IL-2-mediated hepatotoxicity: knowledge gap identification based on the irAOP concept.

Drug-induced hepatotoxicity constitutes a major reason for non-approval and post-marketing withdrawal of pharmaceuticals. In many cases, preclinical models lack predictive capacity for hepatic damage in humans. A vital concern is the integration of immune system effects in preclinical safety assessment. The immune-related Adverse Outcome Pathway (irAOP) approach, which is applied within the Immune Safety Avatar (imSAVAR) consortium, presents a novel method to understand and predict immune-mediated adverse events elicited by pharmaceuticals and thus targets this issue. It aims to dissect the molecular mechanisms involved and identify key players in drug-induced side effects. As irAOPs are still in their infancy, there is a need for a model irAOP to validate the suitability of this tool. For this purpose, we developed a hepatotoxicity-based model irAOP for recombinant human IL-2 (aldesleukin). Besides producing durable therapeutic responses against renal cell carcinoma and metastatic melanoma, the boosted immune activation upon IL-2 treatment elicits liver damage. The availability of extensive data regarding IL-2 allows both the generation of a comprehensive putative irAOP and to validate the predictability of the irAOP with clinical data. Moreover, IL-2, as one of the first cancer immunotherapeutics on the market, is a blueprint for various biological and novel treatment regimens that are under investigation today. This review provides a guideline for further irAOP-directed research in immune-mediated hepatotoxicity.

Parabens promotes invasive properties of multiple human cells: A potential cancer-associated adverse outcome pathway.

Parabens are esters of p-hydroxybenzoic acid, which have been used as preservatives and considered safe for nearly a century, until the last two decades when concerns began to be raised about their association with cancers. Knowledge of the mode of action of parabens on the metastatic properties of different cancer cells is still very limited. In the present study, we investigated the effects of methylparaben (MP) and propylparaben (PP) on cell invasion and/or migration in multiple human cancerous and noncancerous cells, including hepatocellular carcinoma cells (HepG2), cervical carcinoma cells (HeLa), breast carcinoma cells (MCF-7), and human placental trophoblasts (HTR-8/SVneo). MP and PP at concentrations in a range of 5-500 µg/L significantly promoted the invasion of four cell lines, with a minimum effective concentration of 5 µg/L. MP and PP up-regulated the expression levels and enzymatic activities of matrix metalloproteinase 2 and 9 (MMP2 and MMP9), as well as altered the expression of the tissue inhibitors of metalloproteinase 1 and 2 (TIMP1 and TIMP2) in four cell lines, suggesting MMPs/TIMPs as potential key events (KEs) for paraben-induced cell invasion. Activation of the p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun N-terminal protein kinases 1/2 (JNK1/2) signaling pathways was required for MP- and PP-promoted invasion of four cell lines, suggesting MAPK signaling pathways as candidates for KEs in cancer or noncancerous cells response to paraben exposure. This study showed for the first time that the two widely used parabens, MP and PP, promoted invasive capacity of multiple human cells through a common mode of action. This study provides evidence for the establishment of a potential cancer-associated AOP for parabens based on pathway-specific mechanism(s), which contributes towards assessing the health risks of these environmental chemicals.

Exposure estimation and neurotoxicity inhibition of dioxins in sensitive populations near domestic waste incineration plant through adverse outcome pathway.

The neurotoxicity induced by dioxins has been recognized as a serious concern to sensitive population living near waste incineration plants. However, investigating the intracellular neurotoxicity of dioxin in humans and the corresponding mitigation strategies has been barely studied. Thus, a domestic waste incineration plant was selected in this study to characterize the neurotoxicity risks of sensitive populations by estimating the ratio of dioxin in human cells using membrane structure dynamics simulation; and constructing a complete dioxin neurotoxicity adverse outcome pathway considering the binding process of AhR/ARNT dimer protein and dioxin response element (DRE). Six dioxins with high neurotoxicity risk were identified. According to the composite neurotoxicity risk analysis, the highest composite neurotoxicity risk appeared when the six dioxins were jointly exposed. Dietary schemes were designed using 1/2 partial factor experimental design to mitigate the composite neurotoxicity risk of six dioxins and No. 16 was screened as the optimum combination which can effectively alleviate the composite neurotoxicity risk by 29.52%. Mechanism analysis shows that the interaction between AhR/ARNT dimer protein and DRE was inhibited under the optimal dietary scheme. This study provides theoretical feasibility and reference significance for assessing composite toxicity risks of pollutants and safety mitigation measures for toxic effects.

