New Approach Methodologies (NAMs)

J Hazard Mater. 2025 Mar 17;491:137949. doi: 10.1016/j.jhazmat.2025.137949. Online ahead of print.

ABSTRACT

A tiered in vitro/in silico approach was developed to screen 12,654 per- and polyfluoroalkyl substances (PFAS) for their potential to disrupt the thyroid hormone transport. Initially, a set of 45 PFAS was tested using TTR-TRβ-CALUX bioassay, which was subsequently employed to develop a classification model, distinguishing active and inactive PFAS. The model fulfills all good practices for QSAR model validation and can predict whether a given PFAS can disrupt plasma transport of the thyroid hormone (T4). Subsequently, active compounds were used to develop two regression approaches: (i) multiple linear regression MLR, and (ii) second approach aimed at identifying multiple valid QSAR models based on different data-splitting strategies. Finally, a comprehensive virtual screening of a large PFAS dataset was conducted to assess their potency in disrupting thyroid hormone transport. The predictions indicated that more than 7500 compounds were active with over 100 PFAS potentially causing even greater adverse effects than PFOA. These findings highlight the critical role of integrating New Approach Methodologies (NAM)-based in vitro toxicity testing with multifaceted molecular modeling in assessing the risks associated with PFAS contamination in environmental matrices.

PMID:40120279 | DOI:10.1016/j.jhazmat.2025.137949

Regul Toxicol Pharmacol. 2025 Mar 17:105810. doi: 10.1016/j.yrtph.2025.105810. Online ahead of print.

ABSTRACT

Chemical safety assessment includes evaluating the potential to disrupt the endocrine system in humans and wildlife. The thyroid hormone system shows high complexity which is conserved across vertebrates, allowing biological read-across between regulatory important taxa, namely mammals and amphibians. Potential thyroid disruption in aquatic vertebrates is typically investigated by activity assays (Amphibian Metamorphosis Assay (AMA), Xenopus Eleutheroembryo Thyroid Assay). Since neither assay is designed to provide detailed mechanistic information, mode of action analyses often rely on mammalian data, assuming overall cross-vertebrate conservation. This manuscript elaborates on the imperative that, despite overall conservation, the T-modality in metamorphosing amphibians needs to be understood in detail to justify biological read-across between mammals and amphibians. To this end, we revisit the AMA regarding amphibian developmental physiology, and the T-modality regarding mechanistic cross-vertebrate conservation. The importance of a mechanistic understanding for read-across is showcased based on the AMA's apparent insensitivity to at least one category of prototypical liver enzyme inducers. From a regulatory perspective, deeper mechanistic understanding is needed, not only to strengthen the scientific basis for designing testing strategies and interpreting study results, but also to allow the identification of data gaps and thus development of New Approach Methodologies (NAMs) to minimize vertebrate testing.

PMID:40107341 | DOI:10.1016/j.yrtph.2025.105810

Environ Toxicol Chem. 2025 Mar 18:vgaf074. doi: 10.1093/etojnl/vgaf074. Online ahead of print.

NO ABSTRACT

PMID:40100402 | DOI:10.1093/etojnl/vgaf074

Toxicology. 2025 Mar 5;514:154104. doi: 10.1016/j.tox.2025.154104. Online ahead of print.

