In murine species such as laboratory mice and rats, the number of nipples is a sexually dimorphic trait, with males typically not expressing nipples. This is an androgen-sensitive trait, with regression of the nipple anlagen dictated by high androgen levels during development. Nipple/areola retention (NR) in male rodents is thus considered a sensitive marker of disrupted androgen signaling during development and used to identify chemicals with antiandrogenic effects. Here, we have developed an adverse outcome pathway network for NR induced by reduced androgen signaling during critical developmental life stages. The network integrates three adverse outcome pathways covering (a) inhibited testosterone production, (b) reduced conversion to dihydrotestosterone, and (c) direct androgen receptor antagonism. It supports the regulatory application of NR as a measure of endocrine disruption relevant for human health and the use of NR as an indicator of antiandrogenicity in mammals and other vertebrates in the environment. It presents key events and key event relationships that can be leveraged to enhance the use of new approach methodologies, enhancing predictive toxicology and assessment of endocrine disruptors.

The Science in Risk Assessment and Policy (SciRAP) platform has been developed to support the structured and transparent evaluation of data in hazard and risk assessment of chemicals. This work aimed to develop a SciRAP tool for the evaluation of the reliability and relevance of observational epidemiological studies (SciRAPepi tool), including cross-sectional, classical case-control, nested case-control, and cohort studies. A first version of the SciRAPepi tool was created, and an expert testing round was conducted to assess its scientific soundness and user-friendliness. Thirty-seven epidemiologists and risk assessors took part in the testing using the tool to evaluate four epidemiological studies representing the main epidemiological study designs. The majority of experts considered the criteria appropriate, and overall evaluations indicated good consistency across most criteria. Based on the testing results, the tool was further developed and refined. Together with the SciRAPepi tool, guidance and instructions on its use were developed, along with a reporting checklist to support researchers in reporting their studies. The SciRAPepi tool intends to enhance the use of reliable epidemiological data in the hazard and risk assessment of chemicals and other scientific assessments, offering a broader range of options for evaluating these data for epidemiologists and risk assessors.

Minimizing exposure to endocrine disruptors (EDs) is a priority within the European Union's political agenda. These chemical substances interfere with the endocrine system, leading to adverse effects on human health and the environment. Despite extensive research, significant uncertainties remain regarding safe levels of exposure to EDs. This paper highlights the need for cautious risk assessment due to substantial knowledge gaps, limitations in testing and unresolved questions about thresholds, non-monotonic dose-responses and low-dose effects. On this basis, we recommend a precautionary approach to risk assessment of EDs and, in general, support initiatives to reduce exposure by phasing out use and production. In cases where EDs are found in, for instance, food and drinking water, we propose the use of an additional uncertainty factor of 10 (as default) for the nature of ED effects to ensure more protective risk assessments. Our recommendations aim to contribute to the ongoing discussion on how to effectively manage the risks associated with EDs.

Abstract Development of adverse outcome pathways (AOPs) can support the implementation of the EFSA–ECHA Guidance (2018) for identifying endocrine disruptors under EU Regulations 528/2012 and 1107/2009. However, their regulatory utility depends on them being fully developed and OECD‐endorsed, and in many cases their incorporation into more extensive AOP networks (AOPNs). To accelerate this process, EFSA has prioritized the creation of AOPNs spanning estrogenic, androgenic, steroidogenic, and thyroid (EATS) modalities, with an initial focus on mammalian reproductive toxicity driven by estrogenic, androgenic, and steroidogenic (EAS) disruption. In this context, this project developed a foundational AOPN capturing anti‐androgenic mechanisms that converge on key regulatory outcomes, including reduced anogenital distance, nipple retention, hypospadias, and impaired fertility. This AOPN currently includes 10 fully developed AOP. Additional developments under the project includes upstream networks for androgen, estrogen, and steroidogenesis modalties. The work establishes a modular architecture in which upstream molecular signaling pathways feed into shared developmental key events, thereby enabling integration of diverse mechanistic data and supporting transparent tracking of causal evidence. By mapping conserved pathways and identifying mechanistic nodes with high regulatory relevance, the framework also highlights critical knowledge gaps that hinder robust AOP expansion. The project incorporated standardized AOP‐Wiki methodologies to strengthen consistency in reporting and facilitate future network growth, while methodological advances such as refined weight‐of‐evidence procedures, systematic network‐mapping approaches, and strategies for building data‐rich key event relationships enhance reproducibility and evaluative confidence. Together, these developments can increase the practical value of AOPN for chemical evaluation and risk assessment, support alignment with emerging new approach methodologies (NAMs) for endocrine disruptor identification and provide a scalable foundation for future AOP and AOPN development.

