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

The PROPLANET project develops innovative bio-based coatings for textiles, food packaging machinery, and glass implementing a Safe and Sustainable by design (SSbD) approach to replace per- and polyfluoroalkyl substances (PFAS) compounds. A hazard assessment approach is carried out integrating a detailed toxicological in vitro assessment. Key toxicological endpoints, including cytotoxicity, genotoxicity, and pro-inflammatory responses are analysed in the chemicals formulations that replace PFAS-based coatings to ensure human health safety. Additionally, a Life Cycle Assessment (LCA) is conducted to evaluate the environmental impact of these formulations. Combining hazard assessment and LCA criteria enables the development and optimisation of coatings with low health and environmental risks. Furthermore, these assessments support PROPLANET’s PFAS-free coatings in meeting regulatory requirements and market demands by promoting safe, efficient, and eco-friendly solutions for industrial applications. The integration of these methodologies strengthens the transition toward safe and sustainable materials and products in a circular economy framework.

The comet assay is one of the most popular tests for genotoxicity in cell cultures, non-animal species, animals and humans. It has high sensitivity to detect low levels of DNA damage, can be applied to non-proliferating cells, requires relatively few cells, is technically simple, and is low cost. The Organisation for Economic Co-operation and Development (OECD) adopted in 2016 the in vivo comet assay for measurement of DNA strand breaks in animal tissues. There is a desire to expand the comet assay to genotoxicity testing in cell cultures, including the detection of oxidatively damaged DNA by incubation of gel-embedded nucleoids with DNA repair enzymes, especially formamidopyrimidine DNA glycosylase (Fpg) which converts oxidised purines to DNA breaks. Based on available information in the literature, this review provides a retrospective evaluation of the validation status of this assay, focusing on accuracy and reliability in genotoxicity testing in vitro. Information on accuracy is scarce, although limited evidence suggests levels of Fpg-sensitive sites are similar to those obtained by Fpg-linked alkaline unwinding and alkaline elution assays. Several ring studies have shown that estimated background levels of DNA breaks vary within and between laboratories. However, ring studies indicate good intra- and inter-laboratory reproducibility of the standard assay on ionizing radiation-exposed and the Fpg-linked assay on potassium bromate exposed cells. Further studies are needed to assess the reproducibility in multiple laboratories using coded samples of non-genotoxins and genotoxins. Nevertheless, the available results indicate the comet assay is a reliable in vitro genotoxicity test.

The comet assay (single-cell gel electrophoresis) is a simple, cost-efficient, robust, reliable, and user-friendly method for measuring DNA damage. The in vitro comet assay can be applied in advanced in vitro mini-organ and mini-tissue models. Higher-throughput formats of the assay, such as 48/96 mini-gels on GelBond® film, and the 12-mini-gel slide format, in combination with automated scoring, make the comet assay a valuable screening method for the genotoxic potential of chemicals. In compliance with the 3Rs to reduce, refine, and replace animal experiments, the development of new approach methods (NAMs) is an important part of the paradigm shift in toxicology toward Next Generation Risk Assessment (NGRA), based on non-animal hazard identification and characterization of chemicals. In the case of advanced cell models, the cells are often grown in three-dimensional (3D) culture, and multiple cell types representative of different organs can be co-cultivated. We demonstrate the applicability of the comet assay with commonly applied advanced models from liver, lung, breast, gut, skin, and brain. We describe both the in vitro standard alkaline version of the comet assay (ACA) which measures DNA strand breaks, and the enzyme-linked modification (ELCA) that allows detection of specific base alterations by applying lesion-specific endonucleases (e.g., formamidopyrimidine-DNA glycosylase (Fpg) and endonuclease III (Endo III) for oxidized purines and pyrimidines, respectively). We consider basic methodological issues, experimental design including treatment conditions, and the importance of including cytotoxicity testing-all of which could have an impact on and/or give biased results. Protocols are provided for both the standard 2-gel and 12-gel slide formats. Further, we address critical points that need to be taken into consideration when assessing genotoxicity. The adaptation of the comet assay to advanced models such as 3D cell cultures, co-cultures, and air-liquid interface (ALI) exposure systems marks a significant advance in genotoxicity testing. These models offer more biologically relevant contexts for measuring DNA damage and repair, leading to better risk assessment and the development of safer chemicals, such as pharmaceuticals, cosmetics, food additives, as well as influencing environmental policies.