Chemical-induced liver cancer: an adverse outcome pathway perspective.

INTRODUCTION: The evaluation of the potential carcinogenicity is a key consideration in the risk assessment of chemicals. Predictive toxicology is currently switching toward non-animal approaches that rely on the mechanistic understanding of toxicity. AREAS COVERED: Adverse outcome pathways (AOPs) present toxicological processes, including chemical-induced carcinogenicity, in a visual and comprehensive manner, which serve as the conceptual backbone for the development of non-animal approaches eligible for hazard identification. The current review provides an overview of the available AOPs leading to liver cancer and discusses their use in advanced testing of liver carcinogenic chemicals. Moreover, the challenges related to their use in risk assessment are outlined, including the exploitation of available data, the need for semantic ontologies, and the development of quantitative AOPs. EXPERT OPINION: To exploit the potential of liver cancer AOPs in the field of risk assessment, 3 immediate prerequisites need to be fulfilled. These include developing human relevant AOPs for chemical-induced liver cancer, increasing the number of AOPs integrating quantitative toxicodynamic and toxicokinetic data, and developing a liver cancer AOP network. As AOPs and other areas in the field continue to evolve, liver cancer AOPs will progress into a reliable and robust tool serving future risk assessment and management.

Disruption of gonadotropin hormone biosynthesis by parabens: A potential development and reproduction-associated adverse outcome pathway.

Parabens are widely used as antibacterial preservatives in foods and personal care products. The knowledge about the modes of toxic action of parabens on development and reproduction remain very limited. The present study attempted to establish a development and reproduction-associated adverse outcome pathway (AOP) by evaluating the effects of methylparaben (MP), ethylparaben (EP), propylparaben (PP) and butylparaben (BP) on the biosynthesis of gonadotropins, which are key hormones for development and reproduction. MP and BP significantly upregulated the mRNA and protein levels of follicle stimulating hormone (FSH) and luteinizing hormone (LH) in pituitary gonadotropic cells in a concentration-dependent manner. Activation of gonadotropin-releasing hormone receptor (GnRHR) was required for gonadotropin biosynthesis induced by BP, but not MP. Molecular docking data further demonstrated the higher binding efficiency of BP to human GnRHR than that of MP, suggesting GnRHR as a potential molecular initiative event (MIE) for BP-induced gonadotropin production. L-type voltage-gated calcium channels (VGCCs) were found to be another candidate for MIE in gonadotropic cells response to both MP and BP exposure. The calcium-dependent activation of extracellular signal-regulated kinase 1 (ERK1) and ERK2 was subsequently required for MP- and BP-induced activation of GnRHR and L-type VGCCs pathways. In summary, MP and BP promoted gonadotropin biosynthesis through their interactions with cellular macromolecules GnRHR, L-type VGCCs, and subsequent key event ERK1/2. This is the first study to report the direct interference of parabens with gonadotropin biosynthesis and establish a potential AOP based on pathway-specific mechanism, which contributes to the effective screening of environmental chemicals with developmental and reproductive health risks.

Quantitative modeling of in vitro data using an adverse outcome pathway for the risk assessment of decreased lung function in humans.

In the absence of epidemiological data, there is a need to develop computational models that convert in vitro findings to human disease risk predictions following toxicant exposure. In such efforts, in vitro data can be evaluated in the context of adverse outcome pathways (AOPs) that organize mechanistic knowledge based on empirical evidence into a sequence of molecular-, cellular-, tissue-, and organ-level key events that precede an adverse outcome (AO). Here we combined data from advanced in vitro organotypic airway models exposed to combustible cigarette (CC) smoke or Tobacco Heating System (THS) aerosol with an AOP for increased oxidative stress leads to decreased lung function. The mathematical modeling predicted reduced risk of decreased ciliary beating frequency (CBF) based on oxidative stress measurements and reduced risk of decreased mucociliary clearance (MCC) based on CBF measurements in THS aerosol- compared with CC smoke-exposed cultures. To extend the predictions to the AO of decreased lung function, we leveraged human MCC data from current smokers, nonsmokers, former smokers, and users of heated tobacco products. This approach provided a plausible prediction of diminished reduction in lung function in response to THS use compared with continued smoking. The current approach may also present a basis for an integrated approach to testing and assessment of tobacco products for future regulatory decision-making.

