INTRODUCTION:In recent years, the scientific community, together with regulatory authorities, institutions and stakeholders, joined in the common objective of changing the regulatory toxicology paradigm, moving from classical toxicological animal testing towards an exposure-driven risk assessment, based on mechanistic information derived from alternative methods, globally known as new approach methodologies (NAMs). The transition to the next generation risk assessment (NGRA) requires the collective effort of all involved actors. One approach addressing the needs of the NGRA is the adverse outcome pathway (AOP) concept. This construct describes a sequence of temporally and causally linked events at distinct levels of biological organization leading to an adverse health effect. The development, validation and implementation of AOPs at the regulatory level have become key objectives of several authorities. METHODS:In this paper, the AOPs and AOP networks as applied by the European Food Safety Agency are analyzed, alongside the proposed batteries of NAMs. RESULTS AND DISCUSSION:Two case studies, (1) Parkinson's disease and pesticide exposure, and (2) exposure to substances with endocrine-disrupting properties and the development of uterine adenocarcinoma, are described. The analysis of the entire AOP construct for each case and the assembly of the specific NAMs allowed us to identify the strengths and limitations of the AOP strategy, as well as of each proposed test. Their integration and status are assessed within the current regulatory framework.

The transition from animal-based chemical risk assessment to Next-Generation Risk Assessment requires the integration of human-relevant New Approach Methodologies and mechanistic kinetic data to support physiologically based kinetic (PBK) modelling for quantitative in vitro-to-in vivo extrapolation. This study investigated isoform-specific and extrahepatic glutathione (GSH) conjugation of Microcystin-LR (MC-LR), a widespread cyanobacterial toxin of emerging concern in food and feed safety. MC-LR detoxification occurs via spontaneous GSH conjugation or catalysed by glutathione-S-transferases (GSTs). A panel of recombinant human GSTs and pooled human and rat intestinal and kidney cytosols was incubated with 1-60 µM MC-LR to characterise kinetic parameters and intrinsic clearance (Cli). The tested GST isoforms catalysed MC-LR conjugation with efficiencies spanning a 15-fold range, with P1 and T1 showing the highest activity. Isoforms exhibited distinct kinetic behaviours, including positive cooperativity, influencing their relative contribution across substrate concentrations. Human intestinal and renal cytosols showed GST-mediated metabolic efficiencies comparable to hepatic data, attributable to the high expression of GSTP1 and T1. However, when scaling to whole-organ Cli, the hepatic enzymatic conjugation exceeded extrahepatic one. In rats, intestine and kidney detoxification capacity was low with respect to the hepatic one, the latter resulting 3.3-fold more efficient than the human GST-mediated reaction. Although the spontaneous conjugation in vitro accounted for 70-80% of total conjugate formation, when the reaction rate scaled to the total hepatic cytosolic volume, its relative contribution was lower than the GST-mediated one. In addition the enzymatic reaction became dominant even in vitro under GSH depletion, at low MC-LR concentrations, typical of long term exposure. These findings underscore the relevance of GST isoform distribution, polymorphism, and GSH availability in driving inter-organ, interspecies, and inter-individual variability in MC-LR detoxification. Overall, this study fills critical data gaps on MC-LR kinetics and provides quantitative parameters to inform PBK model development.

Bisphenol A (BPA) alternatives are increasingly used in the manufacture of industrial and consumer products, following regulatory restrictions on BPA. However, insufficient safety data on these substitutes raise concern as regards potential regrettable substitutions. Under the EU Partnership for the Assessment of Risks from Chemicals (PARC), Work Package 5 (WP5) addresses this challenge by applying a human-relevant tiered hazard assessment strategy grounded on OECD test guidelines as first tier and expanding the battery to include NAMs (New Approach Methodologies). Eight BPA alternatives were prioritized for studies addressing key toxicological endpoints, namely, endocrine disruption (ED), developmental neurotoxicity (DNT), immunotoxicity, genotoxicity and carcinogenicity and metabolic fate examination (detoxification vs. potential bioactivation), to enable early identification of biological activity and support cross-endpoint prioritization. This manuscript describes the structure and implementation of the testing framework. This integrated testing strategy proposes a structured approach to identify substances of potential concern, guide targeted higher-tier studies, and support regulatory prioritization. PARC WP5 framework is testing whether coordinated NAM-based methods may contribute to next-generation risk assessment and help prevent regrettable substitutions among BPA alternatives for rapid regulatory adoption. Detailed experimental results will be reported separately upon completion of the project.