Assessing the mutagenicity of chemical compounds is essential for ensuring their safe handling and use, thereby minimizing potential health risks. New approach methodologies (NAMs), including computational approaches, provide non-animal alternatives for testing novel materials and chemicals. This study highlights the potential of in silico NAMs, which can contribute to the development of novel Safe and Sustainable by Design (SSbD) chemicals and substances by identifying potentially hazardous ones at an early stage. Emphasis is given to the mutagenicity prediction based on data from the Ames (bacterial gene mutation) test curating them to consider stereo-specific input whenever necessary. A consensus strategy integrating different chemical representations (molecular fingerprints, 2D and 3D descriptors and molecular graphs) and modelling methods, i.e., Quantitative Structure-Activity Relationship (QSAR), read-across and deep learning models, is employed to predict the mutagenic profile of chemical compounds. In this course, a XGBoost model is developed based on molecular descriptors and a graph convolutional neural networks model to classify compounds as mutagens and non-mutagens based on the Ames test data. The devised read-across methodology is based on a guided-k-Nearest Neighbours scheme (guided-kNN) where two different molecular representations (molecular fingerprints and descriptors) are considered for neighbour selection and predictions generation. The mutagenicity predictions from the three models are integrated in a majority voting scheme to enhance the overall predictive accuracy (83% in external validation) and reduce individual model biases. Interpretation of the descriptors involved in prediction is performed through explainable AI (XAI) methods to provide insight to the mutagenicity mechanism. To enhance the interpretability of the XAI-derived insights and reinforce user confidence in the models' predictions, the involved descriptors are mapped to key events leading to mutations within the Adverse Outcome Pathway (AOP) networks. Apart from the development of reliable and interpretable mutagenicity models, emphasis is given on delivering a pipeline for the generation of 3D descriptors that can be used as the basis for future cheminformatics models. To support transparency and reproducibility of the results of our work, the curated mutagenicity dataset used for modelling is disseminated through the ChemPharos database (https://db.chempharos.eu/datasets/Datasets.zul?datasetID=ds18), the modelling steps are documented following the standardized Modelling Data (MODA) guidelines and the consensus model is freely available via the Enalos Cloud platform (https://www.enaloscloud.novamechanics.com/insight/polis/), to facilitate virtual screening of novel compounds.

The toxicity of nanomaterials(NMs) is closely tied to their physicochemical properties, such as size, shape, surface chemistry, stability in biological medium, and state of agglomeration as well to their uptake by cells. Key deficiencies in standardized testing approaches have been identified and tackled in recent years. Within the landscape of new approach methods (NAMs), the aim of this work is to review existing approaches for genotoxicity testing of the NMs under different regulatory domains, with a perspective on the development of NAMs that can solve longstanding difficulties in NMs' risk assessment. It critically examines international and European Union guidelines, highlighting the need for harmonization and the potential of NAMs to drive next-generation risk assessment. However, further collaboration, research and validation are essential to gain wider acceptance and applicability. The contribution of innovative technological approaches based on big data, artificial intelligence and machine learning, may pave powerful comparisons among different sectors and grouping strategies that will furtherance innovation in the nanotoxicology research. The future outlook for the genotoxicity testing of NMs will depend on increased cooperation between regulatory agencies, researchers, and industry stakeholders. Key steps toward overcoming current obstacles include establishing clearer pathways for data sharing, standardizing testing protocols, and fostering greater international collaboration.

