Application of AOPs to assist regulatory assessment of chemical risks - Case studies, needs and recommendations.

While human regulatory risk assessment (RA) still largely relies on animal studies, new approach methodologies (NAMs) based on in vitro, in silico or non-mammalian alternative models are increasingly used to evaluate chemical hazards. Moreover, human epidemiological studies with biomarkers of effect (BoE) also play an invaluable role in identifying health effects associated with chemical exposures. To move towards the next generation risk assessment (NGRA), it is therefore crucial to establish bridges between NAMs and standard approaches, and to establish processes for increasing mechanistically-based biological plausibility in human studies. The Adverse Outcome Pathway (AOP) framework constitutes an important tool to address these needs but, despite a significant increase in knowledge and awareness, the use of AOPs in chemical RA remains limited. The objective of this paper is to address issues related to using AOPs in a regulatory context from various perspectives as it was discussed in a workshop organized within the European Union partnerships HBM4EU and PARC in spring 2022. The paper presents examples where the AOP framework has been proven useful for the human RA process, particularly in hazard prioritization and characterization, in integrated approaches to testing and assessment (IATA), and in the identification and validation of BoE in epidemiological studies. Nevertheless, several limitations were identified that hinder the optimal usability and acceptance of AOPs by the regulatory community including the lack of quantitative information on response-response relationships and of efficient ways to map chemical data (exposure and toxicity) onto AOPs. The paper summarizes suggestions, ongoing initiatives and third-party tools that may help to overcome these obstacles and thus assure better implementation of AOPs in the NGRA.

Assessment of the endocrine disrupting properties of bisphenol AF: a case study applying the European regulatory criteria and guidance.

BACKGROUND: Scientific criteria to identify endocrine disruptors (ED) was recently implemented for plant protection products (PPP) and biocidal products (BP). A guidance document has been published by ECHA and EFSA in the context of ED criteria for PPPs and BPs. METHODS: In the present work, a case study was performed on Bisphenol AF (BPAF) to explore the application of the EU criteria and EFSA/ECHA guidance document for the ED assessment of a non-pesticide chemical regulated under REACH. A data dossier was built by a systematic literature search (Web of Science, Pubmed, Embase; n = 511), title/abstract screening (n = 124) and full text examination (n = 88). All the information was extracted and systematically reported for 309 parameters (100 for adversity; 209 for endocrine activity). The reliability of studies was assessed (SciRAP tool). RESULTS: Data were synthesized into 96 lines of evidence for adversity (n = 57), and endocrine activity (n = 39); and assessed by weight of evidence methodology. The initial analysis of the evidence indicated EATS-mediated adversity in mammals, therefore a mode of action (MoA) was postulated for both male and female adult exposure. Female MoA included estrogen receptor activation and altered steroidogenesis leading to ovarian dysfunction, altered estrous cycling and impaired female fertility. Male MoA was initiated by androgen receptor inhibition and altered steroidogenesis leading to dysfunction of male reproductive organs and impaired male fertility. CONCLUSIONS: The overall conclusion of the ED assessment indicated that BPAF meets the ED criteria for human health. The steps described in the ED guidance document were successfully completed, resulting in a thorough, structured and transparent identification of BPAF as an ED. Advantages and limitations of applying the ED criteria and guidance for a REACH chemical are discussed.

Testing and refining the Science in Risk Assessment and Policy (SciRAP) web-based platform for evaluating the reliability and relevance of in vivo toxicity studies.

The Science in Risk Assessment and Policy (SciRAP) web-based platform was developed to promote and facilitate structure and transparency in the evaluation of ecotoxicity and toxicity studies for hazard and risk assessment of chemicals. The platform includes sets of criteria and a colour-coding tool for evaluating the reliability and relevance of individual studies. The SciRAP method for evaluating in vivo toxicity studies was first published in 2014 and the aim of the work presented here was to evaluate and develop that method further. Toxicologists and risk assessors from different sectors and geographical areas were invited to test the SciRAP criteria and tool on a specific set of in vivo toxicity studies and to provide feedback concerning the scientific soundness and user-friendliness of the SciRAP approach. The results of this expert assessment were used to refine and improve both the evaluation criteria and the colour-coding tool. It is expected that the SciRAP web-based platform will continue to be developed and enhanced to keep up to date with the needs of end-users.