Predicting the Metal Mixture Toxicity with a Toxicokinetic-Toxicodynamic Model Considering the Time-Dependent Adverse Outcome Pathways.

Chemicals mainly exist in ecosystems as mixtures, and understanding and predicting their effects are major challenges in ecotoxicology. While the adverse outcome pathway (AOP) and toxicokinetic-toxicodynamic (TK-TD) models show promise as mechanistic approaches in chemical risk assessment, there is still a lack of methodology to incorporate the AOP into a TK-TD model. Here, we describe a novel approach that integrates the AOP and TK-TD models to predict mixture toxicity using metal mixtures (specifically Cd-Cu) as a case study. We preliminarily constructed an AOP of the metal mixture through temporal transcriptome analysis together with confirmatory bioassays. The AOP revealed that prolonged exposure time activated more key events and adverse outcomes, indicating different modes of action over time. We selected a potential key event as a proxy for damage and used it as a measurable parameter to replace the theoretical parameter (scaled damage) in the TK-TD model. This refined model, which connects molecular responses to organism outcomes, effectively predicts Cd-Cu mixture toxicity over time and can be extended to other metal mixtures and even multicomponent mixtures. Overall, our results contribute to a better understanding of metal mixture toxicity and provide insights for integrating the AOP and TK-TD models to improve risk assessment for chemical mixtures.

Proof of concept for quantitative adverse outcome pathway modeling of chronic toxicity in repeated exposure.

Adverse Outcome Pathway (AOP) is a useful tool to glean mode of action (MOE) of a chemical. However, in order to use it for the purpose of risk assessment, an AOP needs to be quantified using in vitro or in vivo data. Majority of quantitative AOPs developed so far, were for single exposure to progressively higher doses. Limited attempts were made to include time in the modeling. Here as a proof-of concept, we developed a hypothetical AOP, and quantified it using a virtual dataset for six repeated exposures using a Bayesian Network Analysis (BN) framework. The virtual data was generated using realistic assumptions. Effects of each exposure were analyzed separately using a static BN model and analyzed in combination using a dynamic BN (DBN) model. Our work shows that the DBN model can be used to calculate the probability of adverse outcome when other upstream KEs were observed earlier. These probabilities can help in identification of early indicators of AO. In addition, we also developed a data driven AOP pruning technique using a lasso-based subset selection, and show that the causal structure of AOP is itself dynamic and changes over time. This proof-of-concept study revealed the possibility for expanding the applicability of the AOP framework to incorporate biological dynamism in toxicity appearance by repeated insults.

The skin sensitization adverse outcome pathway: exploring the role of mechanistic understanding for higher tier risk assessment.

For over a decade, the skin sensitization Adverse Outcome Pathway (AOP) has served as a useful framework for development of novel in chemico and in vitro assays for use in skin sensitization hazard and risk assessment. Since its establishment, the AOP framework further fueled the existing efforts in new assay development and stimulated a plethora of activities with particular focus on validation, reproducibility and interpretation of individual assays and combination of assay outputs for use in hazard/risk assessment. In parallel, research efforts have also accelerated in pace, providing new molecular and dynamic insight into key events leading to sensitization. In light of novel hypotheses emerging from over a decade of focused research effort, mechanistic evidence relating to the key events in the skin sensitization AOP may complement the tools currently used in risk assessment. We reviewed the recent advances unraveling the complexity of molecular events in sensitization and signpost the most promising avenues for further exploration and development of useful assays.

An integrative data-centric approach to derivation and characterization of an adverse outcome pathway network for cadmium-induced toxicity.