This report describes a novel adverse outcome pathway (AOP) highlighting how the inhibition of aldehyde dehydrogenase 1A (ALDH1A) enzymatic activity can lead to female infertility in mammals through disrupted meiotic entry of fetal germ cells (AOP-Wiki 398). In mammals, all-trans retinoic acid (atRA) can induce germ cell meiosis; during fetal life in females, germ cells enter meiosis prophase I. Reduced levels or absence of atRA disrupts this process, impairing germ cell development and leading to a reduced ovarian reserve in postnatal ovaries. The synthesis of atRA from vitamin A precursors involves an intermediate catalytic conversion of retinal by ALDH1A. Evidence for this AOP, particularly the upstream events, is primarily derived from mouse studies (both genetic models and exposure studies, including explanted ovaries). Human evidence, especially for downstream events, corroborates that the ovarian reserve directly impacts fertility. In reproductive toxicity studies (both animal studies and human epidemiology), fertility is a critical endpoint for chemical safety assessments. Although infertility has multiple causes, this AOP specifically captures events of perturbed meiosis due to reduced atRA signaling during development, thus supporting the use of in silico and in vitro data on nuclear receptor activity of the retinoic acid and retinoid X receptors (RAR/RXR) and atRA synthesis/expression to predict potential in vivo effects.

There is a rapid increase in scientific studies on the environmental and health effects of microplastics and nanoplastics (MNPs), with potential to inform risk assessment. However, regulatory uptake of scientific evidence depends on the reliability and relevance, often referred to as the regulatory adequacy, of the studies. This regulatory adequacy may be challenged by, for example, insufficient characterization of test materials, and lack of transparency and reporting in experimental setup, methods, and analytics. While frameworks exist for evaluating the regulatory adequacy of (eco)toxicity data for chemicals and nanomaterials, they do not fully account for the unique characteristics and challenges associated with MNPs. Therefore, there is a need to provide further guidance specifically tailored to MNP studies. The aim of this paper was to review and synthesize the state of knowledge regarding key physicochemical parameters and experimental considerations critical to evaluating the regulatory adequacy, defined in terms of reliability and relevance, of MNP (eco)toxicity studies. Building on this comparative review, existing criteria were identified, adapted and aligned to support the development of structured tools embedded within the SciRAP and CRED frameworks. A literature review revealed the importance of, for example, polymer identity, size and shape distribution, surface chemistry, additive content, dispersion behaviour, background contamination control, description of sample preparation, and characterization of the test item. Based on these findings, relevant criteria were identified from existing frameworks and literature and subsequently adapted or extended and built into the SciRAP and CRED frameworks, to address the specific challenges of MNP research, supporting structured evaluation of study reliability and relevance. By promoting better reporting practices and attention to MNPs specific test considerations, the proposed frameworks, SciRAPplastic (in vivo and in vitro) and plasticCRED (ecotoxicity), can strengthen the scientific foundation for environmental and health risk assessments of MNPs. Furthermore, they may support the regulatory uptake of data generated in emerging non-standard MNP (eco)toxicity studies.

