulner-
able 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 driv-
ing 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 consid-
ered 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.

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.

In ENDOMIX, we employ a tiered approach to investigate the
effects of chemicals and their mixtures across models of increas-
ing physiological complexity, ranging from cell-based sys-
tems 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). The sec-
ond 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 subsets¹⁷. Within ENDOMIX, we will
further refine these methodologies to characterize how chemi-
cal mixtures influence immune cell subsets, functional param-
eters such as cytokine secretion, and activation status. This
approach also allows us to identify immune cell subpopula-
tions 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.

To address the complexity of linking *in vitro* findings and health outcomes, 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 designed to move beyond associative findings and towards the identification of causal immune targets of EDCs (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 models¹⁸. 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 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 cell- and organ-dependent 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
<figure><img src="image_6.png" /><figcaption>**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.</figcaption></figure>

and policy makers, inform the public and contribute to
improved risk assessment.

ENDOMIX objectives

ENDOMIX aims at unravel the overall immunotoxic or immu-
nomodulatory 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 expo-
sure scenarios, co-exposures' effects (including lifestyle and

socioeconomic factors), target cells and organs, and mecha-
nistic 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 thou-
sands of chemicals in our blood and there are hundreds of
chemicals that potentially cause endocrine disruption and immu-
nomodulation, 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 high-throughput 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

<figure><img src="image_7.png" /><figcaption>**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.</figcaption></figure>
screening data of a number of EDCs: polychlorinated biphenyls, organochlorine pesticides, per- and polyfluorinated compounds, metals, parabens, phthalates, phenols, and organophosphate pesticides. Further, for cohorts with limited availability of 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 per- and 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 project¹⁹ and projects that are part of the European Human Exposome Network (EHEN, including ATHLETE²⁰, LongITools²¹ and Expanse²²). ENDOMIX will use existing targeted screening data of a number of EDCs: polychlorinated biphenyls, organochlorine pesticides, per- and 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, 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, microbiome, 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 immunome 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 patterns²³. 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. In some 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 pathogens²⁴⁻²⁶. 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 barrier²⁷,²⁸, *in vitro* blood-brain-barrier²⁹,³⁰, *in vitro* placenta barrier³¹ and *in vitro* intestinal barrier³²,³³.

Further, for target organ models, we use lung organoids³⁴, *in vitro* heart spheroids³⁵,³⁶, intestinal organoids³⁷,³⁸ and liver spheroids³⁹,⁴⁰. 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 accumulation⁴¹ 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 *in vitro* models will be used to study interactions between different organs (e.g. intestinal-hepatic interaction or liver-adipocyte 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. elegans*⁴²,⁴³. 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-compliant⁴⁴, alternative model that, up to 5 days post-fertilization (dpf), is considered to be a non-protected life stage, according to EU regulations⁴⁵.

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

<figure><img src="image_8.png" /><figcaption>**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.</figcaption></figure>
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 studies⁴⁶⁻⁴⁸. As for *reproductive health*, we will study potential EDC effects on the reproductive performance of female and male offspring of EDC-exposed dams (same exposure protocol as for the other disease models)⁴⁹,⁵⁰. To assess puberty onset in female offspring, we will assess vaginal opening and oestrous cycle analyses, and as ovarian follicle maturation analyses⁵¹,⁵². For male offspring, sperm analyses will provide insights into their fertility status⁵³.

## Objective 5: Engagement with regulators and providing guidance on exposure to the European community

The European public needs access to well-founded information on the effects of complex mixtures of EDCs to avoid disinformation. Citizens need to be empowered to self-regulate and reduce their exposure to EDCs and mixtures of concern in everyday life to increase the number of healthy life years. ENDOMIX will provide guidance on exposure to EDCs and mixtures and information about potential adverse effects and lay the ground work for regulatory changes and preventive measures for citizens, regulators, and relevant stakeholders within the healthcare value chain. The circle will be closed by using the generated information to develop action plans and recommendations to address priority mixtures of concern that impact health and proposing a more harmonized battery of risk assessment with specific toxicological endpoints and well-defined biomarkers.

