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Assessing chemical toxicity in materials like plastic packaging is critical to safeguarding public health. This study presents the development of classification-based machine learning models to predict the toxicity of chemicals associated with plastic packaging. Using an extensive dataset of chemical structures, we trained multiple machine learning models-Random Forest, Support Vector Machine, Linear Discriminant Analysis, and Logistic Regression-targeting endpoints such as Neurotoxicity, Hepatotoxicity, Dermatotoxicity, Carcinogenicity, Reproductive Toxicity, Skin Sensitization, and Toxic Pneumonitis. The dataset was pre-processed by selecting 2D molecular descriptors as feature inputs, with resampling methods (ADASYN, Borderline SMOTE, Random Over-sampler, SVMSMOTE Cluster Centroid, Near Miss, Random Under Sampler) applied to balance classes for accurate classification. A five-fold cross-validation technique was used to optimize model performance, with model parameters fine-tuned using grid search. The model performance was evaluated using accuracy (Acc), sensitivity (Se), specificity (Sp), and area under the receiver operating characteristic curve (AUC-ROC) metrics. In most of the cases, the model accuracy was 0.8 or above for both training and test sets. Additionally, SHAP (SHapley Additive exPlanations) values were utilized for feature importance analysis, highlighting significant descriptors contributing to toxicity predictions. The models were ranked using the Sum of Ranking Differences (SRD) method to systematically select the most effective model. The optimal models demonstrated high predictive accuracy and interpretability, providing a scalable and efficient solution for toxicity assessment compared to traditional methods. This approach offers a valuable tool for rapidly screening potentially hazardous chemicals in plastic packaging., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Developmental toxicity testing is essential to identify substances that may harm embryonic development. This study aimed to establish a protocol for evaluating developmental toxicity using human induced pluripotent stem cells (iPSCs) by analyzing cellular activity and gene expression changes. Two ICH S5(R3) positive substances, valproic acid (VPA), which is a substance previously detected as positive by other test methods, and thalidomide (Thalido), were examined during early trichoderm differentiation without fetal bovine serum. RNA-seq analysis identified seven candidate genes, including TP63 , associated with altered expression following exposure to VPA or Thalido. These genes were implicated in pathways related to tissue development, cell growth, and molecular interactions. While the assay effectively detected VPA and Thalido, its limitations include testing only soluble substances and focusing on early differentiation stages. Nevertheless, the protocol demonstrates potential for the classification and evaluation of emerging modality drugs based on physical properties such as solubility, polarity, and pH. Integration with AI analysis may enhance its capacity to uncover genetic variations and evaluate previously uncharacterized substances. This study provides a foundation for alternative developmental toxicity testing methods, with further refinements in the culture method expected to improve accuracy and applicability in regulatory toxicology.

Early life stage (ELS) toxicity testing in fish is a crucial test procedure used to evaluate the long-term effects of a wide range of chemicals, including pesticides, industrial chemicals, pharmaceuticals, and food additives. This test is particularly important for screening and prioritizing thousands of chemicals under the Registration, Evaluation, Authorization, and Restriction of Chemicals (REACH) regulation. In silico methods can be used to estimate the toxicity of a chemical when no experimental data is available and to reduce the cost, time, and resources involved in the experimentation process. In the present study, we developed predictive Quantitative Structure-Activity Relationship (QSAR) models to assess chronic effects of chemicals on ELS in fish. Toxicity data for ELS in fish was collected from two different sources, i.e. J-CHECK and eChemPortal, which contain robust study summaries of experimental studies performed according to OECD Test Guideline 210. The collected data included two types of endpoints - the No Observed Effect Concentration (NOEC) and the Lowest Observed Effect Concentration (LOEC), which were utilized to develop the QSAR models. Six different partial least squares (PLS) models with various descriptor combinations were created for both endpoints. These models were then employed for intelligent consensus-based prediction to enhance predictability for unknown chemicals. Among these models, the consensus model - 3 (Q 2 F1 = 0.71, Q 2 F2 = 0.71) and individual model - 3 (Q 2 F1 = 0.80, Q 2 F2 = 0.79) exhibited most promising results for both the NOEC and LOEC endpoints. Furthermore, these models were validated experimentally using experimental data from nine different industrial chemicals provided by Global Product Compliance (Europe) AB. Lastly, the models were used to screen and prioritize chemicals obtained from the Pesticide Properties (PPDB) and DrugBank databases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Household chemicals used daily are often combined, leading to inhalation exposure to mixtures. However, methods for assessing their toxic effects are limited. This study proposes an in vitro assay strategy for evaluating household chemical mixtures using benzalkonium chloride (BKC) and didecyldimethylammonium chloride (DDAC), a common disinfectant. Our approach utilizes the mode of action (MOA) of chemicals by applying toxicity units (TU) to assess the key events related to lung disease, such as reactive oxygen species (ROS) production and cell death. The TU (EC 50 ) values for BKC and DDAC were 3.97 µg/mL and 1.89 µg/mL, respectively, from cytotoxicity results. The TU value of the mixture (5:5 ratio of BKC to DDAC) was calculated as 2.56 µg/mL. Using the OpenMRA platform, the TU values were predicted as 2.37 µg/mL with the concentration addition (CA) model and 11.26 µg/mL with the independent action (IA) model, indicating that the mixture effects were additive and closer to that predicted using the CA model. Both BKC and DDAC induced apoptosis and ROS production in human epithelial cells in a dose-dependent manner, suggesting similar modes of action in promoting cell death. Our results suggested that BKC and DDAC exhibited additive toxicity when combined. Our results demonstrate the utility of the TU-based approach, which combines cytotoxicity, ROS induction, and apoptosis measurements to evaluate mixture toxicity. This approach may be beneficial for assessing early key events relevant to lung diseases and offers a practical strategy for evaluating the inhalation toxicity of household chemical mixtures., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 Elsevier B.V. All rights reserved.)

