Enzyme Microb Technol. 2025 Jul 24;191:110724. doi: 10.1016/j.enzmictec.2025.110724. Online ahead of print. ABSTRACT Poly and per fluorinated substances (PFAS) are emerging contaminants of concern that are thought to be involved in causing numerous adverse health effects, such as immunosuppression, increased chance of cancer development, and altered levels of hepatic enzyme levels in humans. However, PFAS are considered highly persistent and resistant to biodegradation given the fact that the C-F bond can have a bond dissociation energy of up to 544 kJ/mol. Though many studies have reported PFAS biodefluorination by bacterial isolates and microbial communities, little is known regarding the molecular foundations for biodefluorination. In this study, a novel defluorinase was identified, that is responsible for the biodefluorination of 6:2 fluorotelomer carboxylic acid (6:2 FTCA) in R.jostii RHA1 using the combination of transposome-based insertional mutagenesis and heterologous expression. From a library of 417 R.jostii RHA1. mutants, 3 individual mutants lost their ability for defluorination when they were exposed to 6:2 FTCA (mutant # 15, 32 and 38 - Table S2). The disruption of the genetic locus in all 3 non-defluorinating mutants was identified coding for a putative MhPC superfamily protein. The MhPC superfamily of proteins is known to harbor other proteins such as fluoroacetate dehalogenase (UniProt - Q6NAM1) that are capable of -C-F bond cleavage. This identified gene was cloned into the heterologous expression host M. smegmatis MC2-155. After induction, the M. smegmatis MC2-155 transformant exhibited the ability to defluorinate 6:2 FTCA at a rate of 13 µmol/h (Vmax = 80.9 µmol/min and Km = 5.04 mM in Michaelis-Menten models). In contrast, defluorination was not observed in either abiotic or biotic controls. Further characterization of the novel defluorinase indicated that it could moderately defluorinate the unsaturated PFAS compound 6:2 FTCUA (4.9 µmol/h fluoride) and minimally defluorinate 5:2 sFTOH (1.3 µmol/h fluoride). The novel defluorinase indicated a maximal specific activity of 12.9 ± 1.9 µmol F/hr/g protein, against its primary PFAS substrate, 6:2 FTCA. However, it showed no activity with 5:3 FTCA or sulfonated PFAS compounds such as 6:2 FTS and 8:2 FTS. The wild-type Rhodococcus could defluorinate 6:2 FTCA at a rate of 2.2 µmol/h. The discovery of this MhPC class novel defluorinase in WT R.jostii RHA1. has substantial value since it is responsible for the critical step that initiates defluorination of PFAS compounds such as 6:2 FTCA. PMID:40714482 | DOI:10.1016/j.enzmictec.2025.110724 ... circular economy. KNAM Marketing plays a key role by sourcing responsibly recovered fiber and stock lots globally, and offering a wide portfolio ... J Toxicol Environ Health B Crit Rev. 2025 Jul 6:1-69. doi: 10.1080/10937404.2025.2521615. Online ahead of print. ABSTRACT Per- and polyfluoroalkyl substances (PFAS) comprise a large class of human-made chemicals that are in widespread use and present concerns for persistence, bioaccumulation and toxicity. Whilst a handful of PFAS have been characterized for their hazard profiles, the vast majority of PFAS have not been extensively studied. A comprehensive evaluation to characterize the hazard profiles of the thousands of available PFAS would require extensive resources in terms of cost, number of animals and time. An alternative and more efficient approach is to develop a structural chemical categorization approach to prioritize which PFAS or categories of PFAS should be subject to additional study. To that end, the U.S. Environmental Protection Agency (EPA), in collaboration with the National Institute of Environmental Health Sciences (NIEHS) Division of Translational Toxicology (DTT), initiated a research project in 2018 to screen approximately 150 PFAS through a battery of alternative model organisms, in vitro cell and biochemical assays, and in vitro toxico kinetic (TK) assays in order to inform chemical category and read-across approaches. The aim of this review summarizes the experimental testing undertaken, how data were processed, what insights were derived from a category perspective and how these might potentially inform subsequent tiered testing. PMID:40619890 | DOI:10.1080/10937404.2025.2521615 Three men have been cleared at Belfast Crown Court of charges relating to misconduct in public office. J Hypertens. 2025 Jun 13. doi: 10.1097/HJH.0000000000004085. Online ahead of print. ABSTRACT OBJECTIVES: Hypertension-induced endothelial damage in cerebral microvessels is a key factor contributing to vascular cognitive impairment (VCI). Endothelial function stabilization considerably depends on the endothelial nitric oxide synthase (eNOS)/nitrogen monoxide (NO) pathway. Furthermore, the eNOS/NO signaling pathway plays a role in stabilizing the vascular endothelium. Although betaine (bet) has been shown to improve cognitive dysfunction, its underlying mechanisms remain unclear. Therefore, this study aimed to determine whether betaine protects cognitive function by regulating eNOS/NO activity. METHODS: Male 7-month-old spontaneously hypertensive rats (SHR) were randomly assigned to four