Scientific Publications

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.

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. 

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.

Human biomonitoring of persistent organic pollutants (POPs) remains essential for tracking long-term exposure, evaluating health risks, and assessing the effectiveness of regulatory bans. For this purpose, an experimental and analytical methodology has been optimized allowing the determination of 47 POPs, encompassing chlorinated cyclodienes, chlorobenzenes, cyclohexanes, polychlorodiphenyl derivatives, several congeners of polychlorobiphenyls (PCBs) and polybromodiphenyl ethers (PBDEs, including the deca-BDE), as well as three non-persistent chemicals (namely quintozene, tecnazene and vinclozolin). The method uses a single liquid-liquid extraction procedure with 500 µL of serum/plasma. Instrumental analysis involves gas chromatography (GC) with electron capture detection (ECD) for the analysis of organochlorines, and a subsequent injection into a GC coupled to mass spectrometry in negative chemical ionization mode (MS-NCI) for the determination of PBDEs. The analytical procedure has been successfully validated, demonstrating high sensitivity (limits of detection: 0.0016–0.012 ng/mL; limits of quantification: 0.0029–0.021 ng/mL), acceptable linearity (R² > 0.9962), good precision (coefficients of variation <25%, except for BDE-209), satisfactory accuracy (recoveries within 70–130% for 41 out of 47 compounds) and minimal matrix effects. Proficiency Testing Materials from the AMAP Ring Test for POPs in human serum confirmed the method’s reliability with results within the accepted reference range. This method represents an efficient and practical approach for human biomonitoring studies, with a simplified workflow suitable for routine application.

Chemical risk assessment can benefit from integrating informative biomarkers in human biomonitoring (HBM). Beyond exposure biomarkers, effect biomarkers inform on biological reactions in the body, potentially leading to adverse effects, while susceptibility biomarkers address inter-individual variability in exposure. DNA methylation of key genes shows promise as an effect biomarker but this epigenetic mark remains underexplored in the context of chemicals. Similarly, although some genetic polymorphisms are linked to increased chemical susceptibility, genetic biomarkers are rarely included in HBM. This mini-review highlights recent literature supporting the inclusion of genetic and epigenetic biomarkers in HBM. Subsequently, we elaborate on how Oxford Nanopore Technologies as sequencing method can efficiently measure these biomarkers simultaneously, even in non-invasive samples like saliva. Widely used in other fields, this experimental set-up could facilitate the design of large-population studies paving the way for a next generation risk assessment (NGRA) of chemicals.

Shinyscreen is an R package and Shiny-based web application designed for the exploration, visualization, and quality assessment of raw data from high resolution mass spectrometry instruments. Its versatile list-based approach supports the curation of data starting from either known or “suspected” compounds (compound list-based screening) or detected masses (mass list-based screening), making it adaptable to diverse analytical needs (target, suspect or non-target screening). Shinyscreen can be operated in multiple modes, including as an R package, an interactive command-line tool, a self-documented web GUI, or a network-deployable service. Shinyscreen has been applied in environmental research, database enrichment, and educational initiatives, showcasing its broad utility. Shinyscreen is available in GitLab (https://gitlab.com/uniluxembourg/lcsb/eci/shinyscreen ↗) under the Apache License 2.0. The repository contains detailed instructions for deployment and use. Additionally, a pre-configured Docker image, designed for seamless installation and operation is available, with instructions also provided in the main repository.

Children’s lead exposure in three mining-impacted residential areas (Črna, Mežica and Žerjav) was modelled using the Integrated Exposure Uptake Biokinetic Model for Lead in Children (IEUBK). Site-specific environmental and dietary Pb source values were determined for modelling. For the first time, dietary exposure from both market and local foods was studied in detail. Children (Group 1: 24–36 and Group 2: 36–48 months) geometric mean blood lead levels (BLLs) were predicted and lead uptake from multiple sources was quantified according to the different dietary exposure scenarios. Biomonitoring data were used for validation. Site-specific soil, house dust and local food Pb contents are higher than legislative and background levels, remaining a cause for concern. Drinking tap water concentrations and outdoor air contents were found in acceptable levels. The determined dietary exposures, ranging from 0.7 to 3.3 µg/kg bw/day, were above the benchmark dose level of 0.5 µg/kg bw/day for developmental neurotoxicity set for Pb in children, indicating a health concern. In general, the estimated BLLs matched reasonably well with the observed BLLs in the Črna and Mežica area for both age groups and in the Žerjav area for Group 2. For Group 1, in the Žerjav area, the output of the IEUBK model overestimated the actual BLLs of the children. For both groups, the primary exposure pathway in Žerjav is from soil/dust, ranging from 55.3 to 84.8%. In Črna and Mežica, soil/dust exposure ranged between 24.2 and 57.8% and between 26.4 and 61.7%, respectively, indicating that dietary exposure predominates when local foods are included. The results of our study also suggest that using the IEUBK default diet value would reduce the dietary exposure up to 25.2% in Črna, 24.2% in Mežica and 8.6% in Žerjav. One of the main findings is that a diet containing local foods can be an important source of lead in mining-contaminated areas.