In 2015, the World Health Organization (WHO) declared Antimicrobial Resistance (AMR) as one of the most critical health issues. It proposed, with the Food and Agriculture Organization (FAO) and OIE (World Organization for Animal Health), to address this by a One Health approach, recognizing the connection between humans, animals, and environmental health. Currently, a hypothesis is developing that cyanobacteria and cyanotoxins may contribute to AMR in water. Recent research appears to suggest: i) an impact of cyanotoxins on antibiotic-resistance gene transfer between bacteria; ii) a role of cyanobacteria as a reservoir of AMR. Finally, cyanotoxin production appears to be stimulated by cyanobacteria exposure to antibiotics. These findings strengthen the importance of considering the environment in its complexity.

Abstract EFSA requested the Panel on Plant Protection Products and their Residues (PPR Panel) to produce a Scientific Opinion on the application of physiologically based kinetic (PBK) modelling for the quantitative in vitro to in vivo extrapolation (QIVIVE) of data from the 17‐assay developmental neurotoxicity in vitro battery (DNT IVB) for pesticide active substances. PBK modelling‐supported QIVIVE is essential for the integration of in vitro data in hazard and risk assessment and may be conducted via forward dosimetry (estimating internal exposure from external exposure to a chemical) or reverse dosimetry (deriving an external exposure from an internal exposure). The request was accomplished via targeted expert discussions and EFSA‐internal and ‐external review. A scientifically robust QIVIVE requires accurate characterisation of two interrelated exposure metrics: the in vitro concentration eliciting a biological response (e.g. free or cellular concentration) and the corresponding in vivo internal concentration predicted by PBK modelling that reflects the same biologically relevant exposure. These metrics are influenced by chemical‐specific properties, assay design and physiological variability, requiring a case‐by‐case assessment of uncertainties. Each QIVIVE assessment should include, as a minimum, a low‐tier PBK model using conservative assumptions to avoid underestimation of internal exposure. Where sufficient kinetic data exist, higher tier models may be applied to enhance prediction accuracy. All modelling parameters, assumptions and validation steps must be transparently documented to facilitate regulatory appraisal. QIVIVE outcomes are to be documented in the overall weight of evidence for the DNT assessment. The PPR Panel identified the following three key uncertainty domains: (a) defining the appropriate in vitro exposure metric, (b) predicting internal exposure metrics via PBK modelling and (c) aligning PBK modelling‐derived internal exposure with the in vitro exposure. Addressing these uncertainties will strengthen regulatory confidence in using DNT IVB data for hazard and risk assessment of pesticide active substances.

Following the occurrence of Tetrodotoxins (TTXs) in Europe—a group of neurotoxins identified in Asia, where fatalities occurred after the ingestion of contaminated pufferfish—the EFSA proposed a limit of 44 µg of TTX/kg of shellfish meat in mollusks in 2017, to protect heavy consumers. The limit was based on an acute reference dose (ARfD) derived from the few available data on TTX toxicity. TTX is expected to increase with sea-surface warming; indeed, it has been found in spring/summer in mollusks in Europe, with concentrations often exceeding this limit. Due to the numerous uncertainties of the EFSA’s ARfD, we conducted a systematic review to provide an update on TTX toxicity. Out of 12,741 articles retrieved from PubMed, Science Direct, and Scopus since 2017, only 17 were eligible for data extraction. Our results show that they are not sufficient to modify the EFSA’s conclusions. Furthermore, our analysis of occurrence data in European seafood, to assess the current risk of exposure to TTX, reveals several gaps, such as different LODs/LOQs and seasonal monitoring not allowing comparisons between areas and too few analyzed sites. However, the presence of positive samples exceeding the EFSA limit indicates a potential risk even for general consumers, highlighting the urgency to address these knowledge gaps.