The Adverse Outcome Pathway (AOP) framework offers a structured approach to organize mechanistic knowledge of toxicological pathways. By describing biological events linking molecular initiating events (MIEs) to an adverse outcome (AO) at the organismal level, it aims to aid regulatory decision-making through predictive toxicology approaches. To serve this purpose, however, it is recognized that AOP networks are required to adequately capture complex biology. Another central feature of the AOP concept is that upstream molecular networks will be shared between numerous downstream AOs. This report focuses on steroidogenesis, a common target of endocrine disrupting chemicals, and the development of an upstream network for reduced steroidogenesis focusing on hormones and enzymes that are particularly relevant to mammalian reproduction. The AOP-Wiki was mapped for existing content related to steroidogenesis and the resulting network expanded by incorporating additional key events (KEs) and KE Relationships (KERs) not yet inventoried. All existing KEs and KERs were evaluated for completeness. Using a pragmatic approach, we developed the identified KEs and KERs by integrating evidence from recent review articles. The focus was particularly on the impact of disrupted cholesterol transport, altered enzyme activities and hormone levels. The resulting upstream AOP network serves as a foundation for developing complete AOPs linking disrupted steroidogenesis with downstream AOs. This upstream network will also contribute to identifying relevant test assays for development and understanding the predictive capabilities of existing in vitro assays, such as the OECD-validated H295R steroidogenesis assay.

Exposure to endocrine disruptors (EDs) are associated with significant risks to human health and the environment. The European Union (EU) thus prioritizes their identification and regulation and is developing a roadmap to phase out animal testing in chemical safety assessments while advancing New Approach Methodologies (NAMs). This review outlines EU's practices for ED identification, focusing on the use of NAMs, as well as Defined Approaches and read-across. We assessed the current EU framework under the Classification, Labelling and Packaging (CLP) Regulation, the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), the Plant Protection Products Regulation (PPPR), and the Biocidal Products Regulation (BPR), evaluating current use of NAMs and reflection on potential future use. We find that EU legislation and guidance documents allow the use of NAMs in ED identification, including for assessment of endocrine activity and adversity. However, guidance on predicting adversity using NAMs remains limited, and ED identifications have largely depended on animal data to assess endocrine-mediated adversity. Continued in vivo testing until reliable methodologies are accepted as alternatives and routinely applied is required. The report concludes with short- and long-term recommendations for updates to the information requirements across regulations and further development of methods to predict endocrine-mediated adversity.

Omics-technologies such as transcriptomics offer valuable insights into toxicity mechanisms. However, integrating this type of data into regulatory frameworks remains challenging due to uncertainties regarding toxicological relevance and links to adverse outcomes. Furthermore, current assessments of endocrine disruptors (EDs) relevant to human health require substantial amounts of data, and primarily rely on standardized animal studies. Identifying EDs is a high priority in the EU, but so are efforts to replace and reduce animal testing. Alternative methods to investigate EDs are needed, and so are health risk assessment methods that support uptake of novel mechanistic information. This study aims to utilize Adverse Outcome Pathways (AOPs) to integrate transcriptomics data for identifying EDs, by establishing a link between molecular data and adverse outcomes. Cadmium (Cd) and 3,3',4,4',5-pentachlorobiphenyl (PCB126) were used as model compounds due to their observed effects on the endocrine system. An AOP network for the estrogen, androgen, thyroid, and steroidogenesis (EATS)-modalities was constructed. RNA sequencing (RNA-Seq) was conducted on zebrafish (Danio rerio) embryos exposed to Cd or PCB126 for 4 days. RNA-Seq data were then linked to the AOP network via Gene Ontology (GO) terms. Enrichment Maps in Cytoscape and the QIAGEN Ingenuity Pathway Analysis (IPA) software were also used to identify potential ED properties and to support the assessment. Potentially EATS-related GO Biological Process (BP) terms were identified for both compounds. A lack of accurate standardized terms in KEs of the AOP network hindered a data-driven mapping approach. Instead, manual mapping of GO BP terms onto the AOP network revealed more connections, underscoring the need for harmonizing AOP development for regulatory use. Both the Enrichment Maps and the IPA results further supported potentially EATS-related effects of both compounds. While AOP networks show promise in integrating RNA-Seq data, several challenges remain.

