Scientific Publications

Background

Uncertainty quantification is central to evidence synthesis for risk assessment and policy-making. Frequentist meta-analysis - currently predominant in epidemiology - quantifies uncertainty through confidence intervals, while Bayesian meta-analysis provides posterior distributions. We performed frequentist and Bayesian meta-analyses to compare uncertainty quantification and applicability for risk assessment using lead exposure and children’s IQ as a case study.

Methods

We systematically searched PubMed and Scopus for epidemiological studies on blood lead level (BLL) and IQ score in children. After screening, risk of bias (RoB) in the identified studies was assessed and regression coefficients were harmonised to the scale. In both meta-analyses, we included the same effect estimates, assumed a multilevel structure and used random-effects models. The Bayesian meta-analysis employed a hierarchical model with weakly-informative priors. Sensitivity analyses examined robustness to RoB and prior specification. and were estimated as measures of between-study heterogeneity.

Results

Twelve studies were included in the meta-analysis. The frequentist model yielded a pooled estimate of -2.45 (95% confidence interval: -3.82 to -1.08) IQ points per unit increase in -transformed BLL (µg/dL) and a 95% predictive interval of -6.53 to 1.53. The Bayesian model yielded a mean pooled estimate of -2.28 (95% credible interval: -3.54 to -1.04) and a 95% predictive interval of -6.28 to 1.69. Between-study heterogeneity was substantial (frequentist: = 3.32, = 85.0%; Bayesian: posterior median = 3.26, = 84.7%). The Bayesian approach provided a full posterior distribution for heterogeneity, directly quantifying uncertainty from between-study variation. Sensitivity analyses showed both meta-analyses were robust to RoB, and Bayesian estimates were insensitive to tested priors. Depending on the specified prior configuration, the Bayesian model was less sensitive to studies reporting substantial uncertainty, resulting in a lower pooled effect compared to the frequentist model. This regularisation is particularly valuable in small meta-analyses, where frequentist heterogeneity estimates can be unstable.

Conclusions

We provide updated pooled estimates for the effect of lead on children’s IQ. The Bayesian framework provides posterior distributions, enabling direct probability statements, e.g. about exceeding risk thresholds, and comparative assessments between pollutants. These features are particularly valuable for risk assessment and policy-making.

Occupational exposure limits (OELs) under occupational safety and health (OSH) legislation, and authorisation and restriction requirements under the chemicals regulation REACH, are the key regulatory tools for managing workplace chemical exposures in the EU. The present study examines how OELs and REACH requirements are interpreted and implemented at the workplace level, and how they impact workplace chemical safety. A mixed-methods study was conducted in Finland, Latvia, Norway, and 
Slovenia. An online survey targeted labor inspectors (n=85), and company OSH managers (n=323) in 3 sectors: chemical industry, metal industry, and motor vehicle maintenance and repair. In addition, semistructured interviews were conducted with labor inspectors (n=19) and OSH experts (n=30), including company and organization representatives and OSH service providers. Survey data were analyzed descriptively, and inductive content analysis was used for the interview data. Awareness of OELs 
and REACH requirements was reported as highly variable across workplaces in all participating countries, with larger companies higher in the supply chain demonstrating greater awareness and implementation capacity. Safety data sheets and chemical suppliers were the most frequently reported information sources. OELs were recognized as reference values for exposure and risk assessment, while REACH restrictions and authorisations were more often associated with discontinuation of substance use. 
Reported barriers to implementation included limited financial resources, insufficient competence in smaller companies, complex legislation, and weak supply chain communication. Positive examples included mandatory training obligations (eg diisocyanate restriction) and proactive inspection practices.

Thyroid hormones (THs) are essential for growth, development, and metabolism across vertebrates. Thyroid hormone system disruption (THSD), as one of the mechanisms of endocrine disruption (ED), is widely recognised as a critical concern for both human and environmental health. Important challenges remain in the availability and regulatory application of methods for THSD assessment. Here we discuss recent methodological advances in the area of THSD, including validation efforts for in vitro assays and THSD-sensitive endpoints as additions to existing Organisation for Economic Co-operation and Development (OECD) Test Guidelines using fish early-life stages. We propose a modular adverse outcome pathway (AOP) network-based framework integrating mechanistic and effect-level information to support regulatory hazard assessment for both human and environmental health under a One Health paradigm. The framework summarises essential elements of the THSD AOP network relevant to human and environmental health and methods were mapped to these mechanism and effect components. In a case study on resorcinol, a chemical of regulatory interest relevant for both human and environmental health, we demonstrate how emerging methods, including in vitro and fish eleutheroembryo (developmental stage between hatching and the onset of independent feeding) assays as new approach methods (NAMs), complement traditional data to strengthen weight-of-evidence for ED classification. By mapping gaps and priorities, this work advances the integration of NAMs into chemical safety assessment, supporting a future update of OECD Guidance Document 150. These efforts align with European Union initiatives to gradually phase out animal testing for chemical hazard assessment and implement Next Generation Risk Assessment.