groups: SHR, Bet, Bet and N(G)-Nitroarginine methyl ester hydrochloride (L-NAME), and L-NAME groups. Male 7-month-old Wistar Kyoto rats (WKY) served as controls. All animals received treatment or saline for 4 weeks. In-vitro experiments were conducted using rat brain microvascular endothelial cells (RBMECs) treated with either homocysteine (Hcy) or betaine. Behavioral experiments, western blotting, pathological tissue staining, Doppler ultrasound technique, and ELISA were employed to assess the impact of hypertension on cognitive and endothelial functions. RESULTS: Hypertension led to cognitive decline in SHR by causing endothelial dysfunction, blood-brain barrier disruption, inflammation, oxidative stress, and apoptosis. Bet administration significantly improved these pathological indicators of cognitive impairment; however, the eNOS inhibitor L-NAME reversed its effects. CONCLUSION: Our findings suggest that betaine protects vascular endothelium and improves VCI by modulating the eNOS/NO signaling pathway. PMID:40530574 | DOI:10.1097/HJH.0000000000004085 Regul Toxicol Pharmacol. 2025 Jun 6;162:105883. doi: 10.1016/j.yrtph.2025.105883. Online ahead of print. ABSTRACT Exposure to endocrine disruptors (EDs) are associated with significant risks to human health and the environment. The European Union (EU) thus prioritizes their identification and regulation and is developing a roadmap to phase out animal testing in chemical safety assessments while advancing New Approach Methodologies (NAMs). This review outlines EU's practices for ED identification, focusing on the use of NAMs, as well as Defined Approaches and read-across. We assessed the current EU framework under the Classification, Labelling and Packaging (CLP) Regulation, the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), the Plant Protection Products Regulation (PPPR), and the Biocidal Products Regulation (BPR), evaluating current use of NAMs and reflection on potential future use. We find that EU legislation and guidance documents allow the use of NAMs in ED identification, including for assessment of endocrine activity and adversity. However, guidance on predicting adversity using NAMs remains limited, and ED identifications have largely depended on animal data to assess endocrine-mediated adversity. Continued in vivo testing until reliable methodologies are accepted as alternatives and routinely applied is required. The report concludes with short- and long-term recommendations for updates to the information requirements across regulations and further development of methods to predict endocrine-mediated adversity. PMID:40484110 | DOI:10.1016/j.yrtph.2025.105883 Regul Toxicol Pharmacol. 2025 Nov;162:105884. doi: 10.1016/j.yrtph.2025.105884. Epub 2025 Jun 6. ABSTRACT Read-across is a widely used adaptation to address information requirements under REACH. However, registrants submitting for the 2010 and 2013 deadline have often failed to satisfy regulatory requirements from ECHA's point of view. Due to their complex composition and unique physicochemical properties, surfactants are posing major challenges in this respect. With the aim to improve future submissions and prevent unnecessary animal testing, read-across-related discussions of 72 ECHA Final Decisions on Compliance Checks and Testing Proposal Evaluations of 24 major surfactant groups were analysed in-depth, and causes for acceptance or rejection were identified. Key drivers of regulatory acceptance/rejection were presence or absence of composition information, considerations on structural similarity as well as availability and nature of bridging studies. Several elements were identified that may be easily improved in future REACH dossiers without the need for additional animal testing. Other cases revealed uncertainty of expectations by ECHA, highlighting the need for improved communication during the dossier preparation. Notably, no example for acceptance of read-across based on non-animal New Approach Methodologies (NAMs) was identified in this analysis. Owing to the benefits that non-animal NAMs may present as supporting information, all stakeholders are encouraged to contribute to an increase of regulatory acceptance. PMID:40484111 | DOI:10.1016/j.yrtph.2025.105884 The American Chemistry Council’s Long-Range Research Initiative (LRI) is proud to announce its participation in the NIH Common Fund’s Complement Animal Research In Experimentation (Complement-ARIE) public-private partnership (PPP) to accelerate the scientific development and evaluation of New Approach Methodologies (NAMs). Sci Total Environ. 