Human biomonitoring (HBM) provides an integrated chemical exposures assessment considering all routes and sources of exposure. The accurate interpretation and comparability of biomarkers of exposure and effect depend on harmonized, quality-assured sampling, processing, and analysis. Currently, the lack of broadly accepted guidance on minimum information required for collecting and reporting HBM data, hinders comparability between studies. Furthermore, it prevents HBM from reaching its full potential as a reliable approach for assessing and managing the risks of human exposure to chemicals.The European Chapter of the International Society of Exposure Science HBM Working Group (ISES Europe HBM working group) has established a global human biomonitoring community network (HBM Global Network) to develop a guidance to define the minimum information to be collected and reported in HBM, called the “Minimum Information Requirements for Human Biomonitoring (MIR-HBM)”. This work builds on previous efforts to harmonize HBM worldwide.The MIR-HBM guidance covers all phases of HBM from the design phase to the effective communication of results. By carefully defining MIR for all phases, researchers and health professionals can make their HBM studies and programs are robust, reproducible, and meaningful. Acceptance and implementation of MIR-HBM Guidelines in both the general population and occupational fields would improve the interpretability and regulatory utility of HBM data. While implementation challenges remain—such as varying local capacities, and ethical and legal differences at the national levels, this initiative represents an important step toward harmonizing HBM practice and supports an ongoing dialogue among policymakers, legal experts, and scientists to effectively address these challenges. Leveraging the data and insights from HBM, policymakers can develop more effective strategies to protect public health and ensure safer working environments.

New Approach Methodologies (NAMs) are gaining significant momentum globally to reduce animal testing and enhance the efficiency and human relevance of chemical safety assessment. Even with substantial EU commitment from regulatory agencies and the academic community, the full regulatory adoption of NAMs remains a distant prospect. This challenge is further complicated by the fact that the academic world, oriented toward NAMs development, and regulatory agencies, focused on practical application, frequently operate in separate spheres. Addressing this disconnect, the present paper, developed within the European Partnership for the Assessment of Risks from Chemicals (PARC), provides a clear overview of both the available non-animal tests and current evaluation practices for genotoxic and carcinogenic hazard assessment, while simultaneously highlighting existing regulatory needs, gaps, and challenges toward greater human health protection and the replacement of animal testing through NAMs adoption.The analysis reveals a complex landscape: while the EU is deeply committed to developing and adopting NAMs, as outlined in its Chemical Strategy for Sustainability and supported by initiatives like PARC, prescriptive regulations such as Classification, Labelling and Packaging (CLP) and Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) still heavily mandate in vivo animal data for hazard classification, particularly for germ cell mutagenicity and carcinogenicity. This reliance creates a “too-short-blanket-problem,” where efforts to reduce animal testing may impact human health protection because of the current in vivo-based classification criteria. In contrast, sectors such as cosmetics and certain European Food Safety Authority (EFSA)-regulated products demonstrate greater flexibility toward progressive integration of NAMs. While the deep mechanistic understanding of genotoxicity and carcinogenicity has significantly advanced the integration of alternatives to animal tests into regulatory chemical hazard assessment, their broader and full implementation faces considerable challenges due to both scientific complexities (i.e., the development and validation of fit-for-purpose NAMs) and existing legislative provisions