ABSTRACT

New Approach Methodologies (NAMs), including cell culture and multi-level omics analyses, are promising alternatives to animal testing. To become useable for risk assessment purposes, they have to be applicable for different substance groups. One important group of substances is food contaminants, including synthetic chemicals, such as perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA), and natural compounds, such as mycotoxins and pyrrolizidine alkaloids. We tested five known contaminants affecting the liver and/or the kidney - PFOS, PFOA, Aflatoxin B₁ (AB₁), lasiocarpine (Las), and cadmium chloride - using HepaRG and RPTEC/TERT1 cells at non-cytotoxic concentrations for 36 and 72 h. Our NAM-based testing protocol included marker protein analysis for cellular functions and targeted transcriptomics followed by bioinformatics pathway analysis. The effects observed were compared with established in vivo results. Protein analysis indicated various affected pathways in HepaRG cells, with generally fewer effects in RPTEC/TERT1 cells. The strongest transcriptional impact was noted for Las in HepaRG cells. This study demonstrated the test protocol's applicability across different substances, revealing significant differences between HepaRG and RPTEC/TERT1 cell lines. RPTEC/TERT1 cells, while expressing renal-specific CYP enzymes, were less suitable for detecting effects of substances requiring hepatic metabolic activation, like Las and AB₁. Our data supports the concept of specific pathway toxicity, with pathway analysis enabling the prediction of effects based on mechanism rather than target organ. Employing multiple omics techniques provided comprehensive insights into various compound effects, including steatosis, reactive oxygen species production and DNA damage, highlighting the potential of an extended omics approach for advancing toxicological assessments.

PMID:40054833 | DOI:10.1016/j.tox.2025.154104

Environ Toxicol Chem. 2025 Mar 1:vgaf056. doi: 10.1093/etojnl/vgaf056. Online ahead of print.

ABSTRACT

The experimental design of New Approach Methodologies (NAMs) might deviate from common ecotoxicological studies, often requiring tailored statistical approaches. For instance, in NAMs developed for the detection of endocrine activity using aquatic vertebrate eleutheroembryos (Xenopus Eleutheroembryonic Thyroid Assay (XETA), Rapid Androgen Disruption Activity Reporter (RADAR) assay and Rapid Estrogen Activity In Vivo (REACTIV) assay), all concentration groups are nested within three independent study repeats, named 'runs' in the relevant Test Guidelines. Here, runs are referred to as replicates to emphasize their role as the repeated, independent entity. By contrast, for most other ecotoxicological studies the replicates are nested in the concentration groups. This leads to a different dependency structure for the XETA, RADAR and REACTIV assays. Disregarding this violates the basic statistical requirement for independence of observations potentially leading to incorrect conclusions. Unfortunately, in the statistical sections of the Test Guidelines of the XETA, RADAR and REACTIV assay, it is not clearly recommended to regard this dependency structure as statistical recommendations using a mixed ANOVA are provided only in the annexes. Here, we present "xeredar", an open-source R-package allowing automated statistical analysis of XETA, RADAR and REACTIV assays where the dependency structure of the data is correctly regarded through a mixed ANOVA. xeredar was validated on 36 XETA ring test studies and further tested on 41 RADAR ring test studies. A power analysis was carried out for the REACTIV assay, demonstrating that ignoring the dependency structure potentially leads to lower power and an increased false positive rate in comparison to the mixed ANOVA approach. The open-source R-package "xeredar" also comes with a Shiny app, making it accessible to everyone and thereby enhancing standardization and reproducibility for the statistical analyses of XETA, RADAR, and REACTIV assays.

PMID:40036949 | DOI:10.1093/etojnl/vgaf056

Aquat Toxicol. 2025 Apr;281:107302. doi: 10.1016/j.aquatox.2025.107302. Epub 2025 Feb 21.

ABSTRACT

Effective and reliable prediction for ecotoxicity, especially when affecting different levels of trophic chains, including humans, is increasingly gaining even more prominence as ecosystems face new threats and challenges, as that posed by the per- and poly-fluoroalkyl substances (PFAS). Toxicological prediction of PFAS in aquatic organisms, such as zebrafish, can be efficiently achieved through computational ecotoxicological approaches which are fully aligned with the state-of-the-art of new approach methodologies (NAMs) and current regulatory recommendations. Specifically in this work, the PFAS toxicodynamics interaction on the zebrafish mitochondrial voltage-dependent anion channel (zfVDAC2) was evaluated, mimicking in silico the PFAS bioaccumulation in low-concentration by integrating structure-based virtual screening (SB-VS) and predictive quantitative structure-activity(mitotoxicity) relationship (QSAR) methodologies (e.g., 2D/3D-QSAR) to address mechanistic aspects of PFAS toxicity. The best ranked PFAS pose docked in zfVDAC2 exhibits a ΔG-binding affinity higher than the ATP, i.e., the native substrate of the zfVDAC2 channel, with prevalence of van der Waal interactions, followed by fluorine (F)-halogen-bonds and finally hydrogen-bonds interactions. Mitochondrial ATP-transport blocking is thus suggested to be linked with local-flexibility perturbations in the zfVDAC2. Similarly, the obtained 2D/3D- QSAR models point out the packing density index as the most significant PFAS molecular descriptor to induce toxicity in the zfVDAC2, and mainly involving van der Waal interactions. The predictive and statistical performance of these models further indicate its NAM relevance regarding PFAS risk assessment while highlighting its interoperability and extrapolation capability for the ecotoxicological evaluation of other families of compounds.

