Next Generation Risk Assessment (NGRA) is an exposure-led approach to safety assessment that uses New Approach Methodologies (NAMs). Application of NGRA has been largely restricted to assessments of consumer use of cosmetics and is not currently implemented in occupational safety assessments, e.g. under EU REACH. By contrast, a large proportion of regulatory worker safety assessments are underpinned by toxicological studies using experimental animals. Consequently, occupational safety assessment represents an area that would benefit from increasing application of NGRA to safety decision making. Here, a workflow for conducting NGRA under an occupational safety context was developed, which is illustrated with a case study chemical; sodium 2-hydroxyethane sulphonate (sodium isethionate or SI). Exposures were estimated using a standard occupational exposure model following a comprehensive life cycle assessment of SI and considering factory-specific data. Outputs of this model were then used to estimate internal exposures using a Physiologically Based Kinetic (PBK) model, which was constructed with SI specific Absorption, Distribution, Metabolism and Excretion (ADME) data. PBK modelling indicated a worst-case plasma maximum concentration (Cmax) of 0.8 μM across the SI life cycle. SI bioactivity was assessed in a battery of NAMs relevant to systemic, reproductive, and developmental toxicity; a cell stress panel, high throughput transcriptomics in three cell lines (HepG2, HepaRG and MCF-7 cells), pharmacological profiling and specific assays relating to developmental toxicity (Reprotracker and devTOX quickPredict). Points of Departure (PoDs) for SI ranged from 104 to 5044 µM. Cmax values obtained from PBK modelling of occupational exposures to SI were compared with PoDs from the bioactivity assays to derive Bioactivity Exposure Ratios (BERs) which demonstrated the safety for workers exposed to SI under current levels of factory specific risk management. In summary, the tiered and iterative workflow developed here represents an opportunity for integrating non animal approaches for a large subset of substances for which systemic worker safety assessment is required. Such an approach could be followed to ensure that animal testing is only conducted as a \"last resort\" e.g. under EU REACH.

Botanical extracts, widely used in cosmetics, pose a challenge to safety assessment due to their complex compositions. The threshold of toxicological concern (TTC) approach, offering a safe exposure level for cosmetic ingredients, proves to be a promising solution for ensuring the safety of cosmetic ingredients with low exposure level. We assessed the safety of Paeonia lactiflora root extract (PLR), commonly used in skin conditioning products, with the TTC. We identified 50 constituents of PLR extract from the USDA database and literature exploration. Concentration of each constituent of PLR extract was determined with the information from USDA references, literature, and experimental analysis. The genotoxicity of PLR and its constituents was assessed in vitro and in silico respectively. Cramer class of the constituents of the PLR extract was determined with Toxtree 3.1 extended decision tree using ChemTunes®. Systemic exposure of each constituent from leave-on type cosmetic products containing PLR at a 1% concentration was estimated and compared with respective TTC threshold. Two constituents exceeding TTC threshold were further analyzed for dermal absorption using in silico tools, which confirmed the safety of PLR extract in cosmetics. Collectively, we demonstrated that the TTC is a useful tool for assessing botanical extract safety in cosmetics.

Next-Generation Risk Assessment (NGRA) aims to implement New Approach Methodologies (NAMs) into risk assessment and to rely on new in vivo testing in animals only as a last resort. However, various technical and regulatory hurdles impede their regulatory implementation. Assumptions about the public\'s expectations could act as barriers to the acceptance of NAMs. This study aimed at investigating public views of animal testing and potential alternatives, namely in vitro and in silico testing. An online survey was conducted (N = 965). The results suggest that people make trade-offs, as they experience negative affect regarding in vivo testing, which partly might explain their openness regarding certain alternatives. In vitro tests were attributed the highest ability to determine harmful effects of chemicals for different endpoints, followed by in vivo and in silico tests. Our results further showed that many people accept chemicals to be only tested with alternatives, with highest acceptance for household consumer products, food contact material or building materials and less accepting for medicines and foods. This article addresses potential challenges that might arise from public perceptions and thus, contributes to the bottom-up initiatives to overcome the hurdles to the implementation of NAMs in regulatory risk assessment.

