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.

Dose addition in mixtures of compounds with dissimilar endocrine modes of action in in vitro receptor activation assays and the zebrafish sexual development test.

Background: Human exposure to pesticides is being associated with feminisation for which a decrease of the anogenital distance (AGD) is a sensitive endpoint. Dose addition for the cumulative risk assessment of pesticides in food is considered sufficiently conservative for combinations of compounds with both similar and dissimilar modes of action (MoA).

Objective: The present study was designed to test the dose addition hypothesis in a binary mixture of endocrine active compounds with a dissimilar mode of action for the endpoint feminisation.

Towards improved screening of toxins for Parkinson's risk.

Parkinson's disease (PD) is a chronic, progressive and disabling neurodegenerative disorder. The prevalence of PD has risen considerably over the past decades. A growing body of evidence suggest that exposure to environmental toxins, including pesticides, solvents and heavy metals (collectively called toxins), is at least in part responsible for this rapid growth.

An adverse outcome pathway for chemical-induced Parkinson's disease: Calcium is key.

Exposure to pesticides is associated with an increased risk of developing Parkinson's disease (PD). Currently, rodent-based risk assessment studies cannot adequately capture neurodegenerative effects of pesticides due to a lack of human-relevant endpoints targeted at neurodegeneration. Thus, there is a need for improvement of the risk assessment guidelines.

New approach methodologies to facilitate and improve the hazard assessment of non-genotoxic carcinogens-a PARC project.

Carcinogenic chemicals, or their metabolites, can be classified as genotoxic or non-genotoxic carcinogens (NGTxCs). Genotoxic compounds induce DNA damage, which can be detected by an established and battery of genotoxicity assays. For NGTxCs, DNA is not the primary target, and the possible modes of action (MoA) of NGTxCs are much more diverse than those of genotoxic compounds, and there is no specific assay for detecting NGTxCs.

Prolonged Differentiation of Neuron-Astrocyte Co-Cultures Results in Emergence of Dopaminergic Neurons.

Dopamine is present in a subgroup of neurons that are vital for normal brain functioning. Disruption of the dopaminergic system, e.g.

Organophosphate insecticides disturb neuronal network development and function via non-AChE mediated mechanisms.

Exposure to organophosphate (OP) insecticides has been related to several adverse health effects, including neurotoxicity. The primary insecticidal mode of action of OP insecticides relies on (irreversible) binding to acetylcholine esterase (AChE), with -oxon metabolites having a much higher potency for AChE inhibition than the parent compounds. However, OP insecticides can also have non-AChE-mediated effects, including changes in gene expression, neuroendocrine effects, disruption of neurite outgrowth and disturbance of the intracellular calcium (Ca) homeostasis.

Acute, sub-chronic and chronic exposures to TiO and Ag nanoparticles differentially affects neuronal function in vitro.

In vivo toxicokinetic studies provide evidence for the translocation and accumulation of nanoparticles (NP) in the brain, thereby causing concern for adverse health effects, particularly for effects following chronic exposure. To date, only few studies investigated the effects of NP exposure on neuronal function in vitro, primarily focusing on short-term effects. The aim of this study was therefore to investigate the effects of two common types of NP, titanium dioxide NP (TiONP) and silver NP (AgNP), on neuronal function following acute (0.

Neuromodulatory and neurotoxic effects of e-cigarette vapor using a realistic exposure method.

The most direct effects of inhaled harmful constituents are the effects on the airways. However, inhaled compounds can be rapidly absorbed and subsequently result in systemic effects. For example, e-cigarette vapor has been shown to evoke local effects in the lung, although little is known about subsequent effects in secondary target organs such as the brain.

Dose Addition in the Induction of Craniofacial Malformations in Zebrafish Embryos Exposed to a Complex Mixture of Food-Relevant Chemicals with Dissimilar Modes of Action.

Background: Humans are exposed to combinations of chemicals. In cumulative risk assessment (CRA), regulatory bodies such as the European Food Safety Authority consider dose addition as a default and sufficiently conservative approach. The principle of dose addition was confirmed previously for inducing craniofacial malformations in zebrafish embryos in binary mixtures of chemicals with either similar or dissimilar modes of action (MOAs).

Effects of subchronic dietary exposure to the engineered nanomaterials SiO and CeO in C57BL/6J and 5xFAD Alzheimer model mice.

Background: There is an increasing concern about the neurotoxicity of engineered nanomaterials (NMs). To investigate the effects of subchronic oral exposures to SiO and CeO NMs on Alzheimer's disease (AD)-like pathology, 5xFAD transgenic mice and their C57BL/6J littermates were fed ad libitum for 3 or 14 weeks with control food pellets, or pellets dosed with these respective NMs at 0.1% or 1% (w/w).

Exploring Neurobehaviour in Zebrafish Embryos as a Screening Model for Addictiveness of Substances.

Tobacco use is the leading cause of preventable death worldwide and is highly addictive. Nicotine is the main addictive compound in tobacco, but less is known about other components and additives that may contribute to tobacco addiction. The zebrafish embryo (ZFE) has been shown to be a good model to study the toxic effects of chemicals on the neurological system and thus may be a promising model to study behavioral markers of nicotine effects, which may be predictive for addictiveness.