Cadmium is a prominent toxic heavy metal that contaminates both terrestrial and aquatic environments. Owing to its high biological half-life and low excretion rates, cadmium causes a variety of adverse biological outcomes. Adverse outcome pathway (AOP) networks were envisioned to systematically capture toxicological information to enable risk assessment and chemical regulation. Here, we leveraged AOP-Wiki and integrated heterogeneous data from four other exposome-relevant resources to build the first AOP network relevant for inorganic cadmium-induced toxicity. From AOP-Wiki, we filtered 309 high confidence AOPs, identified 312 key events (KEs) associated with inorganic cadmium from five exposome-relevant databases using a data-centric approach, and thereafter, curated 30 cadmium relevant AOPs (cadmium-AOPs). By constructing the undirected AOP network, we identified a large connected component of 18 cadmium-AOPs. Further, we analyzed the directed network of 59 KEs and 82 key event relationships (KERs) in the largest component using graph-theoretic approaches. Subsequently, we mined published literature using artificial intelligence-based tools to provide auxiliary evidence of cadmium association for all KEs in the largest component. Finally, we performed case studies to verify the rationality of cadmium-induced toxicity in humans and aquatic species. Overall, cadmium-AOP network constructed in this study will aid ongoing research in systems toxicology and chemical exposome.

Nontargeted Identification of Organic Components in Fine Particulate Matter Related to Lung Tumor Metastasis Based on an Adverse Outcome Pathway Strategy.

Emerging studies implicate fine particulate matter (PM2.5) and its organic components (OCs) as urgent hazard factors for lung cancer progression in nonsmokers. Establishing the adverse outcome pathway (AOP)-directed nontargeted identification method, this study aimed to explore whether PM2.5 exposure in coal-burning areas promoted lung tumor metastasis and how we identify its effective OCs to support traceability and control of regional PM2.5 pollution. First, we used a nude mouse model of lung cancer for PM2.5 exposure and found that the exposure significantly promoted the hematogenous metastases of A549-Luc cells in lung tissues and the adverse outcomes (AOs), with key events (KEs) including the changed expression of epithelial-mesenchymal transition (EMT) markers, such as suppression of E-cad and increased expression of Fib. Subsequently, using AOs and KEs as adverse outcome directors, we identified a total of 35 candidate chemicals based on the in vitro model and nontargeted analysis. Among them, tributyl phosphate (C12H27O4P), 2-bromotetradecane (C14H29Br), and methyl decanoate (C11H22O2) made greater contributions to the AOs. Finally, we clarified the interactions between these OCs and EMT-activating transcription factors (EMT-ATFs) as the molecular initiation event (MIE) to support the feasibility of the above identification strategy. The present study updates a new framework for identifying tumor metastasis-promoting OCs in PM2.5 and provides solid data for screening out chemicals that need priority control in polluted areas posing higher lung cancer risk.

Human biomonitoring and toxicokinetics as key building blocks for next generation risk assessment.

Human health risk assessment is historically built upon animal testing, often following Organisation for Economic Co-operation and Development (OECD) test guidelines and exposure assessments. Using combinations of human relevant in vitro models, chemical analysis and computational (in silico) approaches bring advantages compared to animal studies. These include a greater focus on the human species and on molecular mechanisms and kinetics, identification of Adverse Outcome Pathways and downstream Key Events as well as the possibility of addressing susceptible populations and additional endpoints. Much of the advancement and progress made in the Next Generation Risk Assessment (NGRA) have been primarily focused on new approach methodologies (NAMs) and physiologically based kinetic (PBK) modelling without incorporating human biomonitoring (HBM). The integration of toxicokinetics (TK) and PBK modelling is an essential component of NGRA. PBK models are essential for describing in quantitative terms the TK processes with a focus on the effective dose at the expected target site. Furthermore, the need for PBK models is amplified by the increasing scientific and regulatory interest in aggregate and cumulative exposure as well as interactions of chemicals in mixtures. Since incorporating HBM data strengthens approaches and reduces uncertainties in risk assessment, here we elaborate on the integrated use of TK, PBK modelling and HBM in chemical risk assessment highlighting opportunities as well as challenges and limitations. Examples are provided where HBM and TK/PBK modelling can be used in both exposure assessment and hazard characterization shifting from external exposure and animal dose/response assays to animal-free, internal exposure-based NGRA.

Integrating aggregate exposure pathway and adverse outcome pathway for micro/nanoplastics: A review on exposure, toxicokinetics, and toxicity studies.