Biocidal active substances are of considerable importance in protecting human and animal health against pathogenic or harmful biological agents that may enter in the food chain. Biocidal active substances are various in terms of their chemical structures and their applications (disinfectants, preservatives or pest control), while the same substance may be included in the formulation of different types of biocidal products. To date, the presence of biocidal residues in foodstuffs has been poorly documented. There is still a need to provide data on food contamination by biocidal products containing active substances in order to estimate consumer exposure 
and the associated risk. The aim of the current study was to propose a methodology for a risk ranking of biocidal active substances in the animal food chain using a structured and detailed approach. The established approach is qualitative and uses priority levels assigned to each biocidal active substance based (i) on their main potential sources of contamination of food of animal origin by biocidal products, (ii) the information on biocidal product regulation and (iii) the collection of existing knowledge on the substances hazards. Priority levels assigned for each criteria allowed to draw up a ranking list of 175 biocidal active substances to be screened in foodstuffs of animal origin. Most of the substances on this list (i.e. 117 substances) have been identified, on the basis of reported studies in the literature, as being analyzable by LC-MS and can therefore be investigated to provide new data on the presence of these substances in foodstuffs.

Effective early warning systems for aquatic contamination require monitoring strategies capable of detecting subtle, long-term shifts in mixture-driven biological activity. Suspended particulate matter (SPM) serves as a carrier and reservoir for complex contaminant mixtures, facilitating their transport and persistence in aquatic systems, yet systematic toxicological time series for archived SPM remain scarce. Regulatory monitoring predominantly targets Priority Substances and River Basin Specific Pollutants, leaving the temporal trends of particle-associated mixture toxicity largely unresolved. Leveraging 18 years (2005–2022) of cryogenically archived annual SPM composites from the Rhine River, we conducted a spatiotemporal effect-based assessment integrating receptor-mediated effects, oxidative stress analysis and untargeted Cell Painting phenomics. This integrated toolbox enabled evaluation of pathway-specific responses and multi-compartment cellular perturbations associated with particle-bound contaminant mixtures. Polar SPM-associated chemicals elicited oxidative stress response and caused endocrine disruption through estrogen receptor α (ERα) activation and androgen receptor inhibition (anti-AR). Trend analysis showed spatiotemporal variation along the river, with statistically increasing trends of oxidative stress and anti-AR activity over time at Koblenz, driven by polar chemicals. Both polar and non-polar SPM extracts activated the aryl hydrocarbon receptor (AhR), indicating presence of compounds capable of triggering xenobiotic response pathways. Several subcellular compartments were affected, with mitochondrial features being among the most affected. These findings demonstrate that SPM-associated chemicals elicit diverse toxicological effects by acting on several receptors and impacting diverse cellular structures. Combining targeted and phenomics-based effect approaches provided comprehensive mechanistic insights and valuable information to support the early warning systems for chemical contamination in aquatic environments.

The hazard identification of chemicals is a key step of the ‘Safe and Sustainable by Design’ (SSbD) framework introduced by the European Commission, aiming to eliminate hazardous substances early in innovation. In this context, in silico methods such as (Quantitative) Structure-Activity Relationship ((Q)SAR) models offer rapid, cost-effective, and animal-free alternatives for early-stage hazard screening. The Partnership for the Assessment of Risks from Chemicals (PARC) is developing a toolbox to facilitate SSbD assessments containing numerous (Q)SAR models. Challenges, however, exist in using and combining multiple in silico tools. Here, we developed a workflow to assess chemical hazards using multiple in silico tools within the PARC toolbox. The workflow consists of three phases: 1) the preparation stage, 2) running the models, and 3) the evaluation stage. To demonstrate the approach, we applied it to a case study comparing Bisphenol A, Isosorbide, and Bisphenol AP. Tools from the PARC toolbox were screened for relevance, transparency, and open access availability. Only models aligned with SSbD required endpoints and adequately documented via (Q)SAR Model Reporting Formats were retained. The properties assessed in this study cover carcinogenicity, germ cell mutagenicity, reproductive toxicity, endocrine disruption, persistence, bioaccumulation, and aquatic toxicity. Predictions were filtered using applicability domain criteria and reliability scores. Next, three strategies were applied for integrating different model outputs. Model agreement varied across endpoints and integration methods. This emphasizes the possibility of different SSbD assessment outcomes and thus the need for transparent documentation of the chosen strategy and explicit handling of uncertainty. Our study demonstrates how multiple models can systematically and transparently be integrated via the developed workflow. Key areas for improvement are to refine integration strategies, harmonize the definition and communication of applicability domains across tools, expand in silico coverage for currently underrepresented endpoints, and to develop approaches to consider data gaps in SSbD assessments.