Developmental Neurotoxicity of Environmentally Relevant Pharmaceuticals and Mixtures Thereof in a Zebrafish Embryo Behavioural Test.

Humans are exposed daily to complex mixtures of chemical substances via food intake, inhalation, and dermal contact. Developmental neurotoxicity is an understudied area and entails one of the most complex areas in toxicology. Animal studies for developmental neurotoxicity (DNT) are hardly performed in the context of regular hazard studies, as they are costly and time consuming and provide only limited information as to human relevance.

Evaluation of the neurotoxic effects of engineered nanomaterials in C57BL/6J mice in 28-day oral exposure studies.

In recent years, concerns have emerged about the potential neurotoxic effects of engineered nanomaterials (NMs). Titanium dioxide and silver are among the most widely used types of metallic NMs. We have investigated the effects of these NMs on behaviour and neuropathology in male and female C57BL/6J mice following 28-day oral exposure with or without a 14-day post-exposure recovery.

Relevance of Transcriptomics for Mode of Action Assessment.

Recently, we reported an toxicogenomics comparison approach to categorize chemical substances according to similarities in their proposed toxicological modes of action. Use of such an approach for regulatory purposes requires, among others, insight into the extent of biological concordance between and findings. To that end, we applied the comparison approach to transcriptomics data from the Open TG-GATEs database for 137 substances with diverging modes of action and evaluated the outcomes obtained for rat primary hepatocytes and for rat liver.

The effects of aliphatic alcohols and related acid metabolites in zebrafish embryos - correlations with rat developmental toxicity and with effects in advanced life stages in fish.

The zebrafish embryo toxicity test (ZFET) is a simple medium-throughput test to inform about (sub)acute lethal effects in embryos. Enhanced analysis through morphological and teratological scoring, and through gene expression analysis, detects developmental effects and the underlying toxicological pathways. Altogether, the ZFET may inform about hazard of chemical exposure for embryonal development in humans, as well as for lethal effects in juvenile and adult fish.

An efficient neuron-astrocyte differentiation protocol from human embryonic stem cell-derived neural progenitors to assess chemical-induced developmental neurotoxicity.

Human embryonic stem cell neuronal differentiation models provide promising in vitro tools for the prediction of developmental neurotoxicity of chemicals. Such models mimic essential elements of human relevant neuronal development, including the differentiation of a variety of brain cell types and their neuronal network formation as evidenced by specific gene and protein biomarkers. However, the reproducibility and lengthy culture duration of cell models present drawbacks and delay regulatory implementation.

An ontology for developmental processes and toxicities of neural tube closure.

In recent years, the development and implementation of animal-free approaches to chemical and pharmaceutical hazard and risk assessment has taken off. Alternative approaches are being developed starting from the perspective of human biology and physiology. Neural tube closure is a vital step that occurs early in human development.

Distinguishing mode of action of compounds inducing craniofacial malformations in zebrafish embryos to support dose-response modeling in combined exposures.

Knowledge on mode-of-action (MOA) is required to understand toxicological effects of compounds, notably in the context of risk assessment of mixtures. Such information is generally scarce, and often complicated by the existence of multiple MOAs per compound. Here, MOAs related to developmental craniofacial malformations were derived from literature, and assembled in a MOA network.

Evaluation of neurological effects of cerium dioxide nanoparticles doped with different amounts of zirconium following inhalation exposure in mouse models of Alzheimer's and vascular disease.

Increasing evidence from toxicological and epidemiological studies indicates that the brain is an important target for ambient (ultrafine) particles. Disturbance of redox-homeostasis and inflammation in the brain are proposed as possible mechanisms that can contribute to neurotoxic and neurodegenerative effects. Whether and how engineered nanoparticles (NPs) may cause neurotoxicity and promote neurodegenerative diseases such as Alzheimer's disease (AD) is largely unstudied.

Application of the comparison approach to open TG-GATEs: A useful toxicogenomics tool for detecting modes of action in chemical risk assessment.

Mode of action information is one of the key components for chemical risk assessment as mechanistic insight leads to better understanding of potential adverse health effects of a chemical. This insight greatly facilitates assessment of human relevance and enhances the use of non-animal methods for risk assessment, as it ultimately enables extrapolation from initiating events to adverse effects. Recently, we reported an in vitro toxicogenomics comparison approach to categorize (non-)genotoxic carcinogens according to similarities in their proposed modes of action.

Comparison of different in vitro cell models for the assessment of pesticide-induced dopaminergic neurotoxicity.

Biomedical and (neuro) toxicity research on (neuro) degenerative diseases still relies strongly on animal models. However, the use of laboratory animals is often undesirable for both ethical and technical reasons. Current in vitro research thus largely relies on tumor derived- or immortalized cell lines.

The effect of zirconium doping of cerium dioxide nanoparticles on pulmonary and cardiovascular toxicity and biodistribution in mice after inhalation.

Development and manufacture of nanomaterials is growing at an exponential rate, despite an incomplete understanding of how their physicochemical characteristics affect their potential toxicity. Redox activity has been suggested to be an important physicochemical property of nanomaterials to predict their biological activity. This study assessed the influence of redox activity by modification of cerium dioxide nanoparticles (CeO NPs) via zirconium (Zr) doping on the biodistribution, pulmonary and cardiovascular effects in mice following inhalation.