This study investigates the immunomodulatory effects of BPA substitutes using in silico and in vitro approaches. A comprehensive in silico screening with Endocrine Disruptome of BPA and its 25 substitutes revealed that BPG, BPS-MAE, BPS-MPE, BPPH and PF201 have the highest potential to bind nuclear receptors. Based on the in silico ranking, 12 compounds were selected and tested in vitro to investigate their effects on THP-1 derived macrophages, Jurkat T cells and LCLs. The metabolic activity tests showed that BPA has IC₅₀ values of 99-182 µM, while the values for BPG, BPPH and BPP are 5-10 times lower. Comparison of the IC₅₀ values with the logP of the tested compounds showed that the BPA analogues affect cell viability in proportion to their lipophilicity (R² = 0.9185). The results of the cytokine release assays showed that the BPA substitutes generally stimulated the proinflammatory response in THP-1 macrophages in a statistically significant manner when exposed to the environmentally relevant concentrations and suppressed it in the micromolar range in all cell lines tested. Further analysis using RamosBlue reporter cells indicated that BPAP, BPG, BPP and BPPH alter the NF-κB/AP-1 signalling pathway. Moreover, BPAP, BPG, BPP, BPPH, BPS-MAE, BPS-MPE, BTUM and PF201 were predicted in silico to be TLR4-MyD88 inhibitors. The overall results of this study identify BPG, BPP, BPPH, BPZ and TCBPA as BPA analogues with the highest immunomodulatory potential. In addition, the immunomodulatory potential of the BPA alternatives Pergafast201 and BTUM was confirmed in vitro for the first time. This study emphasises the need for cautious evaluation of BPA substitutes due to their potential immunomodulatory effect, which could significantly affect public health.

New Approach Methodologies (NAMs) in Next-Generation Risk Assessment (NGRA), integrating toxicokinetics (TK) with toxicodynamics (TD), provides an accurate evaluation of combined chemical exposures. This study assesses pyrethroids, which pose regulatory challenges due to their widespread use and cumulative exposure risks. A tiered NGRA framework was compared with conventional risk assessment (RA) to evaluate regulatory applicability. In Tier 1, ToxCast data established gene and tissue bioactivity indicators, facilitating hypothesis-driven hazard identification. Tier 2 examined combined risk assessments, rejecting the hypothesis of the same mode of action and highlighting inconsistencies in in vitro data and NOAEL/ADI correlations. Tier 3 applied Margin of Exposure (MoE) analysis and TK modeling to realistic exposure estimations for risk assessment screening based on internal doses, identifying tissue-specific pathways as critical risk drivers. Tier 4 refined bioactivity indicators using TK approaches to improve the NAM-based effect assessment, including an in vitro vs. in vivo comparison, with coherent results based on interstitial concentrations, though intracellular estimations remained uncertain. Tier 5 confirmed that dietary exposure in healthy adults is close to but below levels of concern, with bioactivity MoE values remaining below concern thresholds, and in vivo MoEs within the standard safety factors. Nevertheless, the MoEs are insufficient for addressing the additional non-dietary exposure expected from other pyrethroid uses such as biocides or pharmaceuticals. Results demonstrate that NGRA with TK-NAM-based TD offers a nuanced, regulatory-relevant framework for risk assessment. The proposed approach integrates the information on individual pyrethroids using bioactivity indicators; and the re-assessment of regulatory toxicity studies to select organ-relevant NOAELs allowed an improved in vitro-in vivo comparison, demonstrating the capacity of bioactivity-based MoEs for combined exposure assessments. This tiered approach provides key insights for regulatory decision-making, establishing a robust model for evaluating pyrethroids and similar chemical classes.