2025 Jul 1;984:179690. doi: 10.1016/j.scitotenv.2025.179690. Epub 2025 May 24. ABSTRACT Aquatic ecosystems provide essential services, yet they face increasing pressures from anthropogenic activities, including land-use change, eutrophication, browning, and contaminant pollution. While the ecological effects of these stressors are documented, the impacts of complex contaminant mixtures, particularly those from wastewater treatment plant (WWTP) effluents, remain poorly understood. Mixtures effects are typically assessed using traditional species-by-species toxicological approaches, which, though the gold standard, are time-intensive, require test animals, and have limited extrapolability. New Approach Methodologies (NAMs), such as environmental DNA (eDNA), offer a non-invasive alternative, enabling broader assessments of taxa responses across trophic levels. Here, we apply an eDNA approach to assess community-wide responses to effluent discharge in the St. Lawrence River, one of North America's most diverse freshwater ecosystems. We sampled water and aquatic communities along the effluent plume of the Montréal WWTP, analyzing taxa-specific responses across trophic levels using high-throughput sequencing. We evaluated the influence of water physico-chemistry and per- and polyfluoroalkyl substances (PFAS) on aquatic beta diversity and network structure. To validate our eDNA results, we compared fish-specific detections with traditional fishing surveys. Our findings highlight how wastewater-derived contaminants influence biodiversity patterns and species interactions, with taxonomic responses varying across trophic levels. Network analyses revealed shifts in ecological stability, with changes in species connectivity and modularity influenced by effluent exposure. This study demonstrates the value of eDNA for characterizing biodiversity responses to anthropogenic stressors and provides insights into the broader implications of point-source pollution for freshwater ecosystem resilience. PMID:40414060 | DOI:10.1016/j.scitotenv.2025.179690 Neurotoxicology. 2025 Jul;109:11-26. doi: 10.1016/j.neuro.2025.05.006. Epub 2025 May 19. ABSTRACT With the societal interest to decrease experimental animal testing for regulatory purposes, the need for reliable new approach methods (NAMs) has become evident. To ensure the continued safe use of chemicals, NAMs should perform ideally at a comparable or better level of sensitivity and specificity as the in vivo modalities that they aim to replace, especially if they are to be used for hazard assessment. The use of relevant reference substances, selected with transparent criteria, forms a cornerstone of developing and validating (in silico and in vitro) NAMs. Claims on sensitivity and specificity should be based on results generated with reference chemicals that were previously scrutinised by independent expert panels on whether the substance has a hazardous property. CLP (Regulation (EC) No 1272/2008 on the classification, labelling and packaging of substances and mixtures) forms a key pillar in EU chemicals management. The evaluation of all available information by the Committee for Risk Assessment (RAC) and their comparison to CLP classification criteria creates the opportunity to objectively compile lists of positive reference substances. We collated a reference list of neurotoxic substances to aid in the development of neurotoxicity NAMs. We screened CLP Annex VI and reflected on existing reference lists and mode of action data. The identified substances included industrial chemicals and active substances in plant protection products and biocidal products. This list of neurotoxicants is not an exhaustive consensus list, which ideally would be the result of combining this list with those generated by other authorities or expert groups. PMID:40398565 | DOI:10.1016/j.neuro.2025.05.006 Deadline: 2025-06-03
Approximate launch date: 05/06/2025 In 2023 EFSA launched a project to promote the use of New Approach Methods (NAMs) into the risk assessment of pesticide residues metabolites (NAMs4PestRes) through the use of the OECD QSAR toolbox, a free available in silico tool managed by ECHA, to enhance connectivity of IUCLID and MetaPath and to implement embedded in silico models. The result will be an automated and standardized workflow for the assessment of mutagenicity (bacterial gene mutation) of pesticide residue metabolites to be implemented in a... Mutat Res Genet Toxicol Environ Mutagen. 2025 May-Jun;904:503867. doi: 10.1016/j.mrgentox.2025.503867. Epub 2025 Mar 30. ABSTRACT 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. PMID:40382189 | DOI:10.1016/j.mrgentox.2025.503867 Environ Sci Technol. 2025 May 13. doi: 10.1021/acs.est.4c12090. Online ahead of print. ABSTRACT Adverse outcome pathway (AOP) has become a key component in next-generation risk assessment (NGRA), as AOPs provide mechanistic and causal basis for linking toxicological responses across multiple levels of biological organization, thus allowing utilization of new approach methodologies (NAMs) to support prioritization, classification, and hazard assessment of chemicals. Quantitative AOPs (qAOPs) further allow estimation of points of departure (PODs) along a cascade of toxicological events and prediction of final adverse outcome(s) based on early key events. The present study developed a zebrafish-based qAOP model for an OECD-endorsed AOP linking aromatase inhibition to reproductive dysfunction (AOPWiki, AOP no. 25). The model was built based on newly generated laboratory data from a prototypical aromatase inhibitor propiconazole (PRO) and further challenged by another chemical tebuconazole (TEB) to evaluate the model's performance and applicability. We found that female and male zebrafish have different effects to establish AOP after exposure to PRO and qAOP