One challenge for current chemical risk assessment is the integration of different in vitro based new approach methodologies (NAMs) to establish a concept for Next-Generation Risk Assessment (NGRA). It is particularly difficult to assess activities of chemicals associated with carcinogenicity that is not mediated by direct effects on genome integrity, known as non-genotoxic carcinogens (NGTxC). NGTxC activities may be induced through various mechanisms and cellular pathways. Thus, it is important to combine readouts, reflecting distinct mechanisms in a single test system, to limit the total number of tests needed. In this study, the previously described E-Morph screening assay (ESA), based on human MCF-7 breast cancer cells, for detection of ER-agonistic chemicals, was expanded for a simultaneous detection of DNA damage, to facilitate the distinction of genotoxic and non-genotoxic nuclear-receptor mediated carcinogens. For assessing the assay performance, we assembled a chemical library including chemicals with genotoxic or NGTxC mode of action and monitor their ER-agonistic and genotoxic activity after application of six different concentrations starting from 100 µM. The same set of chemicals was tested in a human liver cell line (HepG2) with the γH2AX/pH3 genotoxicity method. We observed a strong correlation between the two cell lines for the detection of genotoxic effects. Additionally, we observed a strong correlation between γH2AX induction and ER-agonistic activity in MCF-7 cells and aneugenicity in HepG2 cells. Consequently, the extended ESA is a promising new NAM that might play an important role in future NGRA for identifying and characterizing carcinogenic hazards.

Zebrafish behavioral profiling is increasingly used in environmentally oriented New Approach Methodologies (NAMs) for chemical hazard assessment, including neuroactive chemicals. However, interpreting acoustic/vibrational startle habituation requires understanding how different quantification strategies influence toxicological readouts. In this study, we performed a controlled cross-platform comparison of two common habituation metrics in 5-dpf zebrafish eleutheroembryos following acute chemical exposure. Using embryos from the same spawning and shared stock solutions, we evaluated: (i) a centroid-based workflow (30 fps), quantifying poststimulus displacement as area under the curve (AUC), and (ii) a high-speed kinematic workflow (1000 fps), classifying individual startle events to calculat%habituation. Eight neuroactive reference chemicals were tested to benchmark both approaches and to improve interpretation of an environmentally relevant neurobehavioral screening endpoint. The high-speed kinematic workflow showed high directional concordance with expected modulation patterns for 6/8 compounds and provided detailed resolution of C-start mechanics. Meanwhile, the centroid-based workflow, a robust and widely accessible standard in the field, reproduced expected directions for 3/8 compounds, capturing robust modulations for specific neuroactive profiles while showing lower sensitivity to other chemicals, such as ketamine, despite their marked effect on habituation kinetics. Notably, results for compounds such as nimodipine showed that both assays are essential for identifying complete loss of the motor response, thereby avoiding misinterpretation of performance deficits as altered plasticity. Overall, our findings show that while both platforms capture behavioral shifts, readout choice substantially influences the sensitivity and interpretation of chemical effects. This study supports fit-for-purpose endpoint selection, strengthening environmental hazard assessment and prioritization.

The replacement, reduction, and refinement (3Rs) of animal experiments is a central objective in modern toxicology. Human cell-based in vitro assays have become key tools to implement these principles by providing mechanistically driven and human-relevant New Approach Methodologies (NAMs) for toxicity testing. However, even in approaches that avoid the use of living animals, many protocols still rely on animal-derived cell culture components beyond fetal bovine serum (FBS), such as bovine serum albumin (BSA) in supplements, extracellular matrix (ECM) preparations such as Matrigel, and animal-sourced antibodies, which may introduce variability. This review provides a targeted materials analysis of human cell-based assays within the Developmental Neurotoxicity in vitro Battery (DNT-IVB), examining basal media, supplements, ECM, growth factors, and antibodies. While serum-free media are widely implemented, animalderived components remain in use, particularly in supplements, ECM, and immunodetection workflows. Guidance documents, including OECD Good in vitro Method Practices (GIVIMP), or Good Cell Culture Practice (GCCP), promote replacing undefined components such as FBS, but ECM and BSA are less explicitly addressed. Recommendations from the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) encourage the use of animal-free antibodies where feasible. We propose a two-pronged strategy: (1) New protocols incorporate animal-free design from inception; (2) established DNT-IVB assays evaluate component reduction where feasible, balancing validation requirements with practicality. Manufacturers can contribute through standardized labeling (“serum-free,” “animal-free”, “xeno-free,” “chemically defined”) and expanded animal-free product availability. Updated GCCP/ GIVIMP guidance could explicitly address BSA and ECM as sources of variability alongside serum. By addressing persistent animal-derived reagents, this reagents-focused review advances animal-free DNT-IVB implementation and supports broader 3Rs objectives by providing actionable strategies for animal-free cell culture in human-relevant NAMs.