To combine information from experimental and epidemiological studies conducted in ENDOMIX and existing and future literature, we will adopt a Weight-of-Evidence (WoE) approach. These approaches are commonly used in authoritative risk assessment programs by organizations such as the WHO, EU, and US-EPA³⁷,⁵⁴. In addition, we will utilize novel text mining methodologies to create knowledge graphs that capture the relevant complex biomechanistic insights. Specifically, we will employ the Integrated Network and Dynamical Reasoning Assembler (INDRA) Reach (Reading and Assembling Contextual and Holistic Mechanisms from Text) environment, which uses automated large-scale reading of scientific literature, natural language processing (NLP) approaches, and structured databases to collect and assemble mechanistic understanding of cellular processes and associated health effects³⁹. This approach has already been successfully applied for genotoxic compounds⁵⁵ and will be extended to EDCs in this study. Through INDRA, mechanistic information from multiple sources will be de-duplicated, standardized, and assembled into sets of statements with associated evidence levels. These Statements can then be used to construct executable rule-based models and a variety of network models, such as knowledge graphs⁵⁵. We aim to assess the concordance between the traditional WoE approach and generated Statements to provide an expert-led assessment of the validity of this novel computational approach to evidence synthesis”

## Science to policy translation

The scientific insights and knowledge generated by ENDOMIX will be the basis for subsequent application in policy making and development of new regulations. The consortium aims to develop new hands-on research-based guidance and policy options and will provide policy makers and decision makers with new data and evidence-based information. To strengthen the science-policy interface and promote acceptance and uptake of the developed new knowledge base and guidelines, active stakeholder involvement and a thorough consultation process will be ensured through the set-up of a dedicated Stakeholder Board (SHB). The SHB involves members advocating for future policies, scientific advisors, and a citizen representative
who provide input early on in the process to ensure their perspectives are considered when developing policy recommendations. ENDOMIX will contribute to the EU's goal to ensure EU policy making is based on evidence, provide simple, clear and transparent recommendations and involve stakeholders along the knowledge chain in the development. Further, communication and dissemination activities including educational approaches will facilitate science-to-policy translation of the ENDOMIX findings and guidelines.

The strategy chosen for ENDOMIX to fulfill the objectives is shown in Figure 1 and Figure 4.

Within ENDOMIX, we bring together renowned experts in toxicology, clinical science, environmental immunology, social, environmental and molecular epidemiology, biology, statistics and data science, and risk assessment. Exposure to chemicals

can lead to different adverse effects in humans depending on sex, gender, and age. Population studies have shown different exposure patterns in men and women that show a risk factor for diseases such as those related to the immune, reproductive, and metabolic area. Population-based data from the cohorts will be separately analysed for females and males to understand the possible different impacts of different EDC mixtures depending on the sex and on life stages. As for experimental models, ENDOMIX will incorporate the sex difference dimension into the toolbox by using *in vivo* data from animals of both sexes where available for benchmarking the *in vitro* assays and by adapting methods and protocols using cell lines from males as well as from females (where available) as research has shown the relevance of including both sexes in experiments⁵⁶. Thus, sex differences in sensitivity, sex-specific variables of biomarkers and sex-specific cut-off values will be considered. From a scientific point of view, sex parameters are

<figure><img src="image_9.png" /><figcaption>**Figure 4. The ENDOMIX concept and workflow.** The ENDOMIX project is structured into well-coordinated work packages (WPs) fostering an interdisciplinary approach to understanding the true impact of endocrine disrupting chemical (EDCs) on human health. In addition to the WPs related to the desired research outputs, work dedicated to project coordination and management, clustering activities and ethics requirement is underlying this concept.</figcaption></figure>
specifically considered in our work and attempts to include cell lines and 3D organoids originating from tissues originating from males and females will be made wherever possible. ENDOMIX has already designed organoid studies to contemplate sex differences between female and male, this is particularly relevant for placenta organoids, where the origin of the tissue is often overlooked. Models relying on available cell lines may be a limiting factor for some of the outcomes. Molecular markers are available to determine sex and can be used for retrospective analyses of sex differences. In the epidemiological studies, the models will be adjusted for sex or stratified by sex where applicable.