was expanded and applied to cross-species prediction with fathead minnow. During the environmentally relevant concentrations of PRO and TEB for 21 days, aromatase CYP19, vitellogenin, and steroidogenesis were affected significantly, and the reproduction of zebrafish significantly decreased. The present study aimed to expand the mechanism of aromatase and assembled quantitative AOP in zebrafish and further linked with in vitro, which extended aromatase inhibitor-mediated reproductive dysfunction as a demonstrative case of published qAOP model in fathead minnow Pimephales promelas (AOPWiki, AOP no. 25). PMID:40359424 | DOI:10.1021/acs.est.4c12090 The 2nd Commission Conference on the Roadmap towards Phasing out Animal Testing for Chemical Safety Assessments, held on 25 October 2024, brought together key stakeholders from regulatory bodies, industry, research organisations, and NGOs to discuss the future of chemical safety assessments done using non-animal methods (NAMs) in the EU. This second conference aimed to collect input from various stakeholders on how they address the transition from animal-based testing to innovative, reliable non-animal alternatives and to report progress with the development of the roadmap, which will set out actions and milestones to drive this transformation across chemical safety assessments. Toxicology. 2025 Aug;515:154167. doi: 10.1016/j.tox.2025.154167. Epub 2025 Apr 27. ABSTRACT Chronic liver diseases, including metabolic dysfunction-associated steatosic liver disease (MASLD) and hepatocellular carcinoma (HCC), are on the rise, potentially due to daily exposure to complex mixtures of chemical compounds forming part of the exposome. Understanding the mechanisms involved in hepatotoxicity of these mixtures is essential to identify common molecular targets that may highlight potential interactions at the molecular level. We illustrated this issue with two families of environmental contaminants, per- and polyfluoroalkyl substances (PFAS) and heterocyclic aromatic amines (HAAs), both of which could be involved in the progression of chronic liver diseases, and whose toxicity may be potentiated by interactions at the level of xenobiotic metabolism. In the study of exposome effects on chronic liver disease, New Approach Methodologies (NAMs) including omics analyses and data from various in vitro, in vivo and in silico approaches, are crucial for improving predictivity of toxicological studies in humans while reducing animal experimentation. Additionally, the development of complex in vitro human liver cell models, such as organoids, is essential to avoid interspecies differences that minimize the risk for humans. All together, these approaches will contribute to construct Adverse Outcome Pathways (AOPs) and could be applied not only to PFAS mixtures but also to other chemical families, providing valuable insights into mixture hepatotoxicity prediction in the study of the exposome. A better understanding of toxicological mechanisms will clarify the role of environmental contaminant mixtures in the development of MASLD and HCC, supporting risk assessment for better treatment, monitoring and prevention strategies. PMID:40300710 | DOI:10.1016/j.tox.2025.154167 Toxics. 2025 Apr 11;13(4):297. doi: 10.3390/toxics13040297. ABSTRACT Organophosphate flame retardants (OPFRs) have increasingly replaced polybrominated diphenyl ethers (PBDEs) in various consumer products and raw materials, due to regulatory restrictions on PBDEs. However, concerns about the toxicity and environmental persistence of OPFRs are growing. This review summarizes current research on the toxicity of OPFRs, with a focus on New Approach Methodologies (NAMs) that aim to eliminate the need for animal testing. NAMs, including in vitro models, omics technologies, and computational methods, provide valuable insights into the cellular and molecular effects of OPFR exposure. Evidence suggests that OPFRs may disrupt multiple organ systems, including the nervous, hepatic, pulmonary, reproductive, and endocrine systems. Additionally, the metabolic transformation of OPFRs can increase their toxicity, raising concerns about long-term exposure risks. While NAM studies provide valuable insights, further research is needed to refine risk-assessment frameworks and improve our understanding of the long-term effects of OPFR exposure, particularly at concentrations found in the environment. This new knowledge will help develop more accurate regulatory guidelines and ensure the better protection of public and environmental health. PMID:40278613 | PMC:PMC12031142 | DOI:10.3390/toxics13040297 Front Endocrinol (Lausanne). 