The current testing strategy for the assessment of developmental immunotoxicity (DIT) in European chemicals regulations has well recognised limitations. In response to these limitations, the Partnership for the Assessment of Risks from Chemicals (PARC) initiated a 4-year DIT project in May 2025. This project comprises 14 partner institutions and will tackle two primary objectives: a) to enhance the DIT knowledgebase and refine the understanding of critical phases of immune system development, and b) to facilitate the transition towards the use of New Approach Methodologies (NAMs) in DIT risk assessment. The first objective will be approached by reviews of existing literature and the continued advancement of a physiological map of human immune system development. The reviews and the physiological map will in turn serve as foundational tools to support NAMs and Adverse Outcome Pathway (AOP) development. Addressing the second objective, the project has a short-term aim to promote a testing strategy that leverages current immunotoxicity assays and their modifications to inform regulatory decision processes such as screening, prioritisation and read-across analyses. In the longer term, novel NAMs, encompassing developmental processes, will be developed and assessed for their regulatory applicability. While the primary focus of this PARC project is on the enhancement of human DIT risk assessment, it also aims to contribute to the advancement of ecotoxicological evaluation of immunotoxicity.

This study demonstrates the efficiency and relevance of a direct-injection LC-HRMS approach for the characterization of chemical contamination in distilled spirit beverages. Sixty-six spirit samples were analyzed using a UHPLC-Q/ToF workflow without extraction or pre-concentration, combining targeted analysis (TA) and suspect screening (SS). Despite its analytical simplicity, strong performances were achieved at very low concentration levels. Detection rates reached up to 61% and 84% of the investigated contaminants at 0.1 ng mL⁻¹ and 1 ng mL⁻¹, respectively, depending on the type of spirit. Screening detection limits (SDLs) were established for 58% of the contaminants at a concentration ≤1 ng mL⁻¹. The nature of the sample, particularly aged versus non-aged spirits, was shown to significantly influence analytical performance. TA enabled the monitoring of 960 compounds, leading to the identification of 18 chemicals, mainly pesticide residues, including substances not previously reported in spirits. A risk characterization indicated no health concern for EU adult consumers regarding these 18 compounds. SS finally expanded the chemical scope by annotating 17 additional compounds, some of them being toxic such as pyrogallol. Overall, LC-HRMS stands out as an efficient early-warning tool to improve monitoring of contaminants in beverages, and could be easily used in complement to traditional LC-MS/MS systems to investigate emerging or unprioritized chemicals.

In response to increasing evidence of human and environmental health impacts of endocrine disrupting chemicals (EDCs), screening and testing programs for EDC assessment are being developed, requiring characterization of potential adverse health effects. The development of the lateral line (LL), a neurosensory system in fish, has been suggested as a potential toxicological target of EDCs. The LL contains neuromasts with hair cells (HCs) which convert mechanical stimuli intoneural signals. An exploratory study was performed to assess whether neuromast development is affected by a selection of model EDCs with different modes of action. Zebrafish embryos were exposed to EDCs, targeting estrogen and thyroid pathways, immediately after fertilization. The number of HCs in four neuromasts was counted at 120 h post fertilization. Methimazole and resorcinol (thyroid hormone [TH] synthesis inhibitors) elicited the strongest response, characterized by a reduction in HC numbers, while fulvestrant (anti-estrogen) slightly increased HC numbers. Further investigation confirmed a reduction of HCs and neuromasts after exposure to methimazole during late embryonic development, when TH synthesis is active. Gene transcript level analysis revealed a decreased marker for HC activity and increased markers of support cells, essential for HC regeneration. Taken together, neuromast development appears to be affected by certain EDCs in zebrafish embryos, at concentrations similar to those causing other effects (e.g., impaired swim bladder inflation). However, variability in the responses complicates characterization using the FM1-43 method. Further research, including rescue experiments and more sensitive or functionally relevant methods, is needed to clarify the mechanisms underlying EDC-induced HC disruption.