A call for action: Improve reporting of research studies to increase the scientific basis for regulatory decision-making.

This is a call for action to scientific journals to introduce reporting requirements for toxicity and ecotoxicity studies. Such reporting requirements will support the use of peer-reviewed research studies in regulatory decision-making. Moreover, this could improve the reliability and reproducibility of published studies in general and make better use of the resources spent in research.

Combining web-based tools for transparent evaluation of data for risk assessment: developmental effects of bisphenol A on the mammary gland as a case study.

Different tools have been developed that facilitate systematic and transparent evaluation and handling of toxicity data in the risk assessment process. The present paper sets out to explore the combined use of two web-based tools for study evaluation and identification of reliable data relevant to health risk assessment. For this purpose, a case study was performed using in vivo toxicity studies investigating low-dose effects of bisphenol A on mammary gland development. The reliability of the mammary gland studies was evaluated using the Science in Risk Assessment and Policy (SciRAP) criteria for toxicity studies. The Health Assessment Workspace Collaborative (HAWC) was used for characterizing and visualizing the mammary gland data in terms of type of effects investigated and reported, and the distribution of these effects within the dose interval. It was then investigated whether there was any relationship between study reliability and the type of effects reported and/or their distribution in the dose interval. The combination of the SciRAP and HAWC tools allowed for transparent evaluation and visualization of the studies investigating developmental effects of BPA on the mammary gland. The use of these tools showed that there were no apparent differences in the type of effects and their distribution in the dose interval between the five studies assessed as most reliable and the whole data set. Combining the SciRAP and HAWC tools was found to be a useful approach for evaluating in vivo toxicity studies and identifying reliable and sensitive information relevant to regulatory risk assessment of chemicals. Copyright © 2016 John Wiley & Sons, Ltd.

A proposed framework for the systematic review and integrated assessment (SYRINA) of endocrine disrupting chemicals.

BACKGROUND: The issue of endocrine disrupting chemicals (EDCs) is receiving wide attention from both the scientific and regulatory communities. Recent analyses of the EDC literature have been criticized for failing to use transparent and objective approaches to draw conclusions about the strength of evidence linking EDC exposures to adverse health or environmental outcomes. Systematic review methodologies are ideal for addressing this issue as they provide transparent and consistent approaches to study selection and evaluation. Objective methods are needed for integrating the multiple streams of evidence (epidemiology, wildlife, laboratory animal, in vitro, and in silico data) that are relevant in assessing EDCs. METHODS: We have developed a framework for the systematic review and integrated assessment (SYRINA) of EDC studies. The framework was designed for use with the International Program on Chemical Safety (IPCS) and World Health Organization (WHO) definition of an EDC, which requires appraisal of evidence regarding 1) association between exposure and an adverse effect, 2) association between exposure and endocrine disrupting activity, and 3) a plausible link between the adverse effect and the endocrine disrupting activity. RESULTS: Building from existing methodologies for evaluating and synthesizing evidence, the SYRINA framework includes seven steps: 1) Formulate the problem; 2) Develop the review protocol; 3) Identify relevant evidence; 4) Evaluate evidence from individual studies; 5) Summarize and evaluate each stream of evidence; 6) Integrate evidence across all streams; 7) Draw conclusions, make recommendations, and evaluate uncertainties. The proposed method is tailored to the IPCS/WHO definition of an EDC but offers flexibility for use in the context of other definitions of EDCs. CONCLUSIONS: When using the SYRINA framework, the overall objective is to provide the evidence base needed to support decision making, including any action to avoid/minimise potential adverse effects of exposures. This framework allows for the evaluation and synthesis of evidence from multiple evidence streams. Finally, a decision regarding regulatory action is not only dependent on the strength of evidence, but also the consequences of action/inaction, e.g. limited or weak evidence may be sufficient to justify action if consequences are serious or irreversible.