Non-invasive optical registration and subsequent analysis of heart rate (HR) and heart rate variability (HRV) in transparent aquatic animals have recently been proposed as convenient toxicological endpoints, well-suited for automation data acquisition and processing. This approach was evaluated in experiments involving juvenile Daphnia magna and zebrafish (Danio rerio) embryos exposed to glyphosate solutions (20 mg/L, 2 mg/L, 0.2 mg/L, and 0.02 mg/L) and imidacloprid solutions (30 mg/L, 3 mg/L, 0.3 mg/L, and 0.03 mg/L). The findings indicate that cardiac performance assessment is a promising approach for short-term toxicity evaluation. However, the sensitivity of this physiological endpoint to various external factors may limit its broader application. Results from the two model species highlight their differing sensitivities to the tested substances, emphasizing the need for thorough preliminary studies before establishing this method as a standardized toxicological tool. The potential development and improvement of techniques for assessing heart rate in zebrafish and daphnids are discussed., Competing Interests: Declaration of competing interest The authors declare that the y have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The estrous cycle is a sensitive endpoint from an endocrine disruptor perspective and is included in rodent reproductive toxicity studies. In this paper, the methods for estrous cycle timing and different approaches followed by testing laboratories for evaluating the days of the estrous cycle were reviewed. No major differences are identified for counting 4-day estrous cycle. However, when extended episodes of estrus (E) stages occur, the cycle counting differs between testing laboratories potentially resulting in misinterpretation and inaccurate outcome. Appearance of extended episodes of two to three E in an estrous cycle need explanation. Therefore, the following are proposed, 1) follow OECD guidance document for normal 4/5 day cycles, 2) recommends an update of the OECD document as in the current one it is unclear for a 5-day cycle ending with Proestrus(P) followed by episode(s) of estrus(E), 3) two episodes of E after P in a 5-day cycle and three consecutive episodes of E should be considered abnormal, and 4) stages prior to first E of the first cycle or after last E (last P of a 5-day) of last cycle should not be assumed a complete cycle. Additionally, call for collaboration is made to harmonize the cycle counting approach., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Environmental Safety Assessments (ESA) are mandatory for several regulatory purposes and are an important component of stewardship/sustainability initiatives. Fish testing is used for assessing chemical toxicity and bioaccumulation potential; amphibians are included in some jurisdictions and their use is increasing to assess endocrine disruption. Alternative methods are becoming more available, covering the principles of the 3Rs (i.e., replacing, reducing and refining animal tests), but their regulatory incorporation is still limited. A cross-sector review by the European Partnership for Alternative Approaches to Animal Testing (EPAA), discussed the status and priorities for accelerating the adoption of non-animal approaches in ESA. The lack of an internationally agreed definition for "animal testing" was recognized as a challenge. For example, testing with vertebrate embryos up to specific developmental stages is a suitable refinement alternative only in some jurisdictions. Invertebrate testing offers refinement alternatives to develop tiered approaches using vertebrate testing as a last resort. Aquatic ESA was identified as a common need by all sectors and regulatory areas, while terrestrial ESA is particularly relevant for agrochemicals. The standardization and validation of some alternative methods as OECD test guidelines (TGs) for fish acute toxicity and fish bioaccumulation have not yet triggered the expected replacement in regulatory settings. Priority actions in these areas are needed to generate confidence in the regulatory use of the available OECD TGs designed as alternatives, including the identification of applicability domains and guidance/decision-trees for integrating different lines of evidence. Case studies under the OECD Integrated Approaches to Testing and Assessment (IATA) program could facilitate further global regulatory uptake. Replacement of fish chronic toxicity testing is more complex and less advanced. A dual approach was suggested, in the short-term, exploring lines of evidence that, alone or in combination, could identify when further fish testing is not needed. The second phase should focus on the application of the 3Rs in those cases where chronic information is needed. Another area of increasing interest is endocrine disruption. It represents a challenge but also an opportunity for implementing mechanistic non-animal methods, in addition to integrate human and ESA. This requires a step-by-step approach with continuous dialogue to ensure that technical developments will address regulatory needs. The review also agreed that the long-term aspiration is a new ESA paradigm, mapping the protection goals and providing connectivity between the chemical legislation and environmental protection policies., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Jose V Tarazona reports financial support was provided by EPAA contributed to the preparation of the manuscript through a contact with Instituto de Salud Carlos III, Spain. The authors of this article participated in the workshop that was organized by the EPAA. The authors are affiliated with different public and private organizations.Dr. Adamovsky thanks the RECETOX Research Infrastructure (No LM2023069) financed by the Ministry of Education, Youth and Sports for supportive background. This article was also supported from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 857560 (CETOCOEN Excellence) and from the Horizon Europe programme under grant agreement No. 101057014 (PARC). Views and opinions expressed are however those of the author(s) only and do not necessarily reflect those of the European Union or the European Health and Digital Executive Agency (HADEA). Neither the European Union nor the granting authority can be held responsible for any use that may be made of the information it contains. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 The Authors. Published by Elsevier Inc. All rights reserved.)

New approach methods (NAMs) are required to predict human toxicity effectively, particularly due to limitations in conducting in vivo studies. While NAMs have been established for various industries, such as cosmetics, pesticides, and drugs, their applications in natural products (NPs) are lacking. NPs' complexity (multiple ingredients and structural differences from synthetic compounds) complicates NAM development. In this study, we devised NAMs for NPs using (quantitative) structure-toxicity relationship (Q)STR models and in vitro assays. Validation involved testing each method with single compounds isolated from NPs. A linear regression model was developed for (Q)STR prediction (R 2 on test set: 0.52), with an applicability domain analysis demonstrating its reliability across NPs. This model was applied to predict the LD 50 range of species, aiding in the development of herbal medicine and dietary supplements. In vitro screening employed three reporter cell lines (AP-1, P53, and Nrf2), with Tox scores derived by integrating in silico and in vitro data. Nimbolide exhibited the highest Tox score, with experimental studies corroborating the accuracy and reliability of the predictions made via Tox score analysis. The findings of the study align well with the purpose, as the suggested NAMs, utilizing (Q)STR models and in vitro assays, provide a Tox score to efficiently prioritize NPs for herbal medicine and dietary supplements., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024. Published by Elsevier Inc.)