Enniatins (ENNs), such as ENN A, A1, B, B1, and Beauvericin (BEA) are mycotoxins produced by Fusarium fungi, mainly found in cereal grains and their products. The concern for their potential endocrine disrupting activity is increasing; however, the effects of these compounds on steroidogenesis were never investigated simultaneously, especially by using a validated human in vitro method, which rep resents a gap in their risk assessment. In the frame of the European Partnership for the Assessment of Risks from Chemicals (PARC), we performed the H295R Steroidogenesis Assay (OECD TG 456), to assess effects of ENN A, ENN A1, ENN B, ENN B1 and BEA on steroid hormone synthesis. Cytotoxicity was firstlyevaluated (10 pM– 10 μM); then, conditioned medium of H295R cells, treated with non-cytotoxic concentrations of the studied com pounds, was analysed for the level of 17β-estradiol (E2) and testosterone (T) by ELISA assays. In addition, these same hormones as well as progesterone, aldosterone and cortisol were also analysed by HPLC-MS/ MS. ENN A and A1 were the more cytotoxic compounds, while ENN B was the less toxic. The mycotoxins differently affected hormone synthesis at the highest non-toxic concentrations; in particular, ENN B significantly increased E2 levels whereas ENN A1 induced T synthesis. Progesterone was significantly induced by ENN A1 and BEA but was reduced by ENN B1. Aldosterone levels were increased by ENN A, B1 and BEA, whereas cortisol was induced by ENN B and reduced by ENN A1 and BEA. Overall, the results highlighted different modes of action of these natural toxins, contributing to fill data gaps on their endocrine-disrupting activity.

Obesity is a global pandemic that affects people of all ages. While behavioral, nutritional, and socioeconomic factors all contribute to weight gain, other factors in our immediate environment play an insidious role in the prevalence of obesity. Exposure to environmental and pharmaceutical compounds can be a contributing factor to increased weight gain. Using the zebrafish obesogenic test, our study demonstrates that amiodarone, dibutyl phthalate, rosiglitazone, tributyltin, and triclosan induce a thrifty phenotype under short-term fasting conditions. Diazepam significantly reduces locomotion without exhibiting any obesogenic effect, whereas tributyltin, which has the highest obesogenic potential among the tested compounds, has no effect on locomotion. The obesogen-induced resistance to fat loss is not correlated to inhibition of physical activity and a corresponding reduction in energy expenditure nor to food consumption. Primary prevention measures to fight against the obesity pandemic may include reducing exposure to obesogens that can induce a thrifty phenotype.

The Partnership for the Assessment of Risks from Chemicals (PARC) represents a joint effort among risk assessors, regulatory authorities, and the scientific community to advance the implementation of the Next Generation Risk Assessment (NGRA) in line with the objectives of the EU Chemicals Strategy for Sustainability. Addressing the challenges faced by national and European regulators in integrating data generated by innovative methodologies is central to achieving this goal.

Following an initial phase of the Partnership, in which projects were defined based on a first prioritization of methodologies, a second prioritization round was conducted with input from the Governing Board representatives of all participating entities. This second process also considered the Key Areas of Regulatory Challenge introduced by ECHA in 2023, ensuring that the evolving research agenda within PARC is closely aligned with current and future regulatory needs.

As a result, WP5 Hazard Assessment has updated its project portfolio to include four new projects that bridge identified regulatory gaps and strengthen the implementation phase of PARC. Two projects address newly prioritized endpoints, Developmental and Reproductive Toxicity and Developmental Immunotoxicity, while a third explores Sexual Dimorphism Associated with Hepatotoxicity. In addition, a transversal project, Regulatory Readiness of NAMs, was launched to accelerate the regulatory uptake of promising methods developed under WP5.

This article complements the previous PARC special issue by providing an overview of the updated WP5 project portfolio, illustrating the progression from prioritization to implementation, and highlighting how these new projects respond to evolving regulatory needs and contribute to the effective integration of NAMs into chemical risk assessment.