In vitro transcriptomics holds promise for high-throughput, human-relevant data but is not yet integrated into regulatory decision-making due to the lack of standardized approaches. For genotoxicity assessment, transcriptomic biomarkers such as GENOMARK and TGx-DDI facilitate qualitative and quantitative analysis of complex in vitro transcriptomic datasets. However, advancing their use in quantitative testing requires standardized methods for deriving transcriptomic Points of Departure (tPoDs) and linking them to in vivo responses. Herein, we investigated different approaches to calculate tPoDs and applied in vitro to in vivo extrapolation to obtain administered equivalent doses (AEDs). Human HepaRG cells were exposed for 72 h to 10 known in vivo genotoxicants (glycidol, methyl methanesulfonate, nitrosodimethylamine, 4-nitroquinoline-N-oxide, aflatoxin B1, colchicine, cyclophosphamide, mitomycin C, ethyl methanesulfonate, and N-Nitroso-N-ethylurea) from the highest concentration that induces up to 50 % cytotoxicity through a range of lower concentrations. Gene expression data was generated using a customized version of the TempO-Seq® human S1500 + gene panel. The GENOMARK and TGx-DDI biomarkers produced genotoxic calls for all of these reference genotoxicants. Next, we performed benchmark concentration (BMC) modeling to generate both genotoxicity-specific biomarker (tPoD_biomarkers) and generic tPoDs (tPoD _S1500+). High-throughput toxicokinetic models estimated the human AEDs for these tPoDs, which were compared with (a) previously reported genotoxicity-specific AEDs from other New Approach Methodologies, and (b) in vivo PoDs from animal studies. We found that the generic AEDs were more conservative than genotoxicity-specific biomarker AEDs. For six of the nine genotoxicants, transcriptomic AEDs were lower than the in vivo PoDs; refined kinetic models may improve predictions. Overall, in vitro transcriptomic data in HepaRG cells provide protective estimates of in vivo genotoxic concentrations, consistent with other in vitro genotoxicity testing systems.

Chemicals in the EU are mainly regulated based on their intended use. Each legal framework consists of requirements and guidance for hazard- and risk assessment, along with the associated decision processes e.g., registration or authorisation of chemicals for market access in the EU. As a single chemical may have multiple uses, it may be assessed under more than one framework, potentially leading to different assessment outcomes. To address this, the European Commission has introduced the ‘one substance, one assessment’ approach as part of the Chemicals Strategy for Sustainability. The aims of the approach include streamlining risk assessment processes and reducing duplication of work in assessing the same chemical. This study aimed to map the scope of chemicals subject to assessment in multiple legal frameworks and to illustrate the importance of coordination and communication in chemical assessment processes. This was achieved by identifying chemicals that are either registered or have received specific approval for the EU market, and analysing their presence in different legal frameworks. Our findings showed that almost one-tenth of the substances identified were listed under more than one framework. However, there was a notable lack of coherent chemical identifiers available to accurately identify chemicals across the frameworks. Additionally, we identified the presence of phthalates, bisphenols and PFAS in EU frameworks to illustrate how a group-based approach to chemical assessment could be applied across different legal frameworks.

Nephrotoxicity caused by drug or chemical exposure involves complex mechanisms as well as a temporal integration of injury and repair responses in different nephron segments. Distinct cellular transcriptional programs regulate the time-dependent tissue injury and regeneration responses. Whole kidney transcriptome analysis cannot dissect the complex spatio-temporal injury and regeneration responses in the different nephron segments. Here, we used laser capture microdissection of formalin-fixed paraffin embedded sections followed by whole genome targeted RNA-sequencing-TempO-Seq and co-expression gene-network (module) analysis to determine the spatial–temporal responses in rat kidney glomeruli (GM), cortical proximal tubules (CPT) and outer-medulla proximal tubules (OMPT) comparison with whole kidney, after a single dose of the nephrotoxicant cisplatin. We demonstrate that cisplatin induced early onset of DNA damage in both CPT and OMPT, but not GM. Sustained DNA damage response was strongest in OMPT coinciding with OMPT specific inflammatory signaling, actin cytoskeletal remodeling and increased glycolytic metabolism with suppression of mitochondrial activity. Later responses reflected regeneration-related cell cycle pathway activation and ribosomal biogenesis in the injured OMPT regions. Activation of modules containing kidney injury biomarkers was strongest in OMPT, with OMPT Clu expression highly correlating with urinary clusterin biomarker measurements compared the correlation of Kim1. Our findings also showed that whole kidney responses were less sensitive than OMPT. In conclusion, our LCM-TempO-Seq method reveals a detailed spatial mechanistic understanding of renal injury/regeneration after nephrotoxicant exposure and identifies the most representative mechanism-based nephron segment specific renal injury biomarkers.