New methods are essential to characterize the performance of substitute procedures for detecting therapeutic action(s) of a chemical or key signal of toxicological events. Herein, it was discussed the applications and advantages of using arthropods, worms, and fishes in pharmacological and/or toxicology assessments. First of all, the illusion of similarity covers many differences between humans and mice, remarkably about liver injury and metabolism of xenobiotics. Using invertebrates, especially earthworms (Eisenia fetida), brine shrimps (Artemia salina, Daphnia magna), and insects (Drosophila melanogaster) and vertebrates as small fishes (Oryzias latipes, Pimephales promelas, Danio rerio) has countless advantages, including fewer ethical conflicts, short life cycle, high reproduction rate, simpler to handle, and less complex anatomy. They can be used to find contaminants in organic matters and water and are easier genetically engineered with orthologous-mutated genes to explore specific proteins involved in proliferative and hormonal disturbances, chemotherapy multidrug resistance, and carcinogenicity. As multicellular embryos, larvae, and mature organisms, they can be tested in bigger-sized replication platforms with 24-, 96-, or 384-multiwell plates as cheaper and faster ways to select hit compounds from drug-like libraries to predict acute, subacute or chronic toxicity, pharmacokinetics, and efficacy parameters of pharmaceutical, cosmetic, and personal care products. Meanwhile, sublethal exposures are designed to identify changes in reproduction, body weight, DNA damages, oxidation, and immune defense responses in earthworms and zebrafishes, and swimming behaviors in A. salina and D. rerio. Behavioral parameters also give specificities on sublethal effects that would not be detected in zebrafishes by OECD protocols., Competing Interests: Declarations. Competing interests: The authors declare no competing interests. Ethics approval: Not applicable., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The European regulatory framework on chemicals is at a crossroads. There are calls for the framework to be more effective, by better protecting people and the environment. There is also room for it to be more efficient and cost-effective, by harmonizing assessment practices across sectors and avoiding the need for unnecessary testing. At the same time, there is a political commitment to phase out animal testing in chemical safety assessments. In this commentary, we argue that these needs are not at odds with each other. On the contrary, the European Commission's roadmap to phase out animal testing could also be the transition pathway to a more efficient, effective, and sustainable regulatory ecosystem. Central to our proposal is a framework based on biological reasoning in which biological questions can be answered by a choice of methods, with non-animal methods progressively becoming the only choice. Within this framework, a tiered approach to testing and assessment allows for greater efficiency and effectiveness, while also introducing considerations of proportionality and cost-effectiveness. Testing strategies, and their component methods, should be developed in tandem and judged in terms of their outcomes, and the protection levels they inform, rather than their ability to predict the outputs of animal tests., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society of Toxicology.)

Background: The main challenge in new drug development is accurately predicting the human response in preclinical models., Methods: In this study, we developed three different intestinal barrier models using advanced biofabrication techniques: (i) a manual model containing Caco-2 and HT-29 cells on a collagen bed, (ii) a manual model with a Caco-2/HT-29 layer on a HDFn-laden collagen layer, and (iii) a 3D bioprinted model incorporating both cellular layers. Each model was rigorously tested for its ability to simulate a functional intestinal membrane., Results: All models successfully replicated the structural and functional aspects of the intestinal barrier. The 3D bioprinted intestinal model, however, demonstrated superior epithelial barrier integrity enhanced tight junction formation, microvilli development, and increased mucus production. When subjected to Ibuprofen, the 3D bioprinted model provided a more predictive response, underscoring its potential as a reliable in vitro tool for drug toxicity testing., Conclusion: Our 3D bioprinted intestinal model presents a robust and predictive platform for drug toxicity assessments, significantly reducing the need for animal testing. This model not only aligns with ethical testing protocols but also offers enhanced accuracy in predicting human responses, thereby advancing the field of drug development., Competing Interests: Declarations. Conflict of interest: The author(s) declare no conflict interest. Ethical statement: There are no animal experiments carried out for this article., (© 2025. Korean Tissue Engineering and Regenerative Medicine Society.)

Currently, evaluating respiratory sensitization is challenging with a lack of mechanistic understanding and appropriate testing methods. Given the similarities between skin and respiratory sensitization, using defined approach (DA) in OECD Test Guideline (TG) 497 will be helpful. However, adopting skin sensitization DA is not reliable in predicting respiratory sensitization and has not been validated. To address this limitation, we developed an in vitro respiratory sensitization assay (RS assay) to assess the inflammatory responses associated with respiratory sensitization. Additionally, we investigated the applicability of direct peptide reactivity assay (DPRA) for respiratory sensitization testing. Combined with in silico structure-activity relationship (SAR) predictions derived from the respiratory sensitization reactive domain, respiratory sensitization integrated testing strategy (ITS) was established. RS assay showed 80% sensitivity, 100% specificity, and 90% accuracy. The respiratory sensitization ITS demonstrated more higher predictive capacity for respiratory sensitization than an individual test method, with 90% sensitivity, 100% specificity, and 95% accuracy when using the 20 reference chemicals. When using respiratory sensitization ITS, hazard identification and sub-categorization of potency as strong, moderate/weak, and negative were possible. As a non-animal testing approach, the respiratory sensitization ITS represents a significant milestone for regulating respiratory sensitizers., Competing Interests: Declarations. Conflicts of interest: No conflicts of interest, financial or otherwise, are declared by the authors., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The lack of in vivo accurate human liver models hinders the investigation of liver-related diseases, injuries, and drug-related toxicity, posing challenges for both basic research and clinical applications. Traditional cellular and animal models, while widely used, have significant limitations in replicating the liver's complex responses to various stressors. Liver organoids derived from human pluripotent stem cells, adult stem cells primary cells, or tissues can mimic diverse liver cell types, major physiological functions, and architectural features. Recent advancements in the field have shown that some liver organoids have sufficient accuracy to replicate specific aspects of the human liver's complexity. This review highlights recent progress in liver organoid research, with a particular emphasis on their potential for toxicity assessment and disease modeling. The intrinsic advantages of liver organoids include higher sensitivity and suitability for long-term studies, which enhance the predictive value in drug and nanomaterial toxicity testing. The integration of liver organoids with microfluidic devices enables the simulation of the liver microenvironment and facilitates high-throughput drug screening. The liver organoids also serve as ideal platforms for studying liver diseases such as hepatitis, liver fibrosis, viral liver diseases, and monogenic diseases. Additionally, this review discusses the advantages and limitations of liver organoids along with potential avenues for future research., Competing Interests: Declarations. Consent for publication: All the authors give their consent for publication in Stem Cell Research & Therapy. Ethical approval: Not applicable. AI usage: The authors declare that they have used artificial intelligence to polish the grammar of the manuscript. Conflict of interest: The authors declare that they have no conflicts of interest., (© 2025. The Author(s).)