Research findings suggest that advanced paternal age is associated with an increased risk of autism spectrum disorder (ASD) in children. The biological process behind this father-to-child inheritance of a disease may be driven by sperm epigenetic marks. This has been suggested earlier, but the identification of epigenomic regions responsible for these age-related responses have not been further elaborated. To identify sperm-specific marks, we conducted an epigenome-wide association study in sperm from 63 men, using the Illumina 450K array. Linear regression modeling was applied to identify differentially methylated CpGs (DMCs) by age; we controlled for body mass index, patient status, and multiple testing. We found 14,622 statistically significant age-related DMCs; most (69%) were inversely correlated. We identified 95 imprinted genes and emphasized 747 age-related DMCs adjacent to an imprint control region (ICR). Altered methylation patterns in ICRs may result in disturbed expression of imprinted genes and are suspected to be at the origin of several diseases in offspring, including neurodevelopmental disorders. Mapping our results to other databases revealed the following set of imprinted genes linked to ASD: OTX1, PRDM16, PTPRN2, B4GALNT4, KCNQ1, KCNQ1OT1, DLGAP2, PLAGL1, GNAS, GRB10, MAGEL2, CDH24, and FBRSL1. Further research on these genes could help understand the contribution of paternal age on the development of autism. A change in DNA methylation levels in ICRs before conception may contribute to the heterogeneity and complexity of ASD. Measured DNA methylation effect sizes were subtle, but small epigenetic disturbances in sperm may be important on a population level, especially if men continue delaying fatherhood. Public health would benefit from the development of preventive and educational programs.

The acoustic startle response (ASR) is a conserved sensorimotor reflex widely used to investigate neural plasticity, sensorimotor gating, and neurotoxicity. While zebrafish is an established vertebrate model for ASR analysis, most existing platforms were originally optimized for 6 dpf larvae, which constrains applications requiring reliable assessment of earlier developmental stages. Here, we introduce Zebra_K⁺, a modular extension of the previously developed Zebra_K platform, designed for high-throughput kinematic analysis of ASR in zebrafish embryos (5 days post-fertilization, dpf) and early larvae (6–7 dpf). The system enables simultaneous quantification of ASR kinematics, sensitivity, short-term habituation, and prepulse inhibition (PPI) in up to 25 individuals. Using the NMDA receptor antagonist ketamine, the dopamine receptor agonist apomorphine, and the D₂ receptor antagonist haloperidol, we validated the platform’s ability to detect pharmacologically induced and developmentally specific alterations in startle plasticity. Ketamine reduced habituation and PPI at all developmental stages, whereas apomorphine selectively impaired PPI, an effect that was reversed by haloperidol only at 7 dpf. These results demonstrate the neurodevelopmental progression of glutamatergic and dopaminergic modulation of sensorimotor gating and establish Zebra_K⁺ as a modular technological platform that supports the development of New Approach Methods (NAMs) for neurotoxicological screening and developmental neuropharmacology.

Phospholipidosis (PLD) is the excessive accumulation of phospholipids within cells, which is, for example, observed in the liver following exposure to certain chemicals or drugs. Although the precise molecular mechanisms underlying PLD remain incompletely elucidated, it is documented that PLD is frequently induced by cationic amphiphilic compounds. To facilitate the identification of potential PLD inducers across diverse groups of chemicals, the development of novel high-throughput screening methodologies is imperative. Here, an in vitro human liver cell culture system was established for PLD screening, employing the fluorescence-based LipidTox assay in differentiated HepaRG cells in a 96-well format. Cells were treated with a selection of 35 compounds characterized as either PLD inducers or non-inducers, based on 
previously published in vivo data. Concentration-response curves of phospholipid accumulation were assessed and the results were compared with existing in vivo, in vitro and in silico datasets. The findings confirm the applicability of the LipidTOX assay in differentiated HepaRG cells as a reliable in vitro-based high-throughput screening method for PLD prediction, achieving an overall predictivity of 86%.

A recent study has suggested that plastics may contain more than 16,000 chemicals, including additives, processing aids, starting substances, intermediates and Non-Intentionally Added Substances. Plastic chemicals are released throughout the plastic life cycle, from production, use, disposal and recycling. Most of these chemicals have not been studied for potential hazardous properties for humans and in the environment. To refine the risk assessment of these leachable chemicals, additional hazard data are needed. The PlasticLeach project within the EU co-funded Partnership for the Assessment of Risks from Chemicals (PARC) aims to address this data gap by screening several plastic products in daily use. Leachates will be prepared from a number of these plastic items, and these chemical mixtures will be further tested using several test guideline compliant assays and New Approach Methodologies covering both human health and environmental endpoints. The most toxic leachates will be characterized using a non-targeted analysis pipeline to identify chemicals in the leachate. When single chemicals of concern are identified, these will be further tested to determine hazardous properties and identify the respective potency factors to better understand their specific hazard profiles. A tiered approach for hazard testing will be followed. The experimental work will be complemented by in silico toxicological profiling, using publicly available toxicity databases and tools, including Artificial Intelligence tools that cover both human and environmental endpoints. A comprehensive array of endpoints, including cytotoxicity, endocrine disruption, genotoxicity, immunotoxicity, reproductive toxicity and effects related to ecotoxicity will be evaluated. In this paper, we outline the plastic products to be tested and the battery of assays that will be used to identify hazards relevant to both human health and the environment. Data generated from in silico, in vitro, and in vivo approaches will be reported using standardized formats, stored within a centralized repository, and harmonized to adhere to the FAIR data principles (Findable, Accessible, Interoperable, and Reusable). This integrated strategy will not only advance our understanding of the risks associated with plastic-derived chemicals but will also provide critical support for regulatory decision-making and facilitate the development of safer, and more ecofriendly plastic materials in the future.