In conclusion, the ENDOMIX project aims to unravel the complexity of exposure to EDCs and their impact on human health. In particular, ENDOMIX aims at contributing to the understanding of immune-mediated health outcomes and providing comprehensive insights into the biological mechanisms linking EDC exposure to adverse health outcomes, by incorporating multi-omics data, including immunomics, epigenomics, transcriptomics, metagenomics, and metabolomics so to enhance our understanding of the molecular pathways affected by EDCs. By employing already existing cohorts covering different life phases, ENDOMIX can explore the effects of early life EDC exposure and its influence on long-term health trajectories. The knowledge generated by ENDOMIX will inform evidence-based policy-making aimed at reducing EDC exposure, mitigating associated health risks, raising public

awareness about the risks of EDC exposure and promoting health-promoting behaviours.

## Disclaimer

The views expressed in this article are those of the author(s). Publication in Open Research Europe does not imply endorsement of the European Commission.

## Ethics and consent

Ethical approval and consent were not required for writing this project summary. The ENDOMIX project itself will be conducted on existing prospectively collected data from different European cohort studies. All participating cohorts have been approved by the relevant ethics boards and informed consent was obtained for all participants.

All research that involves human data is conducted under the rules and legislation in place, including the Declaration of Helsinki, the IHC guideline for Good Clinical Practice, as well as the ISO guidelines on good clinical practice ISO 14155:2020, the European Directive 2001/20/EC on Good Clinical Practice, and the EU General Data Protection Regulation (2016/679).

## Data availability statement

No data are associated with this article

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55. Scholten B, Simón LG, Krishnan S, et al.: Automated network assembly of mechanistic literature for informed evidence identification to support cancer risk assessment. *Environ Health Perspect.* 2022; **130**(3): 37002.

PubMed Abstract | Publisher Full Text | Free Full Text

56. Miller I, Diepenbroek C, Rijntjes E, et al.: Gender specific differences in the liver proteome of rats exposed to short term and low-concentration Hexabromocyclododecane (HBCD). *Toxicol Res (Camb).* 2016; **5**(5): 1273-1283.

PubMed Abstract | Publisher Full Text | Free Full Text
# Open Peer Review

Current Peer Review Status: ? ✅ ? ?

## Version 2

Reviewer Report 30 December 2025

https://doi.org/10.21956/openreseurope.23738.r64836

© 2025 Lopez-Rodriguez D. This is an open access peer review report distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

<figure><img src="image_10.png" /></figure>

David Lopez-Rodriguez ID

Predictive Modelling, Pharma Research and Early Development (pRED), F. Hoffmann-La Roche Ltd, Basel, Switzerland

EDCs are clearly a growing concern for the environment, wildlife, and humans around the globe. Despite this, most of the literature in the field has focused on evaluating the same molecules and endpoints. As a consequence, there is an urgent need to better understand the wide range of molecules to which we are all exposed, the health consequences of complex EDC mixtures and the assessment of less commonly investigated endpoints such as the immune system. The ENDOMIX project arrives at a very timely moment to help fill critical gaps in knowledge in the field.

Please find here a series of comments that may help to further improve the manuscript and the project:

1) Despite being a large consortium, the project may be overly ambitious, spanning a multiplicity of methods, topics and expected outcomes. For instance, the core of the project relies on the use (and development) of non-standard assays, mixture assessment, the implementation of NAMs, extremely complex experimental designs - often difficult to interpret - and in silico and statistical/mathematical methods. Contributing to the development of guidance within the timespan of this project, promoting regulatory acceptance of such complex and non-standardised methods or supporting decision-making using complex EDC mixture evidence seems —unfortunately— unrealistic. The authors may consider being more specific in their intended outcomes within the science-to-policy context (e.g., contributing to public awareness via press releases, publishing novel methods and contributing to their integration as an OECD guideline, generating new methods for EDC mixture risk assessment or IVIVE).

2) Following the previous point, this open letter would benefit from a figure schema of the different WPs, their expected outcomes and the broad methodological approaches to be implemented. Additionally, the schema could include an overview of the broad timeline and show how the different WPs will be integrated into a cohesive approach. This figure would provide a more informative big-picture overview of the project than Figure 4, which does not add more
value than the section headers in the main text. The schema could also outline clear actionable steps (for instance, for regulatory relevance).