The increase in production and innovation of chemicals that humans interface with has enhanced the need for rapid toxicity testing of new and existing chemicals. This need, along with efforts to reduce animal testing, has led to the development of high-throughput bioassays typically conducted in microplates. These bioassays offer time and resource advantages over traditional animal models; however, significant chemical losses can occur in microplates. Current methods for measuring chemical losses in microplates require extensive sample preparation and highly sensitive instruments. We propose the use of fluorescence spectroscopy to measure chemical losses in high-throughput bioassays as a low resource alternative to the existing methods. A fluorescent plate reader was used to develop methods for quantifying the aqueous concentrations of two chemicals, 2-hydroxynaphthalene and acridine, in microwells of a 96-well microplate. A high-throughput, 5 day embryonic zebrafish bioassay was used as the model bioassay for method development. Chemical losses were attributed to a combination of photodegradation, sorption, and uptake by the zebrafish embryos, kinetics of which were derived from a pseudo-first order model. Chemical uptake amount was calculated to be approximately 50% and 21% of the total chemical amount added for 2-hydroxynaphthalene and acridine, respectively. Unexpected cranial deformities were observed in the embryonic zebrafish, suggesting further investigation of potential additive toxicity of the ultraviolet radiation exposure from fluorescence measurements and chemical exposure is needed. Nonetheless, this novel method provides a rapid, low resource approach to measuring chemical losses in microplates that can be extended to a variety of autofluorescent chemicals and microplate-based bioassays.

The application of low-toxicity cosolvents in phytotoxicity tests is a common technique to enhance the distribution of non-water-soluble organic pollutants in the aqueous phase. In this study, the physiological and biochemical responses of rice seedlings to four commonly used organic solvents i.e., dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), pyridine, and Tween-80 were investigated using 16 parameters to assess their impacts on rice plants. Results from phytotoxicity tests indicated that these organic solvents caused varying toxic responses in rice plants and significantly affected the uptake and distribution of mineral nutrients within rice tissues. In order to select the best organic solvent for rice-based toxicity tests among the four solvents with optimal concentrations, the technique for order preference by similarity to ideal solution (TOPSIS) was employed to minimize the variations across different treatment groups. Three different weighting coefficients, namely entropy weight coefficients (EWCs), contribution factor coefficients (CFCs), and combination effective coefficients (CECs) were adopted to calibrate the initially created evaluation matrices. All estimations were judged by the relative closeness coefficients, in which the higher relative closeness value means the better solution to the optimal result. Among the four organic solvents evaluated by TOPSIS, pyridine at 1.24 mmol/L emerged as the most effective solution for rice-based phytotoxicity tests. Overall, this study represents the first attempt to comprehensively evaluate the toxic responses of cosolvents to rice plants using the TOPSIS method. It also offers valuable insights into a number of ecotoxicological applications like the analysis of solvent-generated extracts of environmental matrices from soils and sediments, or the detection of organic compounds induced toxicity with cosolvents., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 The Authors. Published by Elsevier Inc. All rights reserved.)

The Hydra vulgaris bioassay is recognized as sensitive invertebrate test species for toxicity assessment of real-life environmental mixtures for enforcement and monitoring investigations. The purpose of this study was to characterize the intra-laboratory variability, study the influence of environmental variables (temperature, luminosity, inter-individual and day of analysis) on ZnSO 4 toxicity, a reference model toxicant for hydra. The sublethal (effect concentration for 50 % of hydra-EC50) and lethal (lethal concentration for 50 % of hydra-LC50) were determined based on characteristic morphological changes for this species. The influence of water hardness, ammonia and dissolved oxygen for over 50 real-life environmental liquid mixtures (effluents and leachates) were examined and compared with rainbow trout and Daphnia magna acute lethality tests. A control chart for Zn was developed from over 40 trials yielding an 96 h LC50 of 0.7 mg/L (0.66-0.77 95 % confidence interval-CI) and EC50 of 0.19 mg/L (0.17-0.21 95 % CI). The influence of 8 different analysts, the trial days and luminosity did not significantly influence the LC50 and EC50. Only higher temperature significantly decreased the toxicity of ZnSO 4 within 15-30 °C range. The hydra bioassay was then practiced on more than 50 real-life effluents/leachates and compared with the 96 h rainbow trout and 48 h Daphnia magna survival tests. The data revealed that water hardness, dissolved oxygen and ammonia were not significantly correlated with either the LC50 or EC50 values. Moreover, the hydra LC50 data predictive (rank correlation of 0.6) the rainbow trout LC50 with the absence of false negatives. The hydra data were at least as if not more sensitive than the rainbow trout LC50 making it a relevant alternative method to reduce fish use for screening potentially toxic environmental mixtures., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest for this article entitled Towards the standardization of Hydra vulgaris bioassay for toxicity assessments of liquid samples., (Crown Copyright © 2024. Published by Elsevier Inc. All rights reserved.)