PMID:40024016 | DOI:10.1016/j.aquatox.2025.107302

J Environ Manage. 2025 Mar;377:124654. doi: 10.1016/j.jenvman.2025.124654. Epub 2025 Feb 20.

ABSTRACT

This study investigates waste management behaviors and public awareness of persistent organic pollutants (POPs) like PBDEs and PFAS in coastal communities of Newfoundland, Canada. Protecting these unique environments requires responsible waste disposal practices. Using an integrated theoretical framework combining the Theory of Planned Behavior (TPB), the Value-Belief-Norm (VBN) theory, and the Norm Activation Model (NAM), we conducted a mixed-methods study employing a pretested survey with open- and closed-ended questions. Although a larger sample was planned, 86 adult residents completed the survey. Our analysis revealed significant differences in waste management behaviors across community types (cities, big towns, and small towns). For example, cities showed higher engagement in e-waste recycling (82%) compared to smaller towns (68%), while smaller towns were more consistent in composting (78% vs. 50% in cities) and hazardous waste disposal (χ² = 33.97, p = 0.0021). Higher education and income levels were positively correlated with increased recycling and proper waste disposal. However, despite a general awareness of environmental issues, knowledge of specific environmental contaminants was limited (45% for PBDEs, 33% for PFAS). These findings highlight the urgent need for targeted public education campaigns and improved waste management services tailored to the unique needs of diverse coastal communities. This study provides valuable insights for policymakers and environmental managers, emphasizing the importance of targeted interventions to promote sustainable practices and protect fragile coastal ecosystems.

PMID:39983581 | DOI:10.1016/j.jenvman.2025.124654

Comput Toxicol. 2024 Jun;30:1-15. doi: 10.1016/j.comtox.2024.100304.

ABSTRACT

Read-across is a well-established data-gap filling technique used within analogue or category approaches. Acceptance remains an issue, mainly due to the difficulties of addressing residual uncertainties associated with a read-across prediction and because assessments are expert-driven. Frameworks to develop, assess and document read-across may help reduce variability in read-across results. Data-driven read-across approaches such as Generalised Read-Across (GenRA) include quantification of uncertainties and performance. GenRA also affords opportunities on how New Approach Method (NAM) data can be systematically incorporated to support the read-across hypothesis. Herein, a systematic investigation of differences in expert-driven read-across with data-driven approaches was pursued in terms of establishing scientific confidence in the use of read-across. A dataset of expert-driven read-across assessments that made use of registration data as disseminated in the public International Uniform Chemical Information Database (IUCLID) (version 6) of Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) Study Results were compiled. A dataset of ~5000 read-across cases pertaining to repeated dose and developmental toxicity was extracted and mapped to content within EPA's Distributed Structure Searchable Toxicity database (DSSTox) to retrieve chemical name and structural identification information. Content could be mapped to ~3600 cases which when filtered for unique cases with curated quantitative structure-activity relationship-ready SMILES resulted in 389 target-source analogue pairs. The similarity between target and the source analogues on the basis of different contexts - from structural similarity using chemical fingerprints to metabolic similarity using predicted metabolic information was evaluated. An attempt was also made to quantify the relative contribution each similarity context played relative to the target-source analogue pairs by deriving a model which predicted known analogue pairs. Finally, point of departure values (PODs) were predicted using the GenRA approach underpinned by data extracted from the EPA's Toxicity Values Database (ToxValDB). The GenRA predicted PODs were compared with those reported within the REACH dossiers themselves. This study offers generalisable insights on how read-across is already applied for regulatory submissions and expectations on the levels of similarity necessary to make decisions.