The aim of the Thyroid Validation Study, coordinated by EURL ECVAM and involving EU-NETVAL laboratories, was to validate selected non-animal methods for the identification of chemicals that can potentially disrupt the thyroid hormone system in humans. The validation study was organized in two parts: Part 1 was to assess method performance and develop standard operating procedures, where needed, and Part 2 was to assess the mechanistic relevance of the methods using a set of vali­dation chemicals. This paper describes the stepwise process to select this validation set of chemicals, mainly based on extensive literature review and expert judgment elicitation to identify chemicals for which there was evidence to show their (lack of) ability to perturb the thyroid hormone signaling mechanisms or modes of action covered by the methods. A unique contribution of the study lies in its mechanistic coverage of molecular targets within the thyroid gland but also regulatory mechanisms in peripheral tissues, reflecting a multifaceted perspective on thyroid hormone action. The validation set consisted of 30 chemicals, providing a balanced representation across a broad chemical space and offering insights into the mechanistic relevance of the selected methods. Once validated, these methods will contribute to advancing the identification and evaluation of endocrine disruptors, informing regulatory decisions, and promoting alternative testing strategies.

Context In vitro toxicity studies are increasingly being included as evidence in systematic reviews and chemical risk assessments. INVITES-IN, a tool for assessing the internal validity of in vitro studies, is under development in a process consisting of four consecutive studies. Study One in the creation of INVITES-IN was the development of an “item bank” database of 405 concepts (“items”) of potential relevance for assessing the internal validity of in vitro toxicity studies. The items were gathered from both focus group discussions and a purposive literature sample. In this paper we present the second study in the creation of INVITES-IN, i.e. the methods and results for identifying items for consideration when assessing the potential for bias in an in vitro study.Method A two-round digital Delphi survey, followed by online Delphi panel discussions guided by a moderator, was performed. The Delphi participants were experienced with both in vitro models and systematic review methods.Results Fifteen experts completed both Delphi rounds, and thirteen participated in a guided Delphi panel discussion. Of the 405 items in the bank, the experts agreed that 372 should be considered when assessing the potential for bias in an in vitro study. Items gathered from both the literature sample and the focus group discussions (Study One) were considered to be important for the assessment of the potential for bias in an in vitro study; 83–100% of the items collected from the literature sample were identified to be important and 91% (127) of the new items discovered in the focus group discussions of Study One were identified to be important.Discussion The 372 retained items will be interpreted into a manageable set of study appraisal criteria and a supporting guidance that will constitute the INVITES-IN study appraisal tool. In terms of lessons for tool development, the high retention of items included in tools designed for assessment of human and animal studies to in vitro studies suggests that many validity concepts are generally applicable across multiple study designs. Therefore, tool development processes should benefit from drawing on assessment tools outside the immediate domain of interest. Tool development would also likely benefit from supplementing literature reviews with focus group discussions, as our results demonstrate that the use of focus group discussions with domain experts was a pragmatic and valuable approach to increasing coverage of items in a tool development process.Conclusion In conclusion, this study demonstrates the value of using rigorous methods to ensure a comprehensive dataset as the starting point for creation of an assessment tool, though the direct application of Delphi methods to item banks may be an unnecessary step in tool development.

The EU recently introduced four new hazard classes to the Classification, Labelling and Packaging Regulation (CLP) (EC) 1272/2008. The classes are endocrine disruption for human health (ED HH) and the environment (ED ENV), persistent, bioaccumulative and toxic (PBT) or very persistent and very bioaccumulative (vPvB), and persistent, mobile and toxic (PMT) or very persistent and very mobile (vPvM). This action was a direct consequence of the EU’s Chemicals Strategy for Sustainability, which aims at strengthening the protection of human health and the environment, as well as reinforcing the CLP Regulation as the central piece of the chemicals legislation. This study examined the regulatory obligations triggered by these new hazard classes, as well as the existing obligations for endocrine disrupters and PBT/vPvB substances identified in other EU regulations. In addition, we compared the CLP criteria for endocrine disruption and PBT/vPvB to criteria existing in other EU regulations and investigated how these criteria are used in the EU chemicals legislation. We found that the implementation of the new hazard classes under the CLP into existing EU chemicals legislation will require the revision of all regulations that rely on the CLP hazard criteria for risk management. Without revision, the immediate impact of the new hazard classes will only extend to six regulations and the regulatory obligations they contain, all of which apply to substances classified under any of the CLP hazard classes. Meanwhile, substances with endocrine disrupting and PBT/vPvB properties are already being identified and regulated using criteria from regulations other than the CLP. When comparing the criteria for identification of endocrine disrupters and PBT/vPvB substances across the chemicals legislation, we found that the criteria differed between regulations. The findings aim to support the efficient implementation of the new CLP hazard classes and harmonization of criteria across regulations, in line with the Chemicals Strategy for Sustainability.