Due to its anatomical and physiological similarities to the human eye, the porcine eye serves as a robust model for biomedical research and ocular toxicity assessment. An air/liquid corneal culture system using porcine eyes was developed, and ex vivo epithelial wound healing was utilized as a critical parameter for these studies. Fresh pig corneas were processed for organ culture, with or without epithelial wounding. The corneas were cultured in a humidified 5% CO2 incubator at 37 °C in MEM, with or without testing agents. Corneal permeability and wound healing rates were measured, and epithelial cells and/or whole corneas can be processed for immunohistochemistry, western blotting, and qPCR for molecular and cellular analyses. This study describes a detailed protocol and presents two studies using this ex vivo system. The data show that porcine corneal organ culture, combined with epithelial wound healing, is a suitable ex vivo model for chemical toxicity testing, studying diabetic keratopathy, and identifying potential therapies.

Exposure of cell cultures at air-liquid interface (ALI), mimicking i.e. human lung surface, is believed to be one of the most realistic means to model toxicity of complex mixtures of pollutants on human health. The complexity of the close cooperation of "emissions source" and toxicology groups and of the instrumentation are among the limiting factors of ALI. In this work, the concepts of ALI exposure and real-world emissions monitoring using portable emissions monitoring systems (PEMS) are combined into a portable emissions or air toxicity system, for field deployment, including operation in moving vehicles. Cell cultures grown on 6 mm inserts are placed in an airtight 17x13x9 cm exposure box, where the sample is symmetrically distributed into 8 wells of a standard Transwell 24-well holder at 25 cm 3 /min/insert. In a 40x35x45 cm inner dimensions incubator, sample and control air are conditioned to 5 % CO 2 , 37 °C and >85 % humidity and drawn through 2-4 exposure boxes. Characterization with silver nanoparticles revealed 50 % particle losses at 15 nm and deposition rate of approximately 1.5 % at both 10 and 21 nm mean diameter. The system has undergone an extensive field validation, including 4 h of exposure and 2 h transport in a vehicle each day for 5 days, 5-day operation outside in vans and tents at -7 to +32 °C, long transport and test on a heavy-duty truck, during which cells were exposed to the diluted exhaust from the truck, this being the first known use of ALI exposure chamber as PEMS. The portable exposure chamber, along with a field-deployable auxiliary mobile base including a small laminar flow box, additional incubator and freezer, can be easily used to study the toxicity of various emissions, effluents and polluted air, aiming for a more relevant toxicity measure than chemical composition alone., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Michal Vojtisek-Lom reports financial support was provided by Czech Science Foundation. Jan Topinka reports financial support was provided by Horizon Europe. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Regulatory dose-response assessments traditionally rely on in vivo data and default assumptions. New Approach Methods (NAMs) present considerable opportunities to both augment traditional dose-response assessments and accelerate the evaluation of new/data-poor chemicals. This review aimed to determine the potential utilization of NAMs through a unified conceptual framework that compartmentalizes derivation of toxicity values into five sequential Key Dose-response Modules (KDMs): (1) point-of-departure (POD) determination, (2) test system-to-human (e.g. inter-species) toxicokinetics and (3) toxicodynamics, (4) human population (intra-species) variability in toxicodynamics, and (5) toxicokinetics. After using several "traditional" dose-response assessments to illustrate this framework, a review is presented where existing NAMs, including in silico , in vitro , and in vivo approaches, might be applied across KDMs. Further, the false dichotomy between "traditional" and NAMs-derived data sources is broken down by organizing dose-response assessments into a matrix where each KDM has Tiers of increasing precision and confidence: Tier 0: Default/generic values, Tier 1: Computational predictions, Tier 2: Surrogate measurements, and Tier 3: Direct measurements. These findings demonstrated that although many publications promote the use of NAMs in KDMs (1) for POD determination and (5) for human population toxicokinetics, the proposed matrix of KDMs and Tiers reveals additional immediate opportunities for NAMs to be integrated across other KDMs. Further, critical needs were identified for developing NAMs to improve in vitro dosimetry and quantify test system and human population toxicodynamics. Overall, broadening the integration of NAMs across the steps of dose-response assessment promises to yield higher throughput, less animal-dependent, and more science-based toxicity values for protecting human health.

Developmental toxicity (DevTox) tests evaluate the adverse effects of chemical exposures on an organism's development. Although current testing primarily relies on large mammalian models, the emergence of new approach methodologies (NAMs) is encouraging industries and regulatory agencies to evaluate novel assays. C. elegans have emerged as NAMs for rapid toxicity testing because of its biological relevance and suitability to high throughput studies. However, current low-resolution and labor-intensive methodologies prohibit its application for sub-lethal DevTox studies at high throughputs. With the recent advent of the large-scale microfluidic device, vivoChip, we can now rapidly collect 3D high-resolution images of ~ 1000 C. elegans from 24 different populations. While data collection is rapid, analyzing thousands of images remains time-consuming. To address this challenge, we developed a machine-learning (ML)-based image analysis platform using a 2.5D U-Net architecture (vivoBodySeg) that accurately segments C. elegans in images obtained from vivoChip devices, achieving a Dice score of 97.80%. vivoBodySeg processes 36 GB data per device, phenotyping multiple body parameters within 35 min on a desktop PC. This analysis is ~ 140 × faster than the manual analysis. This ML approach delivers highly reproducible DevTox parameters (4-8% CV) to assess the toxicity of chemicals with high statistical power., Competing Interests: Declarations. Competing interests: E.H., S.M., and A.B. are co-founders of vivoVerse, LLC and its Associates. A.D., A.S., A.L., E.H., S.M., and A.B. are inventors of several approved and pending patents. A.M. and S.G. at the time of their contribution are employed by vivoVerse, LLC and have no conflict of interest to declare., (© 2025. The Author(s).)

