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.

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.

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.