The CompSafeNano project, a Research and Innovation Staff Exchange (RISE) project funded under the European Union's Horizon 2020 program, aims to advance the safety and innovation potential of nanomaterials (NMs) by integrating cutting-edge nanoinformatics, computational modelling, and predictive toxicology to enable design of safer NMs at the earliest stage of materials development. The project leverages Safe-by-Design (SbD) principles to ensure the development of inherently safer NMs, enhancing both regulatory compliance and international collaboration. By building on established nanoinformatics frameworks, such as those developed in the H2020-funded projects NanoSolveIT and NanoCommons, CompSafeNano addresses critical challenges in nanosafety through development and integration of innovative methodologies, including advanced in vitro models, in silico approaches including machine learning (ML) and artificial intelligence (AI)-driven predictive models and 1st-principles computational modelling of NMs properties, interactions and effects on living systems. Significant progress has been made in generating atomistic and quantum-mechanical descriptors for various NMs, evaluating their interactions with biological systems (from small molecules or metabolites, to proteins, cells, organisms, animals, humans and ecosystems), and in developing predictive models for NMs risk assessment. The CompSafeNano project has also focused on implementing and further standardising data reporting templates and enhancing data management practices, ensuring adherence to the FAIR (Findable, Accessible, Interoperable, Reusable) data principles. Despite challenges, such as limited regulatory acceptance of New Approach Methodologies (NAMs) currently, which has implications for predictive nanosafety assessment, CompSafeNano has successfully developed tools and models that are integral to the safety evaluation of NMs, and that enable the extensive datasets on NMs safety to be utilised for the re-design of NMs that are inherently safer, including through prediction of the acquired biomolecule coronas which provide the biological or environmental identities to NMs, promoting their sustainable use in diverse applications. Future efforts will concentrate on further refining these models, expanding the NanoPharos Database, and working with regulatory stakeholders thereby fostering the widespread adoption of SbD practices across the nanotechnology sector. CompSafeNano's integrative approach, multidisciplinary collaboration and extensive stakeholder engagement, position the project as a critical driver of innovation in NMs SbD methodologies and in the development and implementation of computational nanosafety.

The knowledge on hazards and risks connected to human exposure to engineered nanomaterials (ENMs) is still very limited, despite several decades of research and regulatory efforts at the international level. In particular, sex/gender-related responses to such exposure have not been clearly articulated so far in any of the existing guidance documents or regulatory relevant opinions provided to the parties involved in the risk assessment and risk management of ENMs. We aimed to demonstrate the relevance of the sex/gender dimension for the characterization of the risks and hazards associated with ENMs by analyzing existing scientific data on sex-related differences in response to ENMs exposure. This was achieved by performing an extensive review of in vivo mammalian toxicity studies published in PubMed and Web of Science databases. Further analysis was performed only for data reported in publications that satisfied scientific quality criteria assessed using the GUIDEnano approach. Finally, we demonstrated the importance of the sex/gender dimension for safety testing of ENMs in the future and provided recommendations on how to include the sex/gender dimension in toxicity testing of ENMs to ensure precise, transparent, and reliable conclusions in the process of hazard and risk assessments. This article is categorized under: Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials Toxicology and Regulatory Issues in Nanomedicine > Regulatory and Policy Issues in Nanomedicine.

Protein-nanomaterial interaction is a topic of great interest for nanotechnology research, particularly for advancing strategies in nanomedicine and nanosafety. This study explores the thermodynamic signatures associated with the interactions of six TiO2 nanoforms, (differing in their crystalline structure, surface properties and particle size) with bovine serum albumin as model protein. By integrating findings from electron paramagnetic resonance spectroscopy (EPR) regarding the free radical generation following interaction, together with information on the stability and conformational changes of the protein during adsorption on TiO2 nanomaterials, we aim to elucidate the binding mechanisms and identify the primary factors influencing nanomaterial's reactivity. The effect of the particle size, crystalline structure and surface properties on the binding parameters, protein structural stability and EPR data is discussed. Finally, the relevant parameters suitable for understanding molecular interactions at the bio/nano interface have been corroborated with the toxicological outcomes resulting from the measurements on the viability, proliferation and real time attachment of relevant cell lines, as well as with the detection of DNA strand breaks and oxidized DNA at the single-cell level. Thermodynamic and EPR parameters emerge as key descriptors for determining adsorption/binding processes and toxic effects of nanomaterials. The rankings with respect to cell damage and to oxidative stress inducing potential follow the same ranking seen in nanomaterial's influence on the BSA structural stability, binding affinity and enthalpic character of the interaction. Our findings highlight the intricate relationships between the parameters governing bio-nano interactions and the toxicity of the nanomaterials, and their significance in assessing nanomaterial safety and efficacy.