Micro/nanoplastics (MNPs) have emerged as a significant environmental concern due to their widespread distribution and potential adverse effects on human health and the environment. In this study, to integrate exposure and toxicity pathways of MNPs, a comprehensive review of the occurrence, toxicokinetics (absorption, distribution, and excretion [ADE]), and toxicity of MNPs were investigated using the aggregate exposure pathway (AEP) and adverse outcome pathway (AOP) frameworks. Eighty-five papers were selected: 34 papers were on detecting MNPs in environmental samples, 38 papers were on the ADE of MNPs in humans and fish, and 36 papers were related to MNPs toxicity using experimental models. This review not only summarizes individual studies but also presents a preliminary AEP-AOP framework. This framework offers a comprehensive overview of pathways, enabling a clearer visualization of intricate processes spanning from environmental media, absorption, distribution, and molecular effects to adverse outcomes. Overall, this review emphasizes the importance of integrating exposure and toxicity pathways of MNPs by utilizing AEP-AOP to comprehensively understand their impacts on human and ecological organisms. The findings contribute to highlighting the need for further research to fill the existing knowledge gaps in this field and the development of more effective strategies for the safe management of MNPs.

Combined toxicity and adverse outcome pathways of common pesticides on Chlorella pyrenoidosa.

Pesticides due to their extensive use have entered the soil and water environment through various pathways, causing great harm to the environment. Herbicides and insecticides are common pesticides with long-term biological toxicity and bioaccumulation, which can harm the human body. The concept of the adverse outcome pathway (AOP) involves systematically analyzing the response levels of chemical mixtures to health-related indicators at the molecular and cellular levels. The AOP correlates the structures of chemical pollutants, toxic molecular initiation events and adverse outcomes of biological toxicity, providing a new model for toxicity testing, prediction, and evaluation of pollutants. Therefore, typical pesticides including diquat (DIQ), cyanazine (CYA), dipterex (DIP), propoxur (PRO), and oxamyl (OXA) were selected as research objects to explore the combined toxicity of typical pesticides on Chlorella pyrenoidosa (C. pyrenoidosa) and their adverse outcome pathways (AOPs). The mixture systems of pesticides were designed by the direct equipartition ray (EquRay) method and uniform design ray (UD-Ray) method. The toxic effects of single pesticides and their mixtures were systematically investigated by the time-dependent microplate toxicity analysis (t-MTA) method. The interactions of their mixtures were analyzed by the concentration addition model (CA) and the deviation from the CA model (dCA). The toxicity data showed a good concentration-effect relationship; the toxicities of five pesticides were different and the order was CYA > DIQ > OXA > PRO > DIP. Binary, ternary and quaternary mixture systems exhibited antagonism, while quinary mixture systems exhibited an additive effect. The AOP of pesticides showed that an excessive accumulation of peroxide in green algae cells led to a decline in stress resistance, inhibition of the synthesis of chlorophyll and protein in algal cells, destruction of the cellular structure, and eventually led to algal cell death.

Development of an adverse outcome pathway network for nephrotoxicity.

Adverse outcome pathways (AOPs) were introduced in modern toxicology to provide evidence-based representations of the events and processes involved in the progression of toxicological effects across varying levels of the biological organisation to better facilitate the safety assessment of chemicals. AOPs offer an opportunity to address knowledge gaps and help to identify novel therapeutic targets. They also aid in the selection and development of existing and new in vitro and in silico test methods for hazard identification and risk assessment of chemical compounds. However, many toxicological processes are too intricate to be captured in a single, linear AOP. As a result, AOP networks have been developed to aid in the comprehension and placement of associated events underlying the emergence of related forms of toxicity-where complex exposure scenarios and interactions may influence the ultimate adverse outcome. This study utilised established criteria to develop an AOP network that connects thirteen individual AOPs associated with nephrotoxicity (as sourced from the AOP-Wiki) to identify several key events (KEs) linked to various adverse outcomes, including kidney failure and chronic kidney disease. Analysis of the modelled AOP network and its topological features determined mitochondrial dysfunction, oxidative stress, and tubular necrosis to be the most connected and central KEs. These KEs can provide a logical foundation for guiding the selection and creation of in vitro assays and in silico tools to substitute for animal-based in vivo experiments in the prediction and assessment of chemical-induced nephrotoxicity in human health.