3) Many biomonitoring projects have been conducted in the past 10-15 years in Europe. One of them, HBM4EU, stands out and I am surprised it is not included in Figure 2. Is there a particular reason for this? If the list is not intended to be exhaustive, this should be indicated in the figure caption. Additionally, we live in an ever-changing chemical environment and pollutants found in the 50s may have nothing to do with the ones we are exposed today. How these aspects are considered in the cohorts authors have selected to define the final mixture?

4) From a practical perspective, many different research teams will be working in parallel to achieve ENDOMIX goals. In fact, several ambitious EU-funded projects in the past have struggled to integrate results across partners, ending up by publishing isolated papers within each team's domain of expertise. A concise and short overview of the project collaboration framework, leadership structure and about the integrative strategy that will be performed would definitely strengthen confidence in the feasibility of the project.

5) The concept of uncertainty assessment is not mentioned. While I don't expect a detailed methodology in an open letter, it would be worth mentioning the integration of uncertainty evaluation across all proposed models, specially for the extrapolation to health outcomes and to have a clear quantitative impact on decision making.

6) Objective 6 aims to establish a causal link between EDC exposure and health outcomes. It is unclear how transgenerational in vivo studies will address this question, given that evidence for transgenerational immune effects in humans is largely lacking. Are the authors referring to multigenerational studies rather than germline-mediated effects (true transgenerational effects), which require assessing at least three generations? Unless the intention is to compare germline versus somatic EDC effects (which seems to be out of the scope here), it is not clear how transgenerational studies would support causal associations. Additionally, reproductive transgenerational effects of EDC mixtures have already been investigated and seem outside the scope of this proposal, which focuses primarily on immune outcomes. If the authors intend to retain transgenerational studies, further clarification of the specific endpoints and the definition of causal relationships would be helpful.

7) Broadly, several ideas across the letter are repeated multiple times. Specifically, Page 5 of the manuscript reiterate the knowledge gaps relating EDCs to immune disorders. In this case, the first column of page 5 could be revised to include actual evidence from human studies, for instance, summarising recent findings in the field, for example. Reducing redundancy would also create space for a short section discussing major methodological or conceptual challenges associated to the project, which would be greatly appreciated.

8) Regarding the definition of the chemical mixtures to be evaluated, how will the authors ensure reproducibility across cohorts considering differences in sample types, measurement platforms, time periods, developmental stages, populations, socioeconomic contexts, types of molecules,...?

9) The term "mixture effect drivers" could be briefly defined.

10) Statements such as "The vision of ENDOMIX is to fully elucidate and understand the overall
effects of complex EDC mixtures and the underlying mechanisms" should be toned down.

11) Small typo on page 7: "analysisby" -> "analysis by" (only present in the pdf)

**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)**

Partly

**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 subject-specific 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:** Endocrine disruption, Neurotoxicity, NAMs, computational toxicology, DART, neuroendocrinology, transgenerational EDC effects.

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 02 December 2025

https://doi.org/10.21956/openreseurope.23738.r64634

© 2025 Robert J. This is an open access peer review report distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

<figure><img src="image_11.png" /></figure>

Jacques Robert iD

University of Rochester Medical Center, Rochester, USA

No further comments
**Competing Interests:** No competing interests were disclosed.

**Reviewer Expertise:** 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.

## Version 1

Reviewer Report 29 August 2025

https://doi.org/10.21956/openreseurope.20658.r55593

© 2025 Köhrle J. This is an open access peer review report distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

<figure><img src="image_12.png" /></figure>

Josef Köhrle ID

Charité-Universitätsmedizin Berlin, Berlin, Germany

Acute and chronic exposure to environmental chemicals, especially those targeting the endocrine and reproductive systems and crucial neurodevelopmental processes, has been demonstrated to be associated with predisposition, emergence and deterioration of common diseases, especially if exposure occurs during intrauterine development and sensitive developmental windows of the growing and maturing organism. Mechanistic evidence, clear cause-effect relationships between exposure and adverse outcome, established by using informative animal experimental models, and recently accumulating human epidemiological data provide strong support for adverse, frequently irreversible action of chemicals contained in our nutrition, drinking water, cosmetics and consumer products. Limited information is available so far for their adversity on the metabolic and immune system.