Nucleotide excision repair (NER) is crucial for repairing bulky lesions and crosslinks in DNA caused by exogenous and endogenous genotoxins. The number of studies that have considered DNA repair as a biomarker is limited, and therefore one of the primary objectives of the European COST Action hCOMET (CA15132) was to assemble and analyse a pooled database of studies with data on NER activity. The database comprised 738 individuals, gathered from 5 laboratories that ran population studies using the comet-based in vitro DNA repair assay. NER activity data in peripheral blood mononuclear cells were normalized and correlated with various host-related factors, including sex, age, body mass index (BMI), and smoking habits. This multifaceted analysis uncovered significantly higher NER activity in female participants compared to males (1.08 ± 0.74 vs. 0.92 ± 0.71; P = .002). Higher NER activity was seen in older subjects (>30 years), and the effect of age was most pronounced in the oldest females, particularly those over 70 years (P = .001). Females with a normal BMI (<25 kg/m2) exhibited the highest levels of NER, whereas the lowest NER was observed in overweight males (BMI ≥ 25 kg/m2). No independent effect of smoking was found. After stratification by sex and BMI, higher NER was observed in smoking males (P = .017). The biological implication of higher or lower repair capacity remains unclear; the inclusion of DNA repair as a biomarker in molecular epidemiological trials should elucidate the link between health and disease status.

Genotoxicity assessment is essential for ensuring chemical safety and mitigating risks to human health and the environment. Traditional methods, reliant on animal models, are time-consuming, costly, and raise ethical concerns. New Approach Methods (NAMs) offer innovative, cost-effective, and ethical alternatives, playing a pivotal role in both traditional and next-generation risk assessment (NGRA) by minimizing the need for animal testing, particularly in genotoxicity evaluations. However, the development of NAMs often overlooks the particular physicochemical properties of nanomaterials (NMs), which significantly influence their toxicological behaviour and can interfere with genotoxicity evaluation. This underscores an urgent need for the standardization and adaptation of NAMs to address nano- and advanced material-specific genotoxicity challenges. In this review, we summarize the challenges associated with genotoxicity testing of NMs and highlight the suitability of existing in vitro and in silico NAMs for NMs and advanced materials, enabling genotoxicity testing across various exposure routes and organ systems. Despite considerable progress, regulatory validation remains constrained by the absence of approved test guidelines and standardized protocols. To achieve regulatory acceptance, it is crucial to adapt NAMs to NM-specific exposure scenarios, refine test systems to better mimic human biology, develop tailored in vitro protocols, and ensure thorough characterisation of NMs both in pristine form and dispersed in culture medium. Collaborative efforts among scientists, regulators, industry, and advocacy groups are vital to improving the reliability and regulatory acceptance of NAMs. By addressing these challenges, NAMs have the potential to revolutionize genotoxicity risk assessment, advancing it towards a more sustainable, efficient and ethical framework.

Ensuring data quality, completeness, and interoperability is crucial for progressing safety research, Safe-and-Sustainable-by-Design approaches, and regulatory approval of nanoscale and advanced materials. While the FAIR (Findable, Accessible, Interoperable, and Re-usable) principles aim to promote data re-use, they do not address data quality, essential for data re-use for advancing sustainable and safe innovation. Effective quality assurance procedures require (meta)data to conform to community-agreed standards. Nanosafety data offer a key reference point for developing best practices in data management for advanced materials, as their large-scale generation coincided with the emergence of dedicated data quality criteria and concepts such as FAIR data. This work highlights frameworks, methodologies, and tools that address the challenges associated with the multidisciplinary nature of nanomaterial safety data. Existing approaches to evaluating the reliability, relevance, and completeness of data are considered in light of their potential for integration into harmonized standards and adaptation to advance material requirements. The goal here is to emphasize the importance of automated tools to reduce manual labor in making (meta)data FAIR, enabling trusted data re-use and fostering safer, more sustainable innovation of advanced materials. Awareness and prioritization of these challenges are critical for building robust data infrastructures.