Control list of high-priority chemicals based on 5-HT-RI functionality and the human health interference effects selective CNN-GRU deep learning model.

The antidepressant drug known as 5-HT reuptake inhibitor (5-HT-RI) was commonly detected in biological tissues and result in significant adverse health effects. Homology modeling was used to characterize the functionalities (efficacy and resistance), and the adverse outcome pathway was used to characterize its human health interferences (olfactory toxicity, neurotoxicity, and gut microbial interference). The convolutional neural network coupled with the gated recurrent unit (CNN-GRU) deep learning method was used to construct a comprehensive model of 5-HT-RI functionality and human health interference effects selectivity with small sample data. The architecture with 2 SE, 320 neuronal nodes and 6-folds cross-validation showed the best applicability. The results showed that the confidence interval of the constructed model reached 90 % indicating that the model had reliable prediction ability and generalization ability. Based on the CNN-GRU deep learning model, seven high-priority chemicals with a weak comprehensive effect, including D-VEN, (1R,4S)-SER, S-FLX, CTP, S-CTP, NEF, and VEN, were screened. Based on the molecular three-dimensional structure information, a comprehensive-effect three-dimensional quantitative structure-activity relationship (3D-QSAR) model was constructed to confirm the reliability of the constructed control list of 5-HT-RI high-priority chemicals. Analysis with the ranking of calculated values based on the molecular dynamics method and predicted values based on the CNN-GRU deep learning model, we found that the consistency of the three methods was above 85 %. Additionally, by analyzing the sensitivity, molecular electrostatic potential, polar surface area of the comprehensive-effect CNN-GRU deep learning model, and the electrostatic field of the 3D-QSAR models, we found that the significant effects of five key characteristics (DM, Qyy, Qxz, I, and BP), molecular electronegativity, and polarity significantly affected the high-priority degree of 5-HT-RI. In this study, we provided reasonable and reliable prediction tools and discussed theoretical methods for the risk assessment of functionality and human health interference of emerging pollutants such as 5-HT-RI.

Molecular interactions of paraben family of pollutants with embryonic neuronal proteins of Danio rerio: A step ahead in computational toxicity towards adverse outcome pathway.

The paraben family of endocrine disruptors exhibit persistent behaviours in aquatic matrices, having bio-accumulative effects and necessitating toxicity analysis and safe use, as well as prevention of food web penetration. In this study, the toxicity effects of 9 different parabens (Methyl, Ethyl, Propyl, Butyl, Heptyl, Isopropyl, Isobutyl, benzyl parabens and p-hydroxybenzoic acid) were studied against 17 neuronal proteins (Neurog1, Ascl1a, DLA, Syn2a, Ntn1a, Pitx2, and SoxB1, Her/Hes, Zic family) expressed during the early embryonic developmental stage of Danio rerio. The neuronal genes were selected as a biomarker to study the inhibitory effects on the cascade of genes expressed in the early developmental stage. The study uses trRossetta software to predict protein structures of neuronal genes, followed by structural refinement, energy minimisation, and active site prediction, evaluated using energy value, RC plot and ERRAT scores of PROCHECK and ERRAT programs. Compared to raw structures, highly confident predicted structures and quality scores were observed for refined protein with few exceptions. Based on the polarity and charge of the aminoacids, the probable pockets were identified using active site prediction, which were then used for molecular docking analysis. Further, the ADMET analysis, ligand likeliness and toxicological test revealed the paraben family of compounds as one of the most susceptible toxic and mutagenic compounds. The molecular docking results showed an interesting pattern of increasing binding affinity with increase in the carbon chains of paraben molecules. Benzyl Paraben showed higher binding affinities across all 17 neuronal proteins. Finally, gene co-occurrence/co-expression and protein-protein interaction studies using the STRING database depict that all proteins are functionally related and play essential roles in standard biological processes or pathways, conserved and expressed in diverse organisms. The interaction between paraben compounds and neuronal genes indicates high risks of inhibiting reactions in embryonic stages, emphasising the need for effective treatment measures and strict regulations.