The European Commission’s Safe and Sustainable by Design (EC SSbD) Framework aims to put together safety and sustainability considerations throughout the entire chemical and material innovation processes. Being a voluntary (pre-market) approach, the framework fosters the development of safe and sustainable chemicals, materials, processes, and products while drawing on existing legal frameworks. We explore the relationship between the EC SSbD Framework and current European legislation regarding safety and sustainability. We highlight commonalities and differences to deduce synergies, and identify opportunities for mutual support and benefit. By systematically evaluating each step of assessing safety and sustainability criteria, indicators, and elements in the EC SSbD Framework, we demonstrate how information generated during the innovation process can also support legal compliance while driving pro-active design improvements. Vice versa, we investigate how regulatory data and methodologies can inform SSbD assessment steps, ensuring a reciprocal flow of information between innovation and compliance efforts. Despite notable differences identified, our findings demonstrate that the voluntary EC SSbD Framework has an added value, and it fosters synergies between innovation of chemicals and materials and safety and sustainability provisions of relevant legislation.

With global restrictions on Bisphenol A (BPA), various BPA alternatives are increasingly found in ecosystems, raising concerns. This study focuses on the genotoxic potential of three emerging BPA alternatives — Bisphenol AF (BPAF), Bisphenol AP (BPAP), and Bisphenol PH (BPPH) — using an advanced in vitro 3D model system, spheroids, prepared from a Zebrafish (Danio rerio) liver cell line (ZFL). Their cytotoxicity was evaluated using the CellTiter-Glo® 2.0 assay, while their genotoxic potential was assessed using the comet assay, γH2AX assay, and toxicogenomic analysis. The BPA alternatives were more cytotoxic to ZFL spheroids than BPA. Non-cytotoxic concentrations caused transient DNA damage without a significant increase in DNA double-strand breaks (DSBs). The toxicogenomic analysis confirmed these findings, indicating activation of the TP53 DNA damage response pathway, the nucleotide excision repair (NER) and base excision repair (BER) mechanisms, likely in response to bulky DNA lesions and oxidative DNA damage. In addition, the gene expression analysis indicated the influence of the tested BPs on the endocrine system. Our results indicate that BPAF, BPAP and BPPH have considerable genotoxic potential and pose a significant ecotoxicological risk, underscoring the need for further investigation and careful consideration of these chemicals as BPA replacements. 

Background

Regular monitoring of the air pollutant nitrogen dioxide (NO2), an indicator for traffic-related emissions, is a priority in urban environments. The health impacts associated with NO2 exposure are the result of a combination of factors, including concentration, duration of exposure, and interactions with other pollutants. WHO has established air quality guidelines based on epidemiological studies.

Objective

This study develops a new concept "Health Impact Pathways (HIPs)" using adversity as a probabilistic indicator of health effects. For this purpose, it integrates available toxicological and epidemiological information, using Adverse Outcome Pathways (AOPs), in order to understand chemical-biological interactions and their consequences on health.

Methods

Literature review and meta-analysis of toxicological data supported by expert judgment were performed to establish: a) adversity pathways, b) quantitative criteria for scoring the observed toxicological effects (adversity indicators), c) NO2 exposure - adversity relationship for both long-term (1–36 months) and shortterm (1–7 days). The NO2 daily concentrations from January 2001 to December 2022, were obtained from Madrid city Air Quality network monitoring database. Adversity levels were compared with relative risk levels for all-cause and respiratory mortality estimated using linear equations from WHO 2021 guidelines.