Background: This report addresses the reliability of results from rat Embryo-Fetal Developmental Toxicity (EFDT) studies. Recent literature discusses the roles of reproducibility, replicability, and other influences on scientific reliability. Reproducibility is a re-analysis of the original data, while replicability addresses the same question with a separate study of some type. Concordance of rat and rabbit studies has been addressed previously, but replication of single-species EFDT studies was not found in the literature. A modest modification of the rat study is therefore proposed to assess replicability and possibly enhance reliability., Methods: Regulatory guidelines were consulted and relevant literature was identified through online searches., Results: Each replicate EFDT (r-EFDT) study in rats would consist of half the mated females of the definitive study. Studies would start at the same or different times in one testing facility. Separate shipments of animals (non-littermates) are required. All other procedures would be protocol-driven. The micro- and macro-environments of the animals would be held as constant as possible. Justification, design options, and interpretation methods are discussed., Conclusion: Besides adding reliability, other benefits include reduced animal usage, and potentially reduced cost and time to final reports. By reducing the need for repeated studies due to questionable results, this modified study is viewed as a more efficient use of costly resources. The r-EFDT study design could easily be adapted to assess replicability of rabbit EFDT and some general toxicity studies. Future replicate studies are needed to critically evaluate replicability and the overall impact on study reliability., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Developmental toxicity is key human health endpoint, especially relevant for safeguarding maternal and child well-being. It is an object of increasing attention from international regulatory bodies such as the US EPA (US Environmental Protection Agency) and ECHA (European CHemicals Agency). In this challenging scenario, non-test methods employing explainable artificial intelligence based techniques can provide a significant help to derive transparent predictive models whose results can be easily interpreted to assess the developmental toxicity of new chemicals at very early stages. To accomplish this task, we have developed web platforms such as TIRESIA and TISBE.Based on a benchmark dataset, TIRESIA employs an explainable artificial intelligence approach combined with SHAP analysis to unveil the molecular features responsible for calculating the developmental toxicity. Descending from TIRESIA, TISBE employs a larger dataset, an explainable artificial intelligence framework based on a fragment-based fingerprint encoding, a consensus classifier, and a new double top-down applicability domain. We report here some practical examples for getting started with TIRESIA and TISBE., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The safety and developmental neurotoxicity (DNT) potential of chemicals remain critically understudied due to limitations of current in vivo testing guidelines, which are low throughput, resource-intensive, and hindered by species differences that limit their relevance to human health. To address these issues, robust New Approach Methodologies (NAMs) using deeply characterized cell models are essential. This study presents the comprehensive transcriptomic characterization of two advanced human-induced pluripotent stem cell (hiPSC)-derived models: a 2D adherent and a 3D neurosphere model of human neural progenitor cells (hiNPCs) differentiated up to 21 days. Using high-throughput RNA sequencing, we compared gene expression profiles of 2D and 3D models at three developmental stages (3, 14, and 21 days of differentiation). Both models exhibit maturation towards post-mitotic neurons, with the 3D model maturing faster and showing a higher prevalence of GABAergic neurons, while the 2D model is enriched with glutamatergic neurons. Both models demonstrate broad applicability domains, including excitatory and inhibitory neurons, astrocytes, and key endocrine and especially the understudied cholinergic receptors. Comparison with human fetal brain samples confirms their physiological relevance. This study provides novel in-depth applicability insights into the temporal and dimensional aspects of hiPSC-derived neural models for DNT testing. The complementary use of these two models is highlighted: the 2D model excels in synaptogenesis assessment, while the 3D model is particularly suited for neural network formation as observed as well in previous functional studies with these models. This research marks a significant advancement in developing human-relevant, high-throughput DNT assays for regulatory purposes., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Prof. Ellen Fritsche and Dr. Katharina Koch are shareholders of DNTOX GmbH offering neurotoxicity testing services. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. This output reflects only the authors’ view, and the European Union cannot be held responsible for any use that may be made of the information contained therein., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The development of novel drug candidates is a current challenge in pharmacology where therapeutic benefits must exceed side effects. Toxicology testing is therefore a fundamental step in drug discovery research. Herein, we describe the first line of toxicology testing program, consisting in cell-based high-throughput screening assays, which have the advantage of being easy, rapid, cheap, and reproducible while providing quantitative information. We illustrate MTT and Crystal Violet assays, two common colorimetric tests able to assess both cytostatic and cytotoxic effects, respectively, of a drug candidate. MTT assay allows evaluation of cellular metabolic activity, by which cell viability can be inferred; Crystal Violet staining is directly correlated with attached viable cells, thus allowing direct assessment of cell survival and death. Therefore, combination of the two methodologies represents a useful tool for predicting drug sensitivity and efficacy, the first milestones in pre-clinical toxicology workflow., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The use of adverse effect data from animals as the gold standard in regulatory toxicology has a long tradition dating back to the 1960s. It has also been increasingly criticised, based on both scientific and animal welfare concerns, and yet, animal studies remain the gold standard in most areas of toxicology to this very day. In the 1980s, when the first generation of non-animal methods were evaluated as alternatives to animal testing, it was logical to compare the 'new' data obtained with historical animal data. This worked reasonably well for simple endpoints, such as skin and eye irritation, but became problematic for the more complex systemic endpoints, since in these cases, the in vivo effects are not directly comparable to those observed in in vitro systems. While the need to redefine the gold standard is not new, there is still no consensus on how to do so. We propose a consistent principle that avoids the need for animal reference data, while also ensuring an equivalent or better level of protection. We argue that the gold standard can be redefined, or rather bypassed, by focusing on risk management outcomes rather than the outputs of animal methods. This allows us to more efficiently protect human health and the environment, ensuring the safe use of chemicals while also identifying less hazardous chemicals for use as substitutes. We describe how this might work out for two main contexts of use: classification and labelling, and risk assessment. This has implications for the implementation of the EU Commission Roadmap toward the phasing out of animal testing in chemical safety assessments., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Read-Across (RAx) serves as a strategy to fill a data gap in the toxicological profile of a substance (target) using existing information on similar source substances. The principle is applied also to a category of substances for which similarity may follow a regular trend. Demonstration of similarity is not trivial and requires the analysis of different steps, starting from the precise analytical characterization of both target and source substances and including the analysis of the impact that each minor difference can have on the final outcome. Application of QSARs and performing new experimental tests within the new approach methodologies (NAMs) is necessary to increase confidence in the final prediction and reduce the uncertainty., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