Using systematic reviews for hazard and risk assessment of endocrine disrupting chemicals.

The possibility that endocrine disrupting chemicals (EDCs) in our environment contribute to hormonally related effects and diseases observed in human and wildlife populations has caused concern among decision makers and researchers alike. EDCs challenge principles traditionally applied in chemical risk assessment and the identification and assessment of these compounds has been a much debated topic during the last decade. State of the science reports and risk assessments of potential EDCs have been criticized for not using systematic and transparent approaches in the evaluation of evidence. In the fields of medicine and health care, systematic review methodologies have been developed and used to enable objectivity and transparency in the evaluation of scientific evidence for decision making. Lately, such approaches have also been promoted for use in the environmental health sciences and risk assessment of chemicals. Systematic review approaches could provide a tool for improving the evaluation of evidence for decision making regarding EDCs, e.g. by enabling systematic and transparent use of academic research data in this process. In this review we discuss the advantages and challenges of applying systematic review methodology in the identification and assessment of EDCs.

Facilitating the use of non-standard in vivo studies in health risk assessment of chemicals: a proposal to improve evaluation criteria and reporting.

To improve data availability in health risk assessment of chemicals and fill information gaps there is a need to facilitate the use of non-standard toxicity studies, i.e. studies not conducted according to any standardized toxicity test guidelines. The purpose of this work was to propose criteria and guidance for the evaluation of reliability and relevance of non-standard in vivo studies, which could be used to facilitate systematic and transparent evaluation of such studies for health risk assessment. Another aim was to propose user friendly guidance for reporting of non-standard studies intended to promote an improvement in reporting of studies that could be of use in risk assessment. Requirements and recommendations for the design and execution of in vivo toxicity studies were identified from The Organisation for Economic Co-operation and Development (OECD) test guidelines, and served as basis for the data evaluation criteria and reporting guidelines. Feedback was also collected from experts within the field of toxicity testing and risk assessment and used to construct a two-tiered framework for study evaluation, as well as refine the reporting guidelines. The proposed framework emphasizes the importance of study relevance and an important aspect is to not completely dismiss studies from health risk assessment based on very strict criteria for reliability. The suggested reporting guidelines provide researchers with a tool to fulfill reporting requirements as stated by regulatory agencies. Together, these resources provide an approach to include all relevant data that may fill information gaps and reduce scientific uncertainty in health risk assessment conclusions, and subsequently also in chemical policy decisions.

Exploring a mechanism-based approach for the identification of endocrine disruptors using Adverse Outcome Pathways (AOPs) and New Approach Methodologies (NAMs) : A perfluorooctane sulfonic acid case study.

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.

Methodology for developing data-rich Key Event Relationships for Adverse Outcome Pathways exemplified by linking decreased androgen receptor activity with decreased anogenital distance.

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.

Building an adverse outcome pathway network for estrogen-, androgen- and steroidogenesis-mediated reproductive toxicity.