Results

Non-linear relations were obtained for all long- and short-term NO2 related adversity indicators; for long-term effects, the best fitting was obtained with a modified Haber's law model with an exponential coefficient for the exposure time of 0.25. Estimations are presented for a set of case studies for Madrid city, covering temporal and spatial variability. A clear improvement trend along the two decades was observed, as well as high inter- and intra-station variability; the adversity indicators provided integrated information on the temporal and spatial evolution of population level risk.

Discussion

The proposed HIP conceptual approach offers promising advances for integrating experimental and epidemiological data. The next step is linking the concentration-adversity relationship with population health impacts through probability estimations, the preliminary estimations confirm the need for assessing independently different population groups.

Bisphenol A (BPA) is a well-known endocrine disruptor linked to numerous adverse health outcomes and was, therefore, banned in food-contact materials in the European Union. Numerous alternatives are now in commerce, but their health hazards are often inadequately addressed. This study compared BPA and 26 alternatives in six in vitro bioassays for cytotoxicity, endocrine disruption, xenobiotic metabolism, adaptive stress responses, mitochondrial toxicity, and neurotoxicity. We developed a cumulative specificity ratio score that integrates the degree of specific activation and overall toxicological activity across a test battery, enabling direct comparison of BPA with its alternatives. Several alternatives with close structural resemblance showed similar or stronger activation of the estrogen receptor α (ERα) than BPA. The lack of estrogenicity for several BPA alternatives, e.g., 4-(4-phenylmethoxyphenyl)sulfonylphenol (BPS-MPE), was accompanied by a shift toward peroxisome proliferator-activated receptor γ (PPARγ) activation, a receptor that is not relevant for BPA itself. Some alternatives additionally inhibited mitochondrial functions and caused neurotoxicity. Simulated phase I metabolism reduced the cytotoxicity of all alternatives except for methyl bis(4-hydroxyphenyl)acetate (Bz) and 4-[[4-(allyloxy)phenyl]sulfonyl]phenol (BPS-MAE), while estrogenic activity remained unchanged or decreased. This study demonstrates the utility of bioassays for rapid hazard assessment and comparative evaluation, suggesting that many BPA alternatives are regrettable substitutes, although 2,2,4,4-tetramethyl-1,3-cyclobutanediol (TMCD) is a potentially more benign alternative.

This article reviews the application of multicriteria decision analysis (MCDA) in chemical alternatives assessment (CAA) and presents an overview of how the methodology has been applied within CAA. The study aimed to identify research that uses MCDA to identify the most harmful or least problematic chemicals and evaluate its current use in CAA. The study supports the Partnership for the Assessment of Risks from Chemicals (PARC) in developing a toolbox for safe and sustainable by design (SSbD). 520 studies were analysed, and 21 studies were included. Although MCDA in CAA is still emerging, it shows growth potential in decision analysis and chemical alternatives assessment. The reviewed studies cover various CAA applications and methodological approaches. Multiattribute utility theory (MAUT) is the most often used, followed by Technique for Order Preference by Similarity to Ideal Solution (TOPSIS), ÉLimination Et Choix Traduisant la REalité (ELECTRE), and analytic hierarchy process (AHP). Experimental data and in silico data have been used with roughly equal frequency as input data. Group decision-making involving stakeholders with conflicting interests is rarely addressed, with parameter weighting and problem structuring usually handled by authors, sometimes with expert input. Another little discussed topic is the use of external normalisation of input data. In silico generated predictions on chemical alternatives’ properties come with varying degrees of uncertainty, remaining an issue in CAA with MCDA.

Bisphenol A (BPA) is an endocrine-disrupting chemical with estrogen-like activity, known to impair immune function. BPA may act as a pro-inflammatory agent, reducing immune response efficacy, increasing bacterial load in E. coli infections, and altering immune responses in parasitic infections (Leishmania major, Nippostrongylus brasiliensis, Toxocara canis) through cytokine and regulatory T-cell modulation. Following its ban in food contact materials in Europe, several analogs have been introduced. This study assessed the immunotoxicity of BPA and six analogs, namely BPAP, BPE, BPP, BPS-MAE, BPZ, and TCBPA, by evaluating in vitro the antibody production. Peripheral blood mononuclear cells from healthy male and female donors were exposed to increasing concentrations of each compound for 24 h. After stimulation with rhIL-2 and ODN2006, IgM and IgG secretion were measured on day six. All compounds suppressed antibody production in a concentration-dependent manner, with some sex-related differences. IC₅₀ values showed BPP as the most potent suppressor, and BPE as the weakest. Similarly, IC₂₀ values confirmed these differences in potency, except for BPA being the weakest for IgM in males. Overall, te results do not support the idea that BPA analogs are safer than BPA.