This work presents a case study in applying a systematic review framework (SYRINA) to the identification of chemicals as endocrine disruptors. The suitability and performance of the framework is tested with regard to the widely accepted World Health Organization definition of an endocrine disruptor (ED). The endocrine disrupting potential of triphenyl phosphate (TPP), a well-studied flame retardant reported to exhibit various endocrine related effects was assessed. We followed the 7 steps of the SYRINA framework, articulating the research objective via Populations, Exposures, Comparators, Outcomes (PECO) statements, performed literature search and screening, conducted study evaluation, performed data extraction and summarized and integrated the evidence. Overall, 66 studies, consisting of in vivo, in vitro and epidemiological data, were included. We concluded that triphenyl phosphate could be identified as an ED based on metabolic disruption and reproductive function. We found that the tools used in this case study and the optimizations performed on the framework were suitable to assess properties of EDs. A number of challenges and areas for methodological development in systematic appraisal of evidence relating to endocrine disrupting potential were identified; significant time and effort were needed for the analysis of in vitro mechanistic data in this case study, thus increasing the workload and time needed to perform the systematic review process. Further research and development of this framework with regards to grey literature (non-peer-reviewed literature) search, harmonization of study evaluation methods, more consistent evidence integration approaches and a pre-defined method to assess links between adverse effect and endocrine activity are recommended. It would also be advantageous to conduct more case studies for a chemical with less data than TPP.

The CLP mandates manufacturers and importers to classify substances and mixtures according to hazard criteria, with notifications submitted to the European Chemicals Agency (ECHA). Substances meeting hazard criteria must be appropriately labelled and packaged to communicate hazards effectively. The CLP establishes hazard classification criteria but does not independently prohibit or restrict the use of hazardous chemicals. Instead, it serves as a basis for regulatory obligations in other specific regulations. This study investigates the regulatory implications of meeting hazard criteria under the CLP across EU regulations and directives listed in EU Chemicals Legislation Finder (EUCLEF). The results show that fulfilling criteria for human health hazard classes trigger regulatory obligations in the highest number of regulations/directives, with carcinogenicity, mutagenicity, and reproductive toxicity (CMR) leading to obligations in 19 of 20 pieces of legislation linked to the CLP. Conversely, physical, environmental, and ozone layer hazards are associated with fewer regulations and directives, and lead to fewer prohibitions. The study underscores the pivotal role of the CLP in EU chemical legislation and the need for coherence and consistency across regulations. While regulatory obligations are primarily aimed at substances meeting hazard criteria, the variability in self-classification notifications and limitations in harmonized classification processes were observed. Moreover, the complexity of the regulatory structure poses challenges for stakeholders and policymakers, including inconsistencies, compliance difficulties, and the need for frequent revisions. Addressing these challenges is critical for enhancing regulatory effectiveness and ensuring a more coherent and harmonized approach to chemical management in the EU.

The prevalence of hormone-related health issues caused by exposure to endocrine disrupting chemicals (EDCs) is a significant, and increasing, societal challenge. Declining fertility rates together with rising incidence rates of reproductive disorders and other endocrine-related diseases underscores the urgency in taking more action. Addressing the growing threat of EDCs in our environment demands robust and reliable test methods to assess a broad variety of endpoints relevant for endocrine disruption. EDCs also require effective regulatory frameworks, especially as the current move towards greater reliance on non-animal methods in chemical testing puts to test the current paradigm for EDC identification, which requires that an adverse effect is observed in an intact organism. Although great advances have been made in the field of predictive toxicology, disruption to the endocrine system and subsequent adverse health effects may prove particularly difficult to predict without traditional animal models. The MERLON project seeks to expedite progress by integrating multispecies molecular research, new approach methodologies (NAMs), human clinical epidemiology, and systems biology to furnish mechanistic insights and explore ways forward for NAM-based identification of EDCs. The focus is on sexual development and function, from foetal sex differentiation of the reproductive system through mini-puberty and puberty to sexual maturity. The project aims are geared towards closing existing knowledge gaps in understanding the effects of EDCs on human health to ultimately support effective regulation of EDCs in the European Union and beyond.