While high-throughput (HTP) assays have been proposed as platforms to rapidly assess reproductive toxicity, there is currently a lack of established assays that specifically address germline development/function and fertility. We assessed the applicability domains of yeast (S. cerevisiae) and nematode (C. elegans) HTP assays in toxicity screening of 124 environmental chemicals, determining their agreement in identifying toxicants and their concordance with reproductive toxicity in vivo. We integrated data generated in the two models and compared results using a streamlined, semi-automated benchmark dose (BMD) modeling approach. We then extracted and modeled relevant mammalian in vivo data available for the matching chemicals included in the Toxicological Reference Database (ToxRefDB). We ranked potencies of common compounds using the BMD and evaluated correlation between the datasets using Pearson and Spearman correlation coefficients. We found moderate to good correlation across the three data sets, with r = 0.48 (95 % CI: 0.28-1.00, p<0.001) and r s = 0.40 (p=0.002) for the parametric and rank order correlations between the HTP BMDs; r = 0.95 (95 % CI: 0.76-1.00, p=0.0005) and r s = 0.89 (p=0.006) between the yeast assay and ToxRefDB BMDs; and r = 0.81 (95 % CI: 0.28-1.00, p=0.014) and r s = 0.75 (p=0.033) between the worm assay and ToxRefDB BMDs. Our findings underscore the potential of these HTP assays to identify environmental chemicals that exhibit reproductive toxicity. Integrating these HTP datasets into mammalian in vivo prediction models using machine learning methods could further enhance their predictive value in future rapid screening efforts., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Glyphosate is an acidic herbicide reported to contaminate water sources around the globe. Glyphosate alters the pH of a solution depending upon the concentration and buffering capacity of the solution in which it is present. Hence, toxicity observed in laboratory-based studies could be caused by the chemical or acidic pH if the solution is not adjusted to neutral conditions, confounding toxicity assessments. When reviewing zebrafish glyphosate toxicity studies, major discrepancies were noted among the published literature. Moreover, it was discovered that most of these studies did not mention pH or neutralization of the test solution. Thirty-six articles were identified when restricting the search from January 2009 through April 2024 to studies testing glyphosate toxicity (as glyphosate or glyphosate-based herbicides) in zebrafish and assessed for time of exposure, test concentrations, and mention or assessment of pH in exposure solutions. Additionally, toxicity curves for unadjusted pH and adjusted pH conditions for glyphosate were also determined in developing zebrafish from 1 to 120 hours post fertilization (hpf), to further clarify and support pH influence of glyphosate in these toxicity tests. Furthermore, a pH toxicity curve was established for the same developmental period to address if the divergence noted in the literature was based on glyphosate's influence on acidity of the exposure solution. Results showed that at concentrations greater than 10 ppm (mg/L), the pH of the water used in the experiments at chemistry parameters commonly used in zebrafish toxicity studies reduced to 5.5. As the glyphosate concentration increased, the pH continued to drop as low as 2.98. When comparing unneutralized and neutralized glyphosate solutions, the 120 hpf-LC 50 without neutralization was close to 50 ppm, while minimal lethality was observed up to 1000 ppm in the neutralized solutions. Findings were then compared to the thirty-six zebrafish glyphosate toxicity studies for alignment of findings with glyphosate or pH toxicity. Eighteen of the studies included treatment concentrations less than 10 ppm with pH likely not to influence reported outcomes. Of the 18 remaining studies at higher concentrations likely to influence pH, only one reported neutralizing their exposure solutions. Two additional studies mentioned pH as a potential driving factor but did not repeat in neutral conditions. As a result, 17 of the 36 studies are observing primarily pH toxicity in the glyphosate assessments. Based on these findings, caution is warranted in interpreting results of acidic environmental contaminants in cases where pH of exposure solutions is not stated., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

The Virtual Human Platform for Safety Assessment (VHP4Safety) project aims to build a virtual human platform (VHP) to protect human health and revolutionize the safety assessment of chemicals and pharmaceuticals by transitioning from animal-based to human-based approaches. The goal of this article is to introduce the project and its interdisciplinary approach to co-creation with mul­tiple academic, regulatory, industrial and societal partners covering the entire safety assessment knowledge chain. Three research lines drive the project: 1) building the VHP; 2) feeding the VHP with human data; and 3) implementing the VHP. The project focusses on three case studies that incor­porate human-relevant scenarios not included in current animal-based safety assessment strategies. The VHP is built on tools and services, including pharmacokinetic and computational models, and integrates several data sources within each case study, including data on human physiology, epi­demiology, toxicokinetic and -dynamic parameters, as well as data on chemical characteristics and exposures. In addition, the VHP integrates new data generated within the project using new approach methodologies representing key events within adverse outcome pathways. Implemen­tation of the VHP is investigated using an innovation systems approach, engaging stakeholders, and organizing training and education. Central to the VHP4Safety project is our co-creative approach, which is facilitated by biannual designathons and hackathons that foster active involvement of all project participants from over 30 partner organizations. By integrating technological innovations with transparency and stakeholder collaboration, the VHP4Safety project will help shape the tran­sition to next generation safety assessment in which animal testing becomes redundant.

The workshop titled State of the Science on Assessing Developmental Neurotoxicity Using New Approach Methods was co-organized by University of Maryland’s Joint Institute for Food Safety and Applied Nutrition (JIFSAN) and the U.S. Food and Drug Administration’s (FDA) Center for Food Safety and Applied Nutrition (CFSAN; now called the Human Foods Program), and was hosted by FDA in College Park, MD on November 14-15, 2023. This event convened experts from inter­national organizations, governmental agencies, industry, and academia to explore the transition from traditional in vivo tests to innovative new approach methods (NAMs) in developmental neurotoxicity (DNT) testing. The discussions emphasized the heightened vulnerability of the developing human brain to toxic exposures and the potential of NAMs to provide more ethical, economical, and scientifically robust alternatives to traditional testing. Various NAMs for DNT were discussed, including in silico, in chemico, in vitro, non-mammalian whole organisms, and novel mammalian approaches. In addition to progress in the field, the workshop discussed ongoing chal­lenges such as expectations to perfectly replicate the complex biology of human neurodevelopment and integration of DNT NAMs into regulatory frameworks. Presentations and panel discussions pro­vided a comprehensive overview of the state of the science, assessed the capabilities and limitations of current DNT NAMs, and outlined critical next steps in advancing the field of DNT testing.