Introduction: Adverse Outcome Pathways (AOPs) can support both testing and assessment of endocrine disruptors (EDs). There is, however, a need for further development of the AOP framework to improve its applicability in a regulatory context. Here we have inventoried the AOP-wiki to identify all existing AOPs related to mammalian reproductive toxicity arising from disruption to the estrogen, androgen, and steroidogenesis modalities. Core key events (KEs) shared between relevant AOPs were also identified to aid in further AOP network (AOPN) development. Methods: A systematic approach using two different methods was applied to screen and search the entire AOP-wiki library. An AOPN was visualized using Cytoscape. Manual refinement was performed to remove AOPS devoid of any KEs and/or KERs. Results: Fifty-eight AOPs relevant for mammalian reproductive toxicity were originally identified, with 42 AOPs included in the final AOPN. Several of the KEs and KE relationships (KERs) described similar events and were thus merged to optimize AOPN construction. Sixteen sub-networks related to effects on hormone levels or hormone activity, cancer outcomes, male and female reproductive systems, and overall effects on fertility and reproduction were identified within the AOPN. Twenty-six KEs and 11 KERs were identified as core blocks of knowledge in the AOPN, of which 19 core KEs are already included as parameters in current OECD and US EPA test guidelines. Discussion: The AOPN highlights knowledge gaps that can be targeted for further development of a more complete AOPN that can support the identification and assessment of EDs.

Beyond chemicals: Opportunities and challenges of integrating non-chemical stressors in adverse outcome pathways.

The adverse outcome pathways (AOPs) were developed to accelerate evidence-based chemical risk assessment by leveraging data from new approach methodologies. Thanks to their stressor-agnostic approach, AOPs were seen as instrumental in other fields. Here, we present AOPs that report non-chemical stressors along with the challenges encountered for their development. Challenges regarding AOPs linked to nanomaterials include non-specific molecular initiating events, limited understanding of nanomaterial biodistribution, and needs for adaptations of the in silico modeling and testing systems. Development of AOPs for radiation face challenges in how to incorporate ionizing events type, dose rate, energy deposition, and how to account for targeting multiple macromolecules. AOPs for COVID-19 required the inclusion of SARS-CoV-2-specific replicative steps to capture the essential events driving the disease. Developing AOPs to evaluate efficacy and toxicity of cell therapies necessitates addressing the cellular nature and the therapeutic function of the stressor. Finally, addressing toxicity of emerging biological stressors like microbial pesticides can learn from COVID-19 AOPs. We further discuss that the adaptations needed to expand AOP applicability beyond chemicals are mainly at the molecular and cellular levels while downstream key events at tissue or organ level, such as inflammation, are shared by many AOPs initiated by various stressors. In conclusion, although it is challenging to integrate non-chemical stressors within AOPs, this expands opportunities to account for real-world scenarios, to identify vulnerable individuals, and to bridge knowledge on mechanisms of adversity.

The adverse outcome pathway (AOP) framework was developed to help predict whether chemicals have toxic effects on humans. Structuring available information in an accessible database can reduce animal testing. AOPs usually capture the path from the interaction of a stressor, usually a chemical, with the human body to an adverse outcome, e.g., a disease symptom. The concept of AOPs has now been expanded to include non-chemical stressors such as nanomaterials, radiation, viruses, cells used to treat patients, and microorganisms employed as pesticides. We use discuss how these stressors need to be accommodated within the framework and point out that pathways initiated by these stressors share downstream events like inflammation with chemical stressors. By integrating non-chemical stressors into the framework, real-world scenarios where people may be exposed to different stressor types can be considered, vulnerable individuals can be identified, and knowledge on toxic effects can be compounded.

Applying a modified systematic review and integrated assessment framework (SYRINA) - a case study on triphenyl phosphate.

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.

Enhanced identification of endocrine disruptors through integration of science-based regulatory practices and innovative methodologies: The MERLON Project.

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.

SciRAPnano: a pragmatic and harmonized approach for quality evaluation of in vitro toxicity data to support risk assessment of nanomaterials.