2025 Apr 4;16:1472563. doi: 10.3389/fendo.2025.1472563. eCollection 2025. ABSTRACT INTRODUCTION: The Constitutive Androstane Receptor (CAR) (NR1I3), a pivotal member of the xenosensor family, plays a key role in the hepatic detoxification of xenobiotic and endobiotic chemicals through the induction of the expression of drug-metabolizing enzymes and transporters. CAR's involvement extends beyond detoxification, influencing gluconeogenesis, lipogenesis, bile acid regulation, and cellular processes such as proliferation, tissue regeneration, and carcinogenesis. This review explores CAR regulation by various factors, highlighting its role in mediating metabolic changes induced by environmental contaminants. METHODS: A literature search was conducted to identify all articles on the PubMed website in which the CAR-contaminant and CAR-hepatic steatosis relationship is analyzed in both in vitro and in vivo models. RESULTS: Numerous contaminants, such as perfluorooctanoic acid (PFOA), Zearalenone mycotoxin, PCB, triazole fungicide propiconazole can activate hepatic nuclear receptors contributing to the development of steatosis through increased de novo lipogenesis, decreased fatty acid oxidation, increased hepatic lipid uptake, and decreased gluconeogenesis. Indirect CAR activation pathways, particularly involving PFOA, are discussed in the context of PPARα-independent mechanisms leading to hepatotoxicity, including hepatocellular hypertrophy and necrosis, and their implications in nonalcoholic steatohepatitis (NASH) and nonalcoholic fatty liver disease (NAFLD). The prevalence of NAFLD, a significant component of metabolic syndrome, underscores the importance of understanding CAR's role in its pathogenesis. CONCLUSIONS: Experimental and epidemiological data suggest that endocrine disruptors, especially pesticides, play a significant role in NAFLD's development and progression via CAR-regulated pathways. This review advocates for the inclusion of modern toxicological risk assessment tools, such as New Approach Methodologies (NAMs), Adverse Outcome Pathways (AOPs), and Integrated Approaches to Testing and Assessment (IATA), to elucidate CAR-mediated effects and enhance regulatory frameworks. PMID:40255499 | PMC:PMC12005993 | DOI:10.3389/fendo.2025.1472563 Front Endocrinol (Lausanne). 2025 Apr 4;16:1472563. doi: 10.3389/fendo.2025.1472563. eCollection 2025. ABSTRACT INTRODUCTION: The Constitutive Androstane Receptor (CAR) (NR1I3), a pivotal member of the xenosensor family, plays a key role in the hepatic detoxification of xenobiotic and endobiotic chemicals through the induction of the expression of drug-metabolizing enzymes and transporters. CAR's involvement extends beyond detoxification, influencing gluconeogenesis, lipogenesis, bile acid regulation, and cellular processes such as proliferation, tissue regeneration, and carcinogenesis. This review explores CAR regulation by various factors, highlighting its role in mediating metabolic changes induced by environmental contaminants. METHODS: A literature search was conducted to identify all articles on the PubMed website in which the CAR-contaminant and CAR-hepatic steatosis relationship is analyzed in both in vitro and in vivo models. RESULTS: Numerous contaminants, such as perfluorooctanoic acid (PFOA), Zearalenone mycotoxin, PCB, triazole fungicide propiconazole can activate hepatic nuclear receptors contributing to the development of steatosis through increased de novo lipogenesis, decreased fatty acid oxidation, increased hepatic lipid uptake, and decreased gluconeogenesis. Indirect CAR activation pathways, particularly involving PFOA, are discussed in the context of PPARα-independent mechanisms leading to hepatotoxicity, including hepatocellular hypertrophy and necrosis, and their implications in nonalcoholic steatohepatitis (NASH) and nonalcoholic fatty liver disease (NAFLD). The prevalence of NAFLD, a significant component of metabolic syndrome, underscores the importance of understanding CAR's role in its pathogenesis. CONCLUSIONS: Experimental and epidemiological data suggest that endocrine disruptors, especially pesticides, play a significant role in NAFLD's development and progression via CAR-regulated pathways. This review advocates for the inclusion of modern toxicological risk assessment tools, such as New Approach Methodologies (NAMs), Adverse Outcome Pathways (AOPs), and Integrated Approaches to Testing and Assessment (IATA), to elucidate CAR-mediated effects and enhance regulatory frameworks. PMID:40255499 | PMC:PMC12005993 | DOI:10.3389/fendo.2025.1472563 This editorial provides an update on research & innovation (R&I) needs that can support EFSA's regulatory science in the coming years. The paper presents research needs for EFSA's work in a number of domains: omics technologies; gut microbiome; new approach methodologies (NAMs); allergenicity risk assessment; aggregate exposure assessment and environmental risk assessment (ERA). In briefly describing R&I needs, the document also addresses emerging challenges and opportunities. 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 (Cmax 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
Are New Approach Methodologies (NAMs) the Holy Grail of Toxicology? - August 7, 2025
Identification and recombinant expression of a novel defluorinase from Rhodococcus jostii RHA1, for defluorination and biotransformation of the PFAS compound 6:2 fluorotelomer carboxylic acid - July 27, 2025
De-Risking Seizure Liability: Integrating Adverse Outcome Pathways (AOPs), New Approach Methodologies (NAMs) and In Silico Approaches while Highlighting Knowledge Gaps - July 25, 2025
Refining High-Throughput In Vitro-In vivo Extrapolation Modeling through Incorporation of Intestinal Toxicokinetics - July 22, 2025