Context In vitro toxicity studies are increasingly being included as evidence in systematic reviews and chemical risk assessments. INVITES-IN, a tool for assessing the internal validity of in vitro studies, is under development in a process consisting of four consecutive studies. Study One in the creation of INVITES-IN was the development of an “item bank” database of 405 concepts (“items”) of potential relevance for assessing the internal validity of in vitro toxicity studies. The items were gathered from both focus group discussions and a purposive literature sample. In this paper we present the second study in the creation of INVITES-IN, i.e. the methods and results for identifying items for consideration when assessing the potential for bias in an in vitro study.Method A two-round digital Delphi survey, followed by online Delphi panel discussions guided by a moderator, was performed. The Delphi participants were experienced with both in vitro models and systematic review methods.Results Fifteen experts completed both Delphi rounds, and thirteen participated in a guided Delphi panel discussion. Of the 405 items in the bank, the experts agreed that 372 should be considered when assessing the potential for bias in an in vitro study. Items gathered from both the literature sample and the focus group discussions (Study One) were considered to be important for the assessment of the potential for bias in an in vitro study; 83–100% of the items collected from the literature sample were identified to be important and 91% (127) of the new items discovered in the focus group discussions of Study One were identified to be important.Discussion The 372 retained items will be interpreted into a manageable set of study appraisal criteria and a supporting guidance that will constitute the INVITES-IN study appraisal tool. In terms of lessons for tool development, the high retention of items included in tools designed for assessment of human and animal studies to in vitro studies suggests that many validity concepts are generally applicable across multiple study designs. Therefore, tool development processes should benefit from drawing on assessment tools outside the immediate domain of interest. Tool development would also likely benefit from supplementing literature reviews with focus group discussions, as our results demonstrate that the use of focus group discussions with domain experts was a pragmatic and valuable approach to increasing coverage of items in a tool development process.Conclusion In conclusion, this study demonstrates the value of using rigorous methods to ensure a comprehensive dataset as the starting point for creation of an assessment tool, though the direct application of Delphi methods to item banks may be an unnecessary step in tool development.

The margin of exposure (MOE) is a risk assessment tool used to evaluate the safety of substances in food and feed. Adopted by the European Food Safety Authority (EFSA) in 2005, the MOE is calculated as the ratio between a Reference Point (RP) and the estimated exposure. While some regulatory bodies use 'margin of safety' (MOS) interchangeably with MOE, others define it differently, leading to inconsistencies in interpretation. To address this, EFSA has standardised its terminology, establishing MOE as a primary metric for safety assessments across human and animal health evaluations. In addition, the meaning and interpretation of terms used to qualify a 'concern' is elaborated. The EFSA definitions will come into force from when this statement is published. By refining these definitions and ensuring consistent terminology across sectors, EFSA aims to improve clarity and transparency in its risk assessments, facilitating effective communication.

Essential Oils are innovative products of vegetable origin, whose use can find multifunctional applications in food supplements, aquaculture as a complementary feed, agriculture as plant protection products, topical disinfectants and wellness products. The aims of this study are the development of a multiresidue method to be used with essential oils, the determination of contaminant pesticide levels in twelve different products randomly taken from the Italian market and a preliminary human risk assessment. By using the developed method, fulfilling the EU criteria for pesticide control activity, we found that Citrus EOs showed the highest contamination among the investigated products. Inter-batches variability was also evident. Considering the oral and dermal exposure to different pesticide residues contaminating the analysed EO, based on combined exposure to multiple pesticides, and their hazard potential, no risk for consumers' health seems to be present. However, these preliminary findings underscore the importance of monitoring and quality control measures in the production and distribution of EOs to ensure consumer safety.

Cyanobacteria, often referred to as blue-green algae, are a diverse group of photosynthetic bacteria that play a crucial role in various ecosystems. However, their proliferation and toxin production pose significant challenges to environmental, animal, and human health. In recent years, the interplay between cyanobacteria, climate change, and the production of cyanotoxins has garnered increased attention. This short note aims to delve into the intricate relationship between cyanobacteria and the production of cyanotoxins, the toxicological risks associated with exposure, and strategies for risk assessment and management.