Large amounts of nanotoxicity data from alternative non-animal (in vitro) test methods have been generated, but there is a lack of harmonized quality evaluation approaches for these types of data. Tools for scientifically sound and structured evaluation of the reliability and relevance of in vitro toxicity data to effectively inform regulatory hazard assessment of nanomaterials (NMs), are needed. Here, we present the development of a pragmatic approach to facilitate such evaluation. The tool was developed based on the Science in Risk Assessment and Policy (SciRAP) tool currently applicable to quality evaluation of chemical toxicity studies. The approach taken to develop the tool, referred to as SciRAPnano, included refinement of the original SciRAP in vitro tool through implementation of identified NM-relevant criteria, and further refined based on a set of case studies involving evaluation of 11 studies investigating in vitro toxicity of nano-sized titanium dioxide. Parameters considered cover key physicochemical properties as well as assay-specific aspects that impact NM toxicity, including NM interference with test methods and NM transformation. The final SciRAPnano tool contains 38 criteria for reporting quality, 19 criteria for methodological quality, and 4 guidance items to evaluate relevance. The approach covers essential parameters for pragmatic and harmonized evaluation of NM in vitro toxicity studies and allows for structured use of in vitro data in regulatory hazard assessment of NMs, including transparency on data quality.

Development of a data-driven approach to Adverse Outcome Pathway network generation: a case study on the EATS-modalities.

Adverse Outcome Pathways (AOPs) summarize mechanistic understanding of toxicological effects and have, for example, been highlighted as a promising tool to integrate data from novel in vitro and in silico methods into chemical risk assessments. Networks based on AOPs are considered the functional implementation of AOPs, as they are more representative of complex biology. At the same time, there are currently no harmonized approaches to generate AOP networks (AOPNs). Systematic strategies to identify relevant AOPs, and methods to extract and visualize data from the AOP-Wiki, are needed. The aim of this work was to develop a structured search strategy to identify relevant AOPs in the AOP-Wiki, and an automated data-driven workflow to generate AOPNs. The approach was applied on a case study to generate an AOPN focused on the Estrogen, Androgen, Thyroid, and Steroidogenesis (EATS) modalities. A search strategy was developed a priori with search terms based on effect parameters in the ECHA/EFSA Guidance Document on Identification of Endocrine Disruptors. Furthermore, manual curation of the data was performed by screening the contents of each pathway in the AOP-Wiki, excluding irrelevant AOPs. Data were downloaded from the Wiki, and a computational workflow was utilized to automatically process, filter, and format the data for visualization. This study presents an approach to structured searches of AOPs in the AOP-Wiki coupled to an automated data-driven workflow for generating AOPNs. In addition, the case study presented here provides a map of the contents of the AOP-Wiki related to the EATS-modalities, and a basis for further research, for example, on integrating mechanistic data from novel methods and exploring mechanism-based approaches to identify endocrine disruptors (EDs). The computational approach is freely available as an R-script, and currently allows for the (re)-generation and filtering of new AOP networks based on data from the AOP-Wiki and a list of relevant AOPs used for filtering.

Protocol for designing INVITES-IN, a tool for assessing the internal validity of in vitro studies.

This protocol describes the design and development of a tool for evaluation of the internal validity of in vitro studies, which is needed to include the data as evidence in systematic reviews and chemical risk assessments. The tool will be designed specifically to be applied to cell culture studies, including, but not restricted to, studies meeting the new approach methodology (NAM) definition. The tool is called INVITES-IN (IN VITro Experimental Studies INternal validity). In this protocol, three of the four studies that will be performed to create the release version of INVITES-IN are described. In the first study, evaluation of existing assessment tools will be combined with focus group discussions to identify how characteristics of the design or conduct of an in vitro study can affect its internal validity. Bias domains and items considered to be of relevance for in vitro studies will be identified. In the second study, group agreement on internal validity domains and items of importance for in vitro studies will be identified via a modified Delphi methodology. In the third study, the draft version of the tool will be created, based on the data on relevance and importance of bias domains and items collected in Studies 1 and 2. A separate protocol will be prepared for the fourth study, which includes the user testing and validation of the tool, and collection of users' experience.

Development of an adverse outcome pathway for intrahepatic cholestasis of pregnancy.