How KNAM marketing is driving sustainable growth - July 13, 2025
Insights derived from testing a library of per- and polyfluoroalkyl substances (PFAS) in a battery of new approach methods (NAMs) - July 7, 2025
Jamie Bryson, two others cleared in NAMA misconduct trial - July 3, 2025
Betaine protects cerebral microvascular endothelium and ameliorates hypertension-induced cognitive dysfunction via upregulation of the endothelial nitric oxide synthase/nitric monoxide signaling pathway - June 18, 2025
Bee Venom Disrupts Vascular Homeostasis: Apitoxin and Melittin Trigger Vascular Cell Toxicity and Aortic Dysfunction in Mice - June 11, 2025
Assessment of endocrine disruptors in the European Union: Current regulatory framework, use of new approach methodologies (NAMs) and recommendations for improvements - June 8, 2025
Learning from experience: A retrospective analysis of read-across strategies for surfactants under REACH - June 8, 2025
ACC Research Program Joins Partnership for Validation of New Approach Methodologies (NAMs) - June 3, 2025
Assessing the effects of urban effluent pollution on freshwater biodiversity and community networks using eDNA metabarcoding - May 25, 2025
A reference list of neurotoxicants based on CLP harmonised classifications - May 21, 2025
NP-EFSA-PREV-2025-02 Support from Member States on NAMs4PestRes - May 20, 2025
Deadline to register interest: 03/06/2025
New Approach Methods (NAMs) for genotoxicity assessment of nano- and advanced materials; Advantages and challenges - May 17, 2025
Development and Cross-Validation of a Zebrafish Quantitative Adverse Outcome Pathway Model on Aromatase Inhibition Leading to Reproductive Dysfunction - May 13, 2025
An FDA/CDER nonclinical perspective on the use of hiPSC-CM data for cardiovascular safety assessment and regulatory decisions - May 8, 2025
Conference report : Brussels 25 October 2024 : 2nd Commission conference on the Roadmap Towards Phasing out Animal Testing for Chemical Safety Assessments. - April 29, 2025
Per- and polyfluoroalkyl substances as potentiators of hepatotoxicity in an exposome framework: Current challenges of environmental toxicology - April 29, 2025
Toxicity Assessment of Organophosphate Flame Retardants Using New Approach Methodologies - April 25, 2025
Constitutive androstane receptor, liver pathophysiology and chemical contaminants: current evidence and perspectives - April 21, 2025
Constitutive androstane receptor, liver pathophysiology and chemical contaminants: current evidence and perspectives - April 21, 2025
Advancing EFSA's regulatory science: Updated research and innovation needs - April 16, 2025
Identification of new PFAS for severe interference with thyroid hormone transport: A combined in vitro/silico approach - March 22, 2025
Cross-taxa Extrapolation: Is There a Role for Thyroid Hormone Conjugating Liver Enzymes during Amphibian Metamorphosis? - March 19, 2025
The Xenopus Eleutheroembryo Thyroid Assay (XETA), a New Approach Methodology (NAM) for the regulatory assessment of thyroid activity of chemicals - March 18, 2025
NAM-based analysis of contaminant short-term organ toxicity in HepaRG and RPTEC/TERT1 cells - March 7, 2025
Xeredar: An open-source R-package for the statistical analysis of endocrine new approach methods (NAMs) using fish or amphibian eleutheroembryos - March 4, 2025
Predicting mito-target interactions for per-and poly-fluoroalkyl compounds: Mapping mitochondrial toxicity on zebrafish voltage-dependent anion channel 2 - March 2, 2025
Modeling the developing nervous system: a neuroscience perspective on the use of NAMs in DNT testing - February 27, 2025
Sociodemographic drivers of waste management behaviors and public perceptions of environmental contaminants in coastal communities of Newfoundland, Canada - February 21, 2025
A systematic analysis of read-across within REACH registration dossiers - July 12, 2024
New approach methods to assess developmental and adult neurotoxicity for regulatory use: a PARC work package 5 project - May 14, 2024
Studying mixture effects on uptake and tissue distribution of PFAS in zebrafish (Danio rerio) using physiologically based kinetic (PBK) modelling - November 29, 2023
Analysis of health concerns not addressed by REACH for low tonnage chemicals and opportunities for new approach methodology - September 27, 2023
REACHing for solutions: Essential revisions to the EU chemicals regulation to modernise safety assessment - October 24, 2022
Use of New Approach Methodologies (NAMs) in regulatory decisions for chemical safety: Report from an EPAA Deep Dive Workshop - September 14, 2022
New Approach Methodologies (NAMs)
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Burryman Andrew Taylor meets with local residents as he parades through Queensferry, Scotland, encased in burrs, on August 8, 2025. The Burryman event, held annually on the day before the Ferry Fair, features a man dressed in a suit made from around 11,000 burdock heads, ferns, and flowers. Supported by two attendants, the man makes a seven-mile journey through the town. Residents greet him with donations and serve him whisky through a straw, a tradition dating back to 1746, believed to bring good luck and strengthen community spirit.