PMID:38993812 | PMC:PMC11235147 | DOI:10.1016/j.comtox.2024.100304

Front Toxicol. 2024 Apr 26;6:1359507. doi: 10.3389/ftox.2024.1359507. eCollection 2024.

ABSTRACT

In the European regulatory context, rodent in vivo studies are the predominant source of neurotoxicity information. Although they form a cornerstone of neurotoxicological assessments, they are costly and the topic of ethical debate. While the public expects chemicals and products to be safe for the developing and mature nervous systems, considerable numbers of chemicals in commerce have not, or only to a limited extent, been assessed for their potential to cause neurotoxicity. As such, there is a societal push toward the replacement of animal models with in vitro or alternative methods. New approach methods (NAMs) can contribute to the regulatory knowledge base, increase chemical safety, and modernize chemical hazard and risk assessment. Provided they reach an acceptable level of regulatory relevance and reliability, NAMs may be considered as replacements for specific in vivo studies. The European Partnership for the Assessment of Risks from Chemicals (PARC) addresses challenges to the development and implementation of NAMs in chemical risk assessment. In collaboration with regulatory agencies, Project 5.2.1e (Neurotoxicity) aims to develop and evaluate NAMs for developmental neurotoxicity (DNT) and adult neurotoxicity (ANT) and to understand the applicability domain of specific NAMs for the detection of endocrine disruption and epigenetic perturbation. To speed up assay time and reduce costs, we identify early indicators of later-onset effects. Ultimately, we will assemble second-generation developmental neurotoxicity and first-generation adult neurotoxicity test batteries, both of which aim to provide regulatory hazard and risk assessors and industry stakeholders with robust, speedy, lower-cost, and informative next-generation hazard and risk assessment tools.

PMID:38742231 | PMC:PMC11089904 | DOI:10.3389/ftox.2024.1359507

Sci Total Environ. 2024 Feb 20;912:168738. doi: 10.1016/j.scitotenv.2023.168738. Epub 2023 Nov 27.

ABSTRACT

Per- and polyfluoroalkyl substances (PFAS) are ubiquitously distributed in the aquatic environment. They include persistent, mobile, bioaccumulative, and toxic chemicals and it is therefore critical to increase our understanding on their adsorption, distribution, metabolism, excretion (ADME). The current study focused on uptake of seven emerging PFAS in zebrafish (Danio rerio) and their potential maternal transfer. In addition, we aimed at increasing our understanding on mixture effects on ADME by developing a physiologically based kinetic (PBK) model capable of handling co-exposure scenarios of any number of chemicals. All studied chemicals were taken up in the fish to varying degrees, whereas only perfluorononanoate (PFNA) and perfluorooctanoate (PFOA) were quantified in all analysed tissues. Perfluorooctane sulfonamide (FOSA) was measured at concerningly high concentrations in the brain (C_max over 15 μg/g) but also in the liver and ovaries. All studied PFAS were maternally transferred to the eggs, with FOSA and 6:2 perfluorooctane sulfonate (6,2 FTSA) showing significant (p < 0.02) signs of elimination from the embryos during the first 6 days of development, while perfluorobutane sulfonate (PFBS), PFNA, and perfluorohexane sulfonate (PFHxS) were not eliminated in embryos during this time-frame. The mixture PBK model resulted in >85 % of predictions within a 10-fold error and 60 % of predictions within a 3-fold error. At studied levels of PFAS exposure, competitive binding was not a critical factor for PFAS kinetics. Gill surface pH influenced uptake for some carboxylates but not the sulfonates. The developed PBK model provides an important tool in understanding kinetics under complex mixture scenarios and this use of New Approach Methodologies (NAMs) is critical in future risk assessment of chemicals and early warning systems.