Report of the First ONTOX Stakeholder Network Meeting: Digging Under the Surface of ONTOX Together With the Stakeholders.

The first Stakeholder Network Meeting of the EU Horizon 2020-funded ONTOX project was held on 13-14 March 2023, in Brussels, Belgium. The discussion centred around identifying specific challenges, barriers and drivers in relation to the implementation of non-animal new approach methodologies (NAMs) and probabilistic risk assessment (PRA), in order to help address the issues and rank them according to their associated level of difficulty. ONTOX aims to advance the assessment of chemical risk to humans, without the use of animal testing, by developing non-animal NAMs and PRA in line with 21st century toxicity testing principles. Stakeholder groups (regulatory authorities, companies, academia, non-governmental organisations) were identified and invited to participate in a meeting and a survey, by which their current position in relation to the implementation of NAMs and PRA was ascertained, as well as specific challenges and drivers highlighted. The survey analysis revealed areas of agreement and disagreement among stakeholders on topics such as capacity building, sustainability, regulatory acceptance, validation of adverse outcome pathways, acceptance of artificial intelligence (AI) in risk assessment, and guaranteeing consumer safety. The stakeholder network meeting resulted in the identification of barriers, drivers and specific challenges that need to be addressed. Breakout groups discussed topics such as hazard versus risk assessment, future reliance on AI and machine learning, regulatory requirements for industry and sustainability of the ONTOX Hub platform. The outputs from these discussions provided insights for overcoming barriers and leveraging drivers for implementing NAMs and PRA. It was concluded that there is a continued need for stakeholder engagement, including the organisation of a 'hackathon' to tackle challenges, to ensure the successful implementation of NAMs and PRA in chemical risk assessment.

Per- and polyfluoroalkyl substances and cardiometabolic diseases: A review.

Per- and polyfluoroalkyl substances (PFAS) are a group of persistent and widespread environmental pollutants that represent a high concern for human health. They have been shown to be associated with several important physiological processes such as lipid metabolism and the immune system. Consequently, PFAS are suspected to play a role in cardiometabolic disease development. However, the evidence regarding associations between PFAS and overt cardiovascular disease and type 2 diabetes remains limited and inconsistent. To address this, we conducted a review of the epidemiological evidence. A deeper understanding of potential underlying molecular mechanisms may help to explain inconsistencies in epidemiological findings. Thus, to gain more mechanistic insight, we also summarized evidence from omics and laboratory studies into an adverse outcome pathway framework. Our observations indicate the potential for associations of PFAS with multiple molecular pathways that could have opposite associations with disease risk, which could be further modified by mixture composition, lifestyle factors or genetic polymorphisms. This identifies the need for exposome studies considering mixture effects, the use of multi-omics data to gain insight in relevant pathways and the integration of epidemiological and laboratory studies to enhance mechanistic understanding and causal inference. Improved comprehension is essential for environmental health risk assessments.

Editorial trend: adverse outcome pathway (AOP) and computational strategy - towards new perspectives in ecotoxicology.

The adverse outcome pathway (AOP) has been conceptualized in 2010 as an analytical construct to describe a sequential chain of causal links between key events, from a molecular initiating event leading to an adverse outcome (AO), considering several levels of biological organization. An AOP aims to identify and organize available knowledge about toxic effects of chemicals and drugs, either in ecotoxicology or toxicology, and it can be helpful in both basic and applied research and serve as a decision-making tool in support of regulatory risk assessment. The AOP concept has evolved since its introduction, and recent research in toxicology, based on integrative systems biology and artificial intelligence, gave it a new dimension. This innovative in silico strategy can help to decipher mechanisms of action and AOP and offers new perspectives in AOP development. However, to date, this strategy has not yet been applied to ecotoxicology. In this context, the main objective of this short article is to discuss the relevance and feasibility of transferring this strategy to ecotoxicology. One of the challenges to be discussed is the level of organisation that is relevant to address for the AO (population/community). This strategy also offers many advantages that could be fruitful in ecotoxicology and overcome the lack of time, such as the rapid identification of data available at a time t, or the identification of "data gaps". Finally, this article proposes a step forward with suggested priority topics in ecotoxicology that could benefit from this strategy.