The Adverse Outcome Pathway (AOP) framework has gained widespread acceptance in toxicological disciplines as a tool for aiding chemical hazard assessment. Despite increased activity in AOP development, progress towards a high volume of fully endorsed AOPs has been slow, partly due to the challenging task of constructing complete AOPs according to the AOP Developer’s Handbook. To facilitate greater uptake of new knowledge units onto the open-source AOP-wiki platform, a pragmatic approach was recently proposed. This approach involves considering Key Event Relationships (KERs) for individual development through systematic approaches, as they represent essential units of knowledge from which causality can be inferred; from low complexity test data to adverse outcomes in intact organisms. However, more broadly adopted harmonized methodologies for KER development would be desirable. Using the AOP Developer’s Handbook as a guide, a KER linking 'decreased androgen receptor (AR) activity' with 'reduced anogenital distance (AGD)' was developed to demonstrate a methodology applicable for future developments of KERs requiring systematic literature retrieval approaches.

Endocrine disruptors (EDs) pose a serious threat to human health and the environment and require a comprehensive evaluation to be identified. The identification of EDs require a substantial amount of data, both in vitro and in vivo, due to the current scientific criteria in the EU. At the same time, the EU strives to reduce animal testing due to concerns regarding animal welfare and sensitivity of animal studies to adequately detect adverse effects relevant for human health. Perfluorooctane sulfonic acid (PFOS) is a persistent organic pollutant that is suspected to be an ED based on academic research, however it is not identified as such from a regulatory perspective. It has previously been shown that PFOS has the potential to cause neurotoxicity as well as affect the thyroid system, and it is known that specific thyroid hormone levels are critical in the development of the brain during. In this work, the aim was to evaluate a mechanism-based approach to identify ED properties of PFOS based on the Adverse Outcome Pathway (AOP) framework and using New Approach Methods (NAMs), by comparing this approach to an ED assessment based on the currently available guidance document. An AOP network (AOPN) was generated for the thyroid modality, and AOPs leading to developmental neurotoxicity (DNT) were identified. A literature search and screening process based on the AOPN, and systematic review methodology, was performed, followed by a rigorous Weight-of-Evidence (WoE) assessment. Evidence was mapped back onto the AOPN used for the literature search, to identify possible endocrine Modes-of-Action (MoAs) for PFOS and data gaps in the two assessments. It could be concluded that PFOS fulfils the criteria for ED classification in the standard ED assessment, but not in the mechanism-based assessment. The need for quantitative information, such as quantitative AOPs, for the mechanism-based approach is discussed. The possibility of a directly neurotoxic alternative MoA was also highlighted based on available in vitro data. Opportunities and challenges with implementing AOPs and NAMs into the regulatory assessment of EDs, and assessing hazard in the Next Generation Risk Assessment, is discussed. This case study exploring the mechanism-based approach to ED identification represents an important step toward more accurate and predictive assessment of EDs based on AOPs and NAMs, and to the Next Generation Risk Assessment (NGRA) concept.

The EU Cosmetic Products Regulation requires neither environmental data nor environmental risk assessment for individual ingredients or finished cosmetic products. Instead, it relies on REACH to address environmental risks linked to cosmetic ingredients, including preservatives. We investigated how the environmental risks of cosmetic preservatives are managed by REACH. We identified preservatives of environmental concern and examined if any of these had been selected for Substance Evaluation, proposed for or identified as an SVHC, required authorization or were proposed for, or subject to, restriction under REACH. More than half of the preservatives approved under the Cosmetic Product Regulation, 70 of 137, were identified as being of environmental concern according to the criteria set in this study. Some of the approved preservatives were no longer produced or used in the EU due to their hazardous properties. However, they remained approved and may still enter the EU via the imported products. Our results also indicate that the environmental aspects of cosmetic ingredients, including preservatives, are not efficiently managed by REACH. Besides the known issues in REACH, we identified additional areas in the interface between REACH, CLP and the Cosmetic Products Regulation that call for improvement. Here, we provide practical suggestions in line with the Chemicals Strategy for Sustainability. If implemented, these measures would strengthen the protection of the environment from hazardous cosmetic ingredients.