In this study, we evaluated the genomic stability of oral mucosal epithelial cells (OMECs) cultured in complex media (COM) and xenobiotic-free media (XF) to assess their potential clinical application for limbal stem cell deficiency (LSCD) treatments. OMECs serve as a promising autologous cell source for bilateral LSCD treatment, offering an alternative to limbal epithelial cells (LECs). However, genomic integrity is crucial to ensure the long-term success of transplanted cells. We performed micronucleus (MNi) tests and comet assays to compare DNA damage in OMECs cultured in both media types. The results indicated no significant differences in cell morphology, viability, or size between the two conditions. The MNi frequency was similar, with 5.67 and 6.17 MNi per 1,000 cells in COM and XF conditions, respectively. Comet assay results showed low levels of strand breaks (SBs) and oxidized DNA lesions in both media, with XF showing a slightly lower, albeit statistically insignificant, percentage of tail DNA for net Fpg-sensitive sites. Our findings suggest that OMECs can be effectively cultivated in either COM or XF media without inducing significant DNA damage, supporting the potential use of XF media in clinical settings to reduce contamination risks. This study underscores the importance of genomic stability in cultured cells for ocular surface transplantation, contributing valuable insights into optimizing culture conditions for safer and more effective clinical applications.

Abstract Background Hazard and risk assessment of nanomaterials (NMs) face challenges due to, among others, the numerous existing nanoforms, discordant data and conflicting results found in the literature, and specific challenges in the application of strategies such as grouping and read-across, emphasizing the need for New Approach Methodologies (NAMs) to support Next Generation Risk Assessment (NGRA). Here these challenges are addressed in a study that couples physico-chemical characterization with in vitro investigations and in silico similarity analyses for nine nanoforms, having different chemical composition, sizes, aggregation states and shapes. For cytotoxicity assessment, three methods (Alamar Blue, Colony Forming Efficiency, and Electric Cell-Substrate Impedance Sensing) are applied in a cross-validation approach to support NAMs implementation into NGRA. Results The results highlight the role of physico-chemical properties in eliciting biological responses. Uptake studies reveal distinct cellular morphological changes. The cytotoxicity assessment shows varying responses among NMs, consistent among the three methods used, while only one nanoform gave a positive response in the genotoxicity assessment performed by comet assay. Conclusions The study highlights the potential of in silico models to effectively identify biologically active nanoforms based on their physico-chemical properties, reinforcing previous knowledge on the relevance of certain properties, such as aspect ratio. The potential of implementing in vitro methods into NGRA is underlined, cross-validating three cytotoxicity assessment methods, and showcasing their strength in terms of sensitivity and suitability for the testing of NMs. Graphical abstract Created with BioRender.com (publication license obtained)

Abstract The safety of bisphenol A (BPA) due to its adverse effects on the immune system has led to an increasing concern and a significant regulatory shift. The European Food Safety Authority (EFSA) proposed a reduction in the tolerable daily intake (TDI) of BPA in food in their 2023 scientific opinion, highlighting the need for stricter regulations compared to their previous assessment in 2015. This regulatory action has spurred the production of BPA alternatives, raising concerns about their safety due to insufficient toxicological data. Addressing this gap is crucial for ensuring human and environmental health. In this project, multiple genotoxicity endpoints were applied for testing of two regulatory relevant BPA alternatives, bisphenol E (BPE) and bisphenol P (BPP), in different human models: 2D HepG2 liver cells, 3D liver spheroids and primary human peripheral blood lymphocytes. DNA strand breaks and oxidised base lesions were evaluated by the enzyme‐modified version of the comet assay, while clastogenicity and aneugenicity were analysed by the in vitro micronucleus assay (OECD TG 487, 2016), together with cytotoxicity. Development of new advanced models (NAMs), as 3D spheroids, are essential for next‐generation risk assessment (NGRA) in line with the 3R's to replace, reduce or refine animal experiments. In this aspect, validation and standardisation of NAMs are needed to reach regulatory readiness level and development of OECD Test Guidelines. Therefore, a standardisation and pre‐validation of the advanced 3D liver spheroid model was performed by using multiple genotoxicity endpoints and by comparing the obtained results with standard genotoxicity models.