The nematode Caenorhabditis elegans, widely recognized as a model organism due to its ease of breeding and well-characterized genomes, boasts complete digestive, reproductive, and endocrine systems, as well as conserved signaling pathways shared with mammals. It has become an invaluable resource for metabolomics research, particularly in examining responses to chemical or environmental factors and toxicity assessments. In this article, we provide detailed, step-by-step protocols for cultivating C. elegans and conducting metabolomics analyses, specifically focusing on sample preparation for GC-MS analysis in response to toxic compound treatments. We highlight the critical choice of extraction solvent, introducing two representative systems for extracting metabolites from C. elegans., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

New drug modalities offer life-saving benefits for patients through access to previously undruggable targets. Yet these modalities pose a challenge for the pharmaceutical industry, as side effects are complex, unpredictable, and often uniquely human. With animal studies having limited predictive value due to translatability challenges, the pharmaceutical industry seeks out new approach methodologies. Microphysiological systems (MPS) offer important features that enable complex toxicological processes to be modeled in vitro such as ( a ) an adjustable complexity of cellular components, including immune components; ( b ) a modifiable tissue architecture; ( c ) integration and monitoring of dynamic mechanisms; and ( d ) a multiorgan connection. Here we review MPS studies in the context of four clinical adverse events triggered by new drug modalities: peripheral neuropathy, thrombocytopenia, immune-mediated hepatotoxicity, and cytokine release syndrome. We conclude that while the use of MPS for testing new drug modality-induced toxicities is still in its infancy, we see strong potential going forward.

Given the increased concerns over reproducibility, replicability, and reliability of scientific studies, the embryo-fetal developmental toxicity (EFDT) study was examined from this perspective together with the classic 3Rs. These 6Rs contribute to external validity (i.e., applicability to human safety). Species differences and experimental conditions lower the validity of all preclinical studies to variable extents. The apparent lack/paucity of demonstrated replicate EFDT studies lowers reliability and thus validity. I make suggestions intended to add reliability for the continued use of these studies, which are a cornerstone for assessing safety during human pregnancies. Experienced readers will recognize that the suggestions would also apply to other guideline-mandated developmental and reproductive toxicity and toxicity studies., (© 2024 Wiley Periodicals LLC.)

The use of nanoparticles (NPs) in treatment of diseases have increased exponentially recently, giving rise to the science of nanomedicine. The safety of these NPs in humans has also led to the science of nanotoxicology. Due to a dearth of both readily available models and precise bio-dispersion characterization techniques, nanotoxicological research has obviously been constrained. However, the ensuing years were notable for the emergence of improved synthesis methods and characterization tools. Major advances have been made in linking certain physical variables, paralleling improvements in characterization size, shape, or coating factors to the resulting physiological reactions. Although significant progress has been a contribution to the development of nanotoxicology, however, it faces numerous difficulties and technical constraints distinct from those of conventional toxicological assessment as it attempts to improve the therapeutic effects of medicines. Determining thorough characterization standards, standardizing dosimetry, assessing the kinetics of ions dissolving and enhancing the accuracy of in vitro-in vivo correlation efficiency, also defining restrictions on exposure protection are some of the most important and pressing concerns. This article will explore the past advancement and potential prospects of nanotoxicology, standard models, emphasizing significant findings from earlier studies and examining current challenges, giving insight on the way forward.

Hormone signaling plays an essential role during fetal life and is vital for brain development. Endo­crine-disrupting chemicals can interfere with the hormonal milieu during this critical time-period, disrupting key neurodevelopmental processes. Hence, there is a need for the development of assays that evaluate developmental neurotoxicity (DNT) induced by an endocrine mode of action. Herein, we evaluated the neural progenitor C17.2 cell line as an in vitro test system to aid in the detection of endocrine disruption-induced DNT. For this, C17.2 cells were exposed during 10 days of dif­ferentiation to agonists and antagonists of the thyroid hormone (THR), glucocorticoid (GR), retinoic acid (RAR), retinoic x (RXR), oxysterol (LXR), estrogen (ER), androgen (AR), and peroxisome prolif­erator activated delta (PPARβ/δ) receptors, as well as to the agonist of the vitamin D (VDR) receptor. Upon exposure and differentiation, neuronal morphology (neurite outgrowth and branching) and the percentage of neurons in culture were assessed by immunofluorescence. For this, the cells were stained for βIII-tubulin (neuronal marker). C17.2 cells decreased neurite outgrowth and branching in response to RAR, RXR and PPARβ/δ agonists. Exposure to the GR agonist increased the number of cells differentiating into neurons, while exposure to the RXR agonist had the opposite effect. With this approach, we demonstrate that C17.2 cells are responsive to GR, RAR, RXR, and PPARβ/δ agonists and hence could be useful to develop a test system for hazard assessment of endocrine disruption-induced DNT.

Hepatotoxicity can lead to the discontinuation of approved or investigational drugs. The evaluation of the potential hepatoxicity of drugs in development is challenging because current models assessing this adverse effect are not always predictive of the outcome in human beings. Cell lines are routinely used for early hepatotoxicity screening, but to improve the detection of potential hepatotoxicity, in vitro models that better reflect liver morphology and function are needed. One such promising model is human liver microtissues. These are spheroids made of primary human parenchymal and nonparenchymal liver cells, which are amenable to high throughput screening. To test the predictivity of this model, the cytotoxicity of 152 FDA (US Food & Drug Administration)-approved small molecule drugs was measured as per changes in ATP content in human liver microtissues incubated in 384-well microplates. The results were analyzed with respect to drug label information, drug-induced liver injury (DILI) concern class, and drug class. The threshold IC50ATP-to-Cmax ratio of 176 was used to discriminate between safe and hepatotoxic drugs. "