This work discusses reuse of chemical data across disciplines and the role of various data initiatives and projects including PARC, NORMAN-SLE, MassBank, WorldFAIR, PSDI and NFDI4Chem to facilitate increased data sharing. Improved machine-readable chemical data supports global research and interdisciplinary methodologies crucial for sustainable development and achievement of UNESCO’s Open Science priorities and the UN Sustainability Development Goals. Examples of success and ongoing approaches include integrating toxicology and chemical exposure data using ontologies, linking specialised chemical data collections with larger repositories such as PubChem, and developing IUPAC International Chemicals Identifier (InChI) extensions for nanomaterials and mixtures. National data infrastructure projects in the UK and Germany focus on digitising and standardising chemical research data management workflows, aiding scientists in data collection, storage, processing, analysis, disclosure, and reuse. These global initiatives aim to enhance chemical data interoperability to solve real-world problems, foster collaboration, and promote innovation while considering sustainable data resources beyond individual projects.

Exposure to respiratory sensitizers (RSs) is the leading cause of occupational asthma. Although the prospective identification of RSs is important, there currently exists no OECD-approved test guideline for this endpoint. The adverse outcome pathway for respiratory sensitization consists of key event (KE) 1: binding of the respiratory sensitizer to a protein, KE2: activation of lung epithelial cells, KE3: activation of dendritic cells, and KE4: T-cell response. Here, we focused on KE2 by investigating whether measuring this KE could contribute to prospectively identify respiratory sensitizers. To mimic real-life exposure, cells were exposed via the air. We used an air–liquid interface model comprising the human bronchial epithelial cell line Calu-3. Exposure to the RS piperazine (3, 18, and 100 mg/m3) dose-dependently increased IL-6 production at dose levels that did not affect the other parameters tested (barrier integrity, cell metabolism, cytotoxicity, and IL-8 production). IL-6 has been linked to asthma in humans. Exposure to the RS chloramine-T (30, 300, and 3000 ng/cm2) showed only minor effects on the parameters tested. Exposure to the RS 2,4-toluene diisocyanate (10, 33, and 100 mg/m3) at the highest dose level clearly affected all parameters tested. Disrupted barrier function has been linked to asthma. In conclusion, this study may possibly suggest that different respiratory sensitizers may differentially impact KE2. Further research is needed to elucidate this.

This report summarises the main findings and discussion from the European Commission (EC) workshop on “The Roadmap Towards Phasing Out Animal Testing ↗ for Chemical Safety Assessments” which was held in Brussels on 11–12 December 2023. The aim of the workshop was to gather ideas and opinions from individuals, organisations and institutions, and to discuss potential approaches for incorporating non-animal methods into chemical legislation with all interested stakeholders. The roadmap will be an EC policy document which will outline milestones and specific actions, addressing all relevant pieces of chemical legislation relating to safety assessment. It intends to describe the necessary steps to replace animal testing in pieces of legislation where it is currently required for chemical safety assessments. The roadmap will outline the path to expand and accelerate the development, validation and implementation of non-animal methods as well as means to facilitate their uptake across legislation. The workshop included presentations from a wide variety of stakeholders. The contributors provided examples of how non-animal methods could be applied to replace, reduce or refine animal testing in the assessment of human health and environmental effects. Furthermore, possible guiding principles for establishing a Next-Generation Risk Assessment (NGRA) in European chemicals legislation were also discussed. The workshop provided the basis for further discussion and for structuring the roadmap work.

Enniatins (ENNs) and beauvericin (BEA) are cyclic hexadepsipeptide fungal metabolites which have demonstrated antibiotic, antimycotic, and insecticidal activities. The substantial toxic potentials of these mycotoxins are associated with their ionophoric molecular properties and relatively high lipophilicities. ENNs occur extensively in grain and grain-derived products and are considered a food safety issue by the European Food Safety Authority (EFSA). The tolerable daily intake and maximum levels for ENNs in humans and animals remain unestablished due to key toxicological and toxicokinetic data gaps, preventing full risk assessment. Aiming to find critical data gaps impeding hazard characterization and risk evaluation, this review presents a comprehensive summary of the existing information from in vitro and in vivo studies on toxicokinetic characteristics and cytotoxic, genotoxic, immunotoxic, endocrine, reproductive and developmental effects of the most prevalent ENN analogues (ENN A, A1, B, B1) and BEA. The missing information identified showed that additional studies on ENNs and BEA have to be performed before sufficient data for an in-depth hazard characterisation of these mycotoxins become available.