The authors provide a detailed outline of an EU funded project addressing the potential adverse impact of exposures to realistic mixtures of (environmental) chemicals targeting components of the immune system and related diseases. Currently, information on adverse effect of chemicals (not only EDCs) mainly is based on analyses of mechanisms and endpoints for single compound, while realistic exposure scenarios comprise complex mixtures and multiple routes of exposure. The ENDOMIX team intends a multimodal application and combination of several methodological approaches to identify biomarkers accessible by large cohort studies for various advisory policies.

As the immune system is involved in many crucial processes relevant for health and disease the planned studies might significantly contribute to clarify which chemical mixtures and which of their main components are associated with or even causally involved with adverse outcome of developmental or chronic impact of mixture exposures. Currently, it is still unclear whether underlying mechanisms result in immuno-, neuro-, repro- or endocrino-toxic effects or their combination. Obviously, statements (chapter ENDOMIX objectives) like "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 ...",
graphically summarized in Fig.1, are not only 'ambitious', but maybe considered as overambitious,
boastful and probably unrealistic for the given time frame, team composition and budget
restrictions and in the context of many research teams who worldwide work on this complex
thematic with serious efforts!

It is also questionable whether the goal will be achieved that "ENDOMIX will deliver evidence-based guidelines and advice to citizens on improving human health". Guidelines is a very clearly defined term in biomedicine. At its best, if successful, ENDOMIX might delineate some **guidance** information for individuals, risk groups, regulatory authorities or policy makers. But consolidation of such information will still need to be confirmed and validated by implementation of properly powered prospective (mid-/long-term) studies which might generate such clinically-relevant evidence for clinical practice and best patient care, including prevention of disease. Authors should be more cautious in their terminology and tone down expectations of the outcome of this challenging project.

The chosen conventional and innovative state of the art models (8!) for testing the selected
mixtures are suitable for the intended purpose and probably will allow identification of novel EDCs
and prioritization of drivers of adverse mixture effects with the focus on the immune system. Less
clear remains the selection of the chemical mixtures to be tested in the various models based on
the exposome data that will be collected from the various cohorts studied. Probably, the inclusion
of some already established EDC reference mixtures (e.g. ovarian fluid, amniotic fluid along
pregnancy trimesters) that have been studied in recent EU-funded projects would provide
benchmarks and allow comparability with existing data. Whether ENDOMIX team will take
advantage the extensive HBM4EU exposure information gathered during the last decade and
exploit this data remains unclear.

ENDOMIX will have access to chemical exposure data from observational studies from several cohorts across Europe which cover various life phases from pregnancy to adulthood (Fig. 2) and will provide rather complex data (microbiome, epigenome, transcriptome and metabolome) on various chemicals contained in real-life mixtures in context of a broad spectrum of disease parameters. Nevertheless, the complexity of this data and the complexity of the immunotoxicity focus of this project will remain a tremendous challenge for its exploitation.

Whether the 5 main immune system-related health outcome goals of this project (Objective 2) can be achieved with the chosen strategy, selected models and method spectrum provided by the multidisciplinary but very heterogeneously composed team of experts remains to be seen.

The team also expects to fill the gap between in vitro model systems to the immune system
operating in an intact organism (e.g. from stem cell-derived organoids to the intact organism
across its life phases) by exploiting the *C. elegans* innate immune response and zebrafish embryo
models. As mentioned before this seems more than ambitious in its extent of goals, parameters
and endpoints to be analyzed. Couldn't they decide to leave something out and to prioritize and
focus on some key aspects instead of presenting a panoptic scenario?

In objective 4 the ENDOMIX consortium intends to establish causality of EDC mixture exposure on
health outcomes and realistically also plans to use experimental mouse models when the chosen
in vitro approaches will reach their predictive and informative limits required for extrapolation and interpretation of in vivo conditions. However, they do not elaborate in detail for which diseases which proper mouse models are existing and for which they are still lacking and where their limitations are in context of immunotoxic EDC effects and mechanisms involved.

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 subject-specific 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.

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](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

<figure><img src="image_13.png" /></figure>

Jacques Robert

<figure><img src="image_14.png" /></figure>

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 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 subject-specific 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 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 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 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.

<figure><img src="image_15.png" /></figure>
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 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 low-level 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

**Is the Open Letter written in accessible language? (Please consider whether all subject-specific 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 low-level 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 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 high-throughput 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
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.