PMID:38030006 | DOI:10.1016/j.scitotenv.2023.168738

Arch Toxicol. 2023 Dec;97(12):3075-3083. doi: 10.1007/s00204-023-03601-5. Epub 2023 Sep 27.

ABSTRACT

In Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) the criterion for deciding the studies that must be performed is the annual tonnage of the chemical manufactured or imported into the EU. The annual tonnage may be considered as a surrogate for levels of human exposure but this does not take into account the physico-chemical properties and use patterns that determine exposure. Chemicals are classified using data from REACH under areas of health concern covering effects on the skin and eye; sensitisation; acute, repeated and prolonged systemic exposure; effects on genetic material; carcinogenicity; and reproduction and development. We analysed the mandated study lists under REACH for each annual tonnage band in terms of the information they provide on each of the areas of health concern. Using the European Chemicals Agency (ECHA) REACH Registration data base of over 20,000 registered substances, we found that only 19% of registered substances have datasets on all areas of health concern. Information limited to acute exposure, sensitisation and genotoxicity was found for 62%. The analysis highlighted the shortfall of information mandated for substances in the lower tonnage bands. Deploying New Approach Methodologies (NAMs) at this lower tonnage band to assess health concerns which are currently not covered by REACH, such as repeat and extended exposure and carcinogenicity, would provide additional information and would be a way for registrants and regulators to gain experience in the use of NAMs. There are currently projects in Europe aiming to develop NAM-based assessment frameworks and they could find their first use in assessing low tonnage chemicals once confidence has been gained by their evaluation with data rich chemicals.

PMID:37755502 | PMC:PMC10567824 | DOI:10.1007/s00204-023-03601-5

Regul Toxicol Pharmacol. 2022 Dec;136:105278. doi: 10.1016/j.yrtph.2022.105278. Epub 2022 Oct 21.

ABSTRACT

The Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) regulation was created to protect human health and the environment through the better and earlier identification of harmful intrinsic properties of chemical substances on the European market. One of its central aims was the promotion of alternatives to animal testing, yet it has instead become a long tick-box list of in vivo experiments questionable relevance to human health outcomes despite a global trend towards new approach methods (NAMs) in chemical safety assessment. The Chemicals Strategy for Sustainability (CSS), proposed by the European Commission in 2020, is a golden opportunity to revise REACH in a significant and impactful way, yet proposals presented so far have significant negative animal welfare consequences. There is still time to correct the course of the ongoing REACH revision - proposals made herein offer a path towards the promising future intended by the CSS. These proposals are anchored in three vectors of action, varying in level of complexity - from changes that ECHA can implement to improve existing processes, through technical changes aimed at minimizing animal testing and increasing NAM acceptance, to deeper structural changes to establish non-animal testing strategies as the basis for risk assessment.

PMID:36280152 | DOI:10.1016/j.yrtph.2022.105278

Regul Toxicol Pharmacol. 2022 Nov;135:105261. doi: 10.1016/j.yrtph.2022.105261. Epub 2022 Sep 11.

ABSTRACT

New Approach Methodologies (NAMs) are considered to include any in vitro, in silico or chemistry-based method, as well as the strategies to implement them, that may provide information that could inform chemical safety assessment. Current chemical legislation in the European Union is limited in its acceptance of the widespread use of NAMs. The European Partnership for Alternative Approaches to Animal Testing (EPAA) therefore convened a 'Deep Dive Workshop' to explore the use of NAMs in chemical safety assessment, the aim of which was to support regulatory decisions, whilst intending to protect human health. The workshop recognised that NAMs are currently used in many industrial sectors, with some considered as fit for regulatory purpose. Moreover, the workshop identified key discussion points that can be addressed to increase the use and regulatory acceptance of NAMs. These are based on the changes needed in frameworks for regulatory requirements and the essential needs in education, training and greater stakeholder engagement as well the gaps in the scientific basis of NAMs.

PMID:36103951 | DOI:10.1016/j.yrtph.2022.105261