The focus on implementation of systematic review (SR) principles in chemical risk assessments (CRAs) is growing as it has the potential to advance the rigour and transparency of the CRAs. However, the SR and CRA communities use their own specific terminologies. Understanding the meaning of core SR and CRA terms and where they overlap is critical for application of SR methods and principles in CRAs. Moreover, it will increase the possibility for cross-sectorial collaboration, avoid misunderstandings, and improve communication among risk assessors, researchers, and policy makers. We present a process for the cross-mapping of core CRA terms and core SR terms. Core terms for study appraisal, evidence synthesis and integration used in the SR and CRA communities will be included. The outcome will be an overview of how core SR terms map onto core CRA terms and vice versa, and a description of the relationship and conceptual overlap between the terms. The cross-mapping is divided in three phases, where in the first phase the core SR and CRA terms will be identified. In the second phase, existing SR and CRA definitions will be mapped. In the third phase, descriptions of the relationship and conceptual overlap between the terms will be derived. The third phase will include weekly one-hour online meetings for SR and CRA experts.

Bisphenol A (BPA), a synthetic chemical widely used in the production of polycarbonate plastic and epoxy resins, has been associated with a variety of adverse effects in humans including metabolic, immunological, reproductive, and neurodevelopmental effects, raising concern about its health impact. In the EU, it has been classified as toxic to reproduction and as an endocrine disruptor and was thus included in the candidate list of substances of very high concern (SVHC). On this basis, its use has been banned or restricted in some products. As a consequence, industries turned to bisphenol alternatives, such as bisphenol S (BPS) and bisphenol F (BPF), which are now found in various consumer products, as well as in human matrices at a global scale. However, due to their toxicity, these two bisphenols are in the process of being regulated. Other BPA alternatives, whose potential toxicity remains largely unknown due to a knowledge gap, have also started to be used in manufacturing processes. The gradual restriction of the use of BPA underscores the importance of understanding the potential risks associated with its alternatives to avoid regrettable substitutions. This review aims to summarize the current knowledge on the potential hazards related to BPA alternatives prioritized by European Regulatory Agencies based on their regulatory relevance and selected to be studied under the European Partnership for the Assessment of Risks from Chemicals (PARC): BPE, BPAP, BPP, BPZ, BPS-MAE, and TCBPA. The focus is on data related to toxicokinetic, endocrine disruption, immunotoxicity, developmental neurotoxicity, and genotoxicity/carcinogenicity, which were considered the most relevant endpoints to assess the hazard related to those substances. The goal here is to identify the data gaps in BPA alternatives toxicology and hence formulate the future directions that will be taken in the frame of the PARC project, which seeks also to enhance chemical risk assessment methodologies using new approach methodologies (NAMs).

Context:In vitro toxicology studies are increasingly being included as evidence in systematic reviews and chemical risk assessments. INVITES-IN, a tool for assessing the internal validity of in vitro studies, is currently under development. The first step in developing INVITES-IN involves the creation of an "item bank," an overview of study assessment concepts that may be relevant to evaluating the internal validity of in vitro toxicology studies. The item bank and methodology for its creation presented in this manuscript are intended to be a general resource for supporting the development of appraisal tools for in vitro toxicology studies and potentially other study designs. Methods:We derived the item bank from seven literature sources (one existing item bank created from a systematic review of assessment criteria for in vitro studies, and six purposively sampled study appraisal tools) and the transcripts of three focus groups. Assessment criteria plausibly relating to internal validity were abstracted from the literature sources and focus group transcripts, disaggregated into individual criteria, then normalised to express in the simplest achievable language the core issue in each criterion - an "item bank" of assessment concepts. The items were then mapped onto a set of bias domains. We conducted simple descriptive statistical analyses and visualisations to describe patterns in the dataset and developed recommendations for the use and development of the item bank. Results:The item bank contains 405 items of potential relevance to evaluating the internal validity of in vitro toxicology studies. Discussion:To our knowledge, this is the second item bank of any kind to have been created for toxicology studies, and the first to use focus groups as a data source alongside literature analysis. The large number of items contributed by focus group discussions suggests this is an efficient method for capturing internal validity issues that are not easily identifiable in the literature. We believe our item bank and methodology for its creation will be a useful resource for supporting the development of appraisal tools. Due to the broad applicability of many items in the item bank, it may be informative for study designs beyond the in vitro domain.