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China’s Shasha Liu takes a shot during the women’s pool 10-ball bronze-medal match against Germany’s Ina Kaplan, during the 2025 World Games, in Chengdu, China, on August 13, 2025.
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A decorated elephant at the Buddhist Temple of the Tooth, as part of celebrations to mark the festival of Esala Perahera, in Kandy, Sri Lanka, on August 8, 2025
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A woman watches the sunset, which was shrouded in smoke from distant wildfires, at a park in Kansas City, Missouri, on August 7, 2025.
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A wildfire burns on Mount Underwood near Port Alberni, on Vancouver Island, British Columbia, Canada, on August 12, 2025.
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William Gregorio and his son Yamry pose for a portrait where their ancestral home used to stand, now submerged in seawater amid rising tides, on August 12, 2025, in Pugad Island, Hagonoy, Philippines. In the Philippines’ coastal communities, water has been rising for years due to climate change. But residents say the sharpest surge came because of large-sc
Abstract
New Approach Methodologies (NAMs), including organoids, microphysiological systems and computer modeling, are gaining increased popularity for toxicological testing and even mechanistic research. With the use of human cells, the primary objectives of NAMs are to develop more human-relevant test systems and to reduce, and ultimately eliminate, animal experiments. There are many advantages of using NAMs for biological research. For example, NAMs can be used to test the dose- and time-dependent toxicity of numerous chemicals and mixtures in a cost-effective way and reduce animal use. Although these are worthwhile goals when considering the big picture, the problems are in the details. First, in vivo insight is needed to build and refine NAMs, including computer modeling. Second, primary human cells are difficult to obtain reliably and in sufficient quantities; substitutes such as immortalized cell lines or induced pluripotent stem cells (iPSCs) have the advantage of being more robust and available in unlimited numbers, but their basal and stress-induced gene expression profiles are quite different compared to primary cells. Third, critical aspects such as metabolic competency, presence of various cell types in an organ, spatial aspects, oxygen gradients, role of inflammatory cells are very difficult to replicate in vitro. Therefore, in vivo experiments are necessary to verify results obtained with NAMs. Importantly, the results of both NAMs and the in vivo animal experiments need to be translatable to human disease processes. The advantages and limitations of NAMs are being discussed using the challenges of investigating mechanisms of drug hepatotoxicity as an example. Abstract
Animal studies are commonly used in drug development, chemical, and environmental toxicology to predict human toxicity, but their reliability, particularly in the central nervous system (CNS) is limited. For example, animal models often fail to predict drug-induced seizures, leading to unforeseen convulsions in clinical trials. Evaluating environmental compounds, such as pesticides, also poses challenges due to time and resource constraints, resulting in compounds remaining untested. To address these limitations, a government-industry collaboration identified 25 biological target families linked to seizure mechanisms by combining key events from adverse outcome pathways (AOPs) with drug discovery data. Over a hundred vitro assay endpoints were identified, covering 24 of the target families, including neurotransmitter receptors, transporters, and voltage-gated calcium channels. A review of reference compounds identified 196 seizure-inducing and 34 seizure-negative chemicals revealed that fewer than 30% of the targets had been tested, highlighting significant data gaps. This proof-of-concept study demonstrates how mechanistic seizure liability can be assessed using an AOP framework and in vitro data. It underscores the need for expanded screening panels to include additional seizure-relevant targets. By integrating mechanistic insights into early drug development and environmental risk assessment, this approach enhances compound prioritization, complements animal studies, and optimizes resource use. Ultimately, this strategy refines CNS safety evaluation in drug development, improves public health protection to neurotoxicants, and bridges knowledge gaps. Abstract
New approach methods (NAMs) that combine high-throughput toxicity and toxicokinetic data have gained prominence as federal entities attempt to evaluate tens of thousands of commercial chemicals for human health hazard. In vitro-in vivo extrapolation employing a generic high-throughput toxicokinetic (HTTK) model to convert in vitro points of departure (POD) to human equivalent doses (ie, PODNAMs) has proven successful translating in vitro data to real-world exposures; however, conservative assumptions, including consideration of only hepatic metabolism, has resulted in PODNAMs that are 10 to 100-fold more conservative when compared to available in vivo-based PODs. This effort evaluates the impact of incorporating intestinal metabolism through consideration of CYP3A4, a cytochrome P450 isozyme responsible for over 80% of intestinal clearance. For 11 chemicals, intrinsic clearance rates were derived in human liver and intestinal microsomes with and without inhibition of CYP3A4 to quantitate relative CYP3A4 contribution. Physiologically-based TK simulations were conducted using Simcyp Simulator to 1) recapitulate the HTTK approach and 2) incorporate CYP3A4 contribution into the elimination model, which by extension incorporates intestinal clearance occurring via CYP3A4. CYP3A4 contribution ranged from 0 to 71% across the chemicals tested, and estimates of oral bioavailability, steady-state concentration, and fraction escaping gut metabolism typically decreasing with increasing CYP3A4 involvement. Further, incorporation of in vitro PODs with this refined model showed a concomitant increase in PODNAMs, indicating that incorporating such information into HTTK provides more predictive risk-based prioritization of the commercial chemical space. Abstract
