Scientific Publications

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.

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.