Adverse Outcome Pathways (AOPs) are a research synthesis tool, used primarily by toxicologists for numerous applications including: hypothesis generation, data integration, biomarker determination, and identification of gaps in current knowledge. The AOP model provides a means for evaluating critical interactions between stressors and biological systems which result in adversity, meaning there is significant potential value in using this model in clinical research. However, AOPs have so far not been applied in this context, which may be attributable to the fact that the method is not yet streamlined with established practices in evidence-based medicine, such as systematic review. Here, we present one approach to developing a clinically focused AOP for intrahepatic cholestasis of pregnancy; aiming to enhance understanding of the mechanistic link between this common, gestational liver disease and its association with preterm birth. Mechanistic aspects of the disease pathogenesis, and use of AOPs to broaden inclusion and improve integration of in vitro and in vivo data in clinical research are discussed. We also demonstrate for the first time how central components of systematic review can be integrated into the development of an AOP.

Systematic evaluation of the evidence for identification of endocrine disrupting properties of Bisphenol F.

Identification of endocrine disruptors (EDs) is a highly prioritized issue in the EU. However, scientific criteria to identify EDs have so far only been implemented for biocidal and plant protection products. The European Commission is working on developing a horizontal approach to the identification of EDs across legislations, based on these scientific criteria. With this study, our aim was to evaluate evidence on endocrine disrupting properties of Bisphenol F (BPF) by applying the process set out for biocidal and plant protection products in Europe. BPF is not registered under REACH and therefore assumed not to be produced in the EU > 1 ton/year, yet the substance has been detected in urine, serum, and breast milk in populations from different countries in Europe. BPF is raising concern since it is an analogue of the known ED and reproductive toxicant Bisphenol A. Relevant evidence on endocrine disrupting properties of BPF from the open literature was collected using systematic review methodology. Pre-defined inclusion criteria were developed to select relevant studies, and data was extracted. The reliability of included studies was evaluated by the Science in Risk Assessment and Policy tool, and results were converted into Klimisch categories to allow for categorization of study reliability. A weight-of-evidence approach was used to analyze the evidence and draw conclusions on endocrine-related activity and/or endocrine adversity. We found that there is sufficient evidence to conclude on an endocrine mechanism, and while evidence for adversity was not considered sufficient, we still conclude that BPF could also cause endocrine-mediated adversity. Two modes of action were postulated based on the collected data for BPF. Challenges of performing the ED assessment for data-poor chemicals and the importance of adequate reporting of studies in the open literature, especially for new approach methods, are discussed.

Assessment of endocrine disruptive properties of PFOS: EFSA/ECHA guidance case study utilising AOP networks and alternative methods.

Endocrine disruptors (EDs) are chemical substances that interfere with the endocrine system, adversely affecting human health and environment. Legislation with aim to eliminate and ban EDs have been introduced in EU, but the identification of EDs remains challenging and crucial step towards regulation and risk management. A guidance for ED assessment has been recently established for pesticides and biocides in the EU, which heavily relies on traditional toxicological testing in vivo. Most notably lacking mechanistic methods for some ED modalities and not covering many other modalities that might be affected by EDs. In this project, we focus on the ED assessment according to the valid legislation and explore the possibility to employ alternative methods to bolster the mechanistic understanding of the ED effects and eventually decrease the need for in vivo testing. We selected a well-studied industrial chemical perfluorooctanesulfonic acid (PFOS) to be a model compound in a case study for ED assessment where the EU criteria were applied in the frame of human health risk assessment with focus on thyroid disruption and developmental neurotoxicity. A systematic literature review has been conducted for these effects (Scopus, Pubmed, Embase), and relevant studies were selected by title/abstract screening (RAYYAN) and full-text examination. Selected studies were assessed for reliability (SciRAP), and all relevant data were extracted into a database and assessed by Weight of Evidence (WoE) approach. The initial analysis showed potential endocrine adverse effects and endocrine activity, meeting the ED criteria. The use of mechanistic and alternative methods enhanced the outcomes of WoE assessment. Also, the study provides a great hands-on experience with the most up-to-date development in the area of risk assessment and EDs.