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.

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.

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.

Environmental risk assessment traditionally relies on a wide range of in vivo testing to assess the potential hazards of chemicals in the environment. These tests are often time-consuming and costly and can cause test organisms\' suffering. Recent developments of reliable low-cost alternatives, both in vivo- and in silico-based, opened the door to reconsider current toxicity assessment. However, many of these new approach methodologies (NAMs) rely on high-quality annotated genomes for surrogate species of regulatory risk assessment. Currently, a lack of genomic information slows the process of NAM development. Here, we present a phylogenetically resolved overview of missing genomic resources for surrogate species within a regulatory ecotoxicological risk assessment. We call for an organized and systematic effort within the (regulatory) ecotoxicological community to provide these missing genomic resources. Further, we discuss the potential of a standardized genomic surrogate species landscape to enable a robust and nonanimal-reliant ecotoxicological risk assessment in the systems ecotoxicology era.

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.

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. Specifically, a mechanistic assessment strategy, based on human physiology and (patho)biology is needed, which can be applied in next generation risk assessment. The Adverse Outcome Pathway (AOP) framework is particularly well-suited to provide the mechanistic basis for such a strategy. Here, we conducted a semi-systematic review in Embase and MEDLINE, focused on neurodegeneration and pesticides, to develop an AOP network for parkinsonian motor symptoms. Articles were labelled and included/excluded using the online platform Sysrev. Only primary articles, written in English, focused on effects of pesticides or PD model compounds in models for the brain were included. A total of 66 articles, out of the 1700 screened, was included. PD symptoms are caused by loss of function and ultimately death of dopaminergic neurons in the substantia nigra (SN). Our literature review highlights that a unique feature of these cells that increases their vulnerability is their reliance on continuous low-level influx of calcium. As such, excess intracellular calcium was identified as a central early Key Event (KE). This KE can lead to death of dopaminergic neurons of the SN, and eventually parkinsonian motor symptoms, via four distinct pathways: 1) activation of calpains, 2) endoplasmic reticulum stress, 3) impairment of protein degradation, and 4) oxidative damage. Several receptors have been identified that may serve as molecular initiating events (MIEs) to trigger one or more of these pathways. The proposed AOP network provides the biological basis that can be used to develop a mechanistic testing strategy that captures neurodegenerative effects of pesticides.

Omics techniques have been increasingly recognized as promising tools for Next Generation Risk Assessment. Targeted metabolomics offer the advantage of providing readily interpretable mechanistic information about perturbed biological pathways. In this study, a high-throughput LC-MS/MS-based broad targeted metabolomics system was applied to study nitrofurantoin metabolic dynamics over time and concentration and to provide a mechanistic-anchored approach for point of departure (PoD) derivation. Upon nitrofurantoin exposure at five concentrations (7.5 µM, 15 µM, 20 µM, 30 µM and 120 µM) and four time points (3, 6, 24 and 48 h), the intracellular metabolome of HepG2 cells was evaluated. In total, 256 uniquely identified metabolites were measured, annotated, and allocated in 13 different metabolite classes. Principal component analysis (PCA) and univariate statistical analysis showed clear metabolome-based time and concentration effects. Mechanistic information evidenced the differential activation of cellular pathways indicative of early adaptive and hepatotoxic response. At low concentrations, effects were seen mainly in the energy and lipid metabolism, in the mid concentration range, the activation of the antioxidant cellular response was evidenced by increased levels of glutathione (GSH) and metabolites from the de novo GSH synthesis pathway. At the highest concentrations, the depletion of GSH, together with alternations reflective of mitochondrial impairments, were indicative of a hepatotoxic response. Finally, a metabolomics-based PoD was derived by multivariate PCA using the whole set of measured metabolites. This approach allows using the entire dataset and derive PoD that can be mechanistically anchored to established key events. Our results show the suitability of high throughput targeted metabolomics to investigate mechanisms of hepatoxicity and derive point of departures that can be linked to existing adverse outcome pathways and contribute to the development of new ones.