As part of its response to the European Citizens’ Initiative “Save Cruelty-Free Cosmetics – Commit to a Europe Without Animal Testing”, the European Commission committed to developing a roadmap to eliminate the use of animals for the safety testing of chemicals. Five animal protection non-govern­mental organizations arranged a roundtable in June 2024 to support this effort, bringing together experts from different fields. The roundtable aimed to identify important elements and organizational structures for shaping the roadmap and explored practical steps to move to a regulatory system that does not rely on animal testing. Discussions covered revising current chemicals legislation, accel­erating the acceptance of non-animal methods, creating EU databases to improve data sharing, strengthening cooperation and communication, building skills and expertise in non-animal methods, monitoring progress, and increasing funding. This report summarizes the main ideas and recommendations from the roundtable to help guide the Commission’s work on the roadmap.

The persistent nature of the environmental contaminants per- and polyfluoroalkyl substances (PFAS) has recently received considerable attention, particularly because of their adverse effects on immune system functionality in the context of vaccine responses to infectious diseases. Following COVID-19 vaccination, some studies have shown a significant negative correlation between serum PFAS concentrations and the humoral immune response to the SARS-CoV-2 spike protein vaccination. However, the influence of PFAS on the cell-mediated immune response to SARS-CoV-2 spike protein post-COVID-19 vaccination remains underexplored. In the present study, we investigated the impact of a human blood-relevant PFAS mixture, containing perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA), perfluorohexane sulfonate (PFHxS), perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), and perfluoroundecanoic acid (PFUnDA) on innate (monocytes and NK cells), cell-mediated (T cells) and B cells adaptive immune responses in COVID-19-vaccinated female and male healthy donors. Human peripheral blood mononuclear cells (PBMCs) were exposed to a mixture of the six PFAS at real life concentrations and subsequently stimulated with the SARS-CoV-2 spike peptide. We report a significant upregulation of IFNγ production in T and NK cells, particularly among male donors exposed to high concentrations of the PFAS mixture. Conversely, we observed a decrease in the total B-cell population, particularly among female donors. A significant reduction in the secretion of the pro-inflammatory chemokines MIP-1α (CCL3) and MIP-3α (CCL20) was observed at high PFAS mixture concentrations. Overall, these findings suggest that high PFAS exposure may differentially affect immune responses in a sex-specific manner, with a potential impact on vaccine efficacy.

Under evolutionary pressure, the kinematic and energetic characteristics of animal locomotion have been optimized for survival. We investigated the kinematics and energetic performance of zebrafish eleutheroembryo escape swims triggered by electrical stimuli in fluids of increasing viscosity. Eleutheroembryos exhibited a decrease in both tail movement frequency and swimming velocity in more viscous environments, while the amplitude of body curvature remains constant. We then combined experimental imaging of freely swimming eleutheroembryos with Navier-Stokes numerical simulations. The results showed that the mechanical power output was initially maximal and remained essentially stable with increasing viscosity, while the cost of transport was linearly correlated with viscosity. Eleutheroembryos maximize neuromuscular power output during the fast-start escape response, enabling them to potentially escape predators under all circumstances in a natural environment. This model may be used to identify genetic and toxicological factors that reduce the mechanical power developed by the neuromuscular system or induce a loss of efficiency in its use.

Parkinson’s disease (PD), the second most common neurodegenerative disorder, is characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta, leading to motor and non-motor symptoms. The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been extensively used in different animal species to develop chemical models of PD. This study aimed to evaluate the effects of acute exposure to MPTP (3 × 150 mg/kg, intraperitoneally) on adult zebrafish by assessing the neurochemical, transcriptional, and motor changes associated with PD pathogenesis. MPTP treatment resulted in a significant decrease in brain catecholamines, including dopamine, norepinephrine, and normetanephrine. Additionally, a trend towards decreased levels of dopamine precursors (tyrosine and L-DOPA) and degradation products (3-MT and DOPAC) was also observed, although these changes were not statistically significant. Gene expression analysis showed the downregulation of dbh, while the expression of other genes involved in catecholamine metabolism (th1, th2, mao, comtb) and transport (slc6a3 and slc18a2) remained unaltered, suggesting a lack of dopaminergic neuron degeneration. Behavioral assessments revealed that MPTP-exposed zebrafish exhibited reduced motor activity, consistent with the observed decrease in dopamine levels. In contrast, the kinematic parameters of sharp turning were unaffected. A significant impairment in the sensorimotor gating of the ASR was detected in the MPTP-treated fish, consistent with psychosis. Despite dopamine depletion and behavioral impairments, the absence of neurodegeneration and some hallmark PD motor symptoms suggests limitations in the validity of this model for fully recapitulating PD pathology. Further studies are needed to refine the use of MPTP in zebrafish PD models.