The present report describes the work performed in the EFSA-project ‘Data collection, update and further development of biologically-based models for humans and animal species to support transparency in food and feed safety’. Here, Focus is given to case studies for food and feed chemicals to predict kinetic parameters and profiles using generic and substance-specific physiologically-based kinetic (PBK) models for humans, including human subgroups, laboratory animal species, farm animals and a kinetic-dynamic model in salmon. For humans, five case studies were conducted to compare kinetic predictions using the human generic PBK 6-compartment COSMOS/TKPlatewith i) in vivo data from human clinical or biomonitoring studies, ii) substance-specific model predictions using molecules relevant to food safety. Another five case studies assessed the impact of physiological variability (including pregnancy, renal excretion, metabolism variability, or ontogeny) and their impact on biomarkers of exposure. Case studies on laboratory and farm animals focused on theophylline, caffeine, cannabinoids, alkaloids and mycotoxins using the generic 11/12 PBK compartment models integrated in EFSA's TKPlate to assess predicted and experimental parameters i.e. plasma concentrations, excretion via milk or eggs. Overall, predictions from the human generic and substance-specific PBK models for parameters of chronic exposure were similar and robust compared to the available experimental data. For test species and farm animals, model predictions from the generic TKPlate PBK models also performed well and were mostly within 2-fold compared to available experimental in vivo data. In addition, 3D molecular modelling case studies were also conducted to investigate transport of chemicals (ochratoxin A, perfluoroalkyls) and cytochrome P450 metabolism (ochratoxin A, safrole and other alkenylbenzenes) as a useful tool to generate metabolism information at the molecular level. Conclusions and recommendations for future work are formulated to further develop generic PBK models for parent compounds and metabolites and further guidance to use and parameterise these models in next generation risk assessment.

Organophosphorothioates (OPT) are pesticides impacting human, animal and environmental health. They enter the environment worldwide, primarily due to their application as insecticides. OPTs are mainly neurotoxic upon bioactivation and inhibition of brain and serum acetylcholinesterase (AChE). Although OPTs are meant to target insects, they are potentially toxic to many other species (including humans), posing risks to non-target organisms and ecosystems. Certain cytochromes P450 (CYP) promote OPTs bioactivation, forming the corresponding oxon metabolites, while others catalyse their detoxification. Understanding the molecular basis of such a bivalent fate may help to clarify the toxicity of OPTs in living organisms, with far-reaching consequences to understand their impact on living organisms and improve risk assessment, to cite but a few. However, although crucial, the underpinning mechanisms still lay unclear. Here, a validated computational pipeline revealed the molecular reasons underlying the differential metabolism of chlorpyrifos in humans by CYP2C19, a primal route of detoxification, and its bioactivation by CYP2B6. The analysis drew the diverse occupancy of the CYP pocket and orientation to the heme group as a convincing evidence-based explanation for the opposite transformation. Moreover, this study explored the impact of CYP2C19 mutational landscape giving a blueprint to unveil the molecular basis of OPTs metabolism and toxicological implications from an inter-individual perspective. Taken together, the outcome described for the first time to the best of our knowledge a structural rationale for the bioactivation/detoxification of OPTs improving the current understanding of their toxicity from a molecular standpoint.

IntroductionCommunities affected by large scale and long lasting industrial contamination are often keen to understand whether their health has been impaired by such contamination. This requires answers that integrate environmental public health and environmental justice perspectives. At these sites, exposure scenarios from environmental contamination over time by multiple chemicals, often involving different environmental matrices, are complex and challenging to reconstruct.MethodsAn approach for describing the health of such communities in association with environmental contamination is presented, with the methods applied across the three domains of environmental contamination, population exposure and toxicology, environmental and social epidemiology, and environmental public health communication. The approach is described with examples from its application to the case study of Porto Torres, a town with a substantial industrially conditioned evolution.ResultsActivities in the field of environmental contamination, population exposure and toxicology focus on the collection and systematization of available contamination data, the identification of priority pollutants based on their toxicological profiles, the qualitative assessment of the likelihood of exposure for the population to priority pollutants and their known health effects. Environmental and social epidemiology methods are applied to describe the health profiles and socioeconomic conditions of the local population, taking into account multiple health outcomes from local information systems and considering specific diseases based on exposure and toxicological assessments. The environmental public health communication methods are directed to produce a communication plan and for its implementation through interaction with local institutional and social actors. The interpretation of health profiles benefits from a transdisciplinary analysis of the results.DiscussionThe proposed approach combines the needs of environmental public health and environmental justice allowing the integration of multidisciplinary knowledge to define recommendations for reducing and/or preventing hazardous environmental exposures and adverse health effects, stimulating the interactions between stakeholders, and making the study results more accessible to citizens.