Bee venom (apitoxin) is a mixture of bioactive molecules, with melittin as its principal component. While its therapeutic potential is increasingly recognized, its toxic effects on vascular homeostasis remain underexplored. We investigated the impact of apitoxin and melittin on vascular cell viability and mouse aortic function. Cytotoxicity was assessed in cultured endothelial and smooth muscle cells using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. Aortic function was evaluated by mounting thoracic aortas from young male and female C57BL/6J mice in tissue baths. Isometric tension was measured during phenylephrine-induced contractions, as well as endothelium-dependent (acetylcholine) and -independent (sodium nitroprusside) relaxations. To evaluate the roles of nitric oxide (NO) and oxidative stress, we used the NO synthase inhibitor Nω-nitro-L-arginine methyl ester (L-NAME) and the antioxidant superoxide dismutase (SOD), respectively. HPLC analysis revealed that melittin comprised 43.80% of apitoxin. Both apitoxin and melittin exhibited concentration-dependent cytotoxicity, significantly reducing endothelial cell viability at concentrations ≥5 µg/mL, while smooth muscle cells were affected at lower concentrations (≥2.5 µg/mL for apitoxin; ≥1.5 µg/mL for melittin). In functional experiments, apitoxin enhanced phenylephrine-induced contractions at 1 µg/mL and impaired both endothelium-dependent and -independent relaxations at ≥ 0.1 µg/mL, particularly in males. Although melittin mimicked these effects, higher concentrations (≥5 µg/mL) were required, suggesting that other venom components contribute to the vascular functional toxicity of apitoxin. L-NAME and SOD prevented apitoxin-induced vascular impairments, implicating the NO pathway and oxidative stress. These findings demonstrate that apitoxin impairs vascular cell viability and aortic function at clinically relevant concentrations, underscoring both its vascular risks and therapeutic potential. Abstract
Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a New Approach Methodology (NAM) used in regulatory submissions to the U.S Food and Drug Administration (FDA). This article builds on the previous FDA analysis using a new search strategy to provide an updated landscape of hiPSC-CM studies submitted to the FDA for review. The current search method is more comprehensive than the previous ones, emphasizing the importance of standardized keywords in study titles for easier identification of NAMs submitted to FDA. Here the authors report an increase in hiPSC-CM studies submitted to FDA, with most using the multielectrode array (MEA) platform. In this new analysis, the authors observed that the study methodology, context of use (COU), and reasons for submission are often unclear, despite their importance for regulatory acceptance and review. hiPSC-CM study results are not discussed in many archived reviews, suggesting limited impact on regulatory decisions. Detailed reporting to characterize the clinical relevance of findings and systematic submission of hiPSC-CM studies to better understand their predictivity compared to familiar nonclinical assessment methods are key components from a Pharmacology/Toxicology perspective to increase regulatory use of this subset of NAMs. Abstract
There is widespread concern that environmental exposures constitute an underappreciated but significant contribution to rising rates of neurodevelopmental disorders (NDDs). There is also international consensus that regulatory frameworks for developmental neurotoxicity (DNT) testing are woefully inadequate, prompting reappraisal of DNT testing methods. One approach aims to make testing more efficient, less animal-intensive, and higher throughput, through in vitro evaluation of DNT. These new approach methodologies (NAMs) promise to accelerate and standardize DNT testing through interrogation of fundamental mechanisms of neurodevelopment. While in the early stages of development, they have significant, well-publicized shortcomings, including little to no accounting for cellular or genetic diversity, cell extrinsic signaling molecules, sex as a biological variable, developmental stage, or relevance to NDDs. One of the most advanced NAM platforms is a collection of 17 in vitro assays termed the DNT in vitro battery (IVB). While it models some aspects of neurodevelopmental processes, it fails to capture others. Proper brain ontogeny, and consequently normal behavior and cognition, relies on the integrity of fundamental mechanisms, their temporal/spatial fidelity, and the magnitude of their expression. These fundamental mechanisms are regulated by factors not considered by the DNT IVB including diverse cell types and neurotransmitters. While the DNT IVB could prove to be an important tool in DNT hazard detection, we identify key areas, including cell-extrinsic neurotransmitter signaling, diversity of neural progenitors, interneurons, and biological sex, that should be prioritized for development and inclusion in future refinements to meaningfully enhance biological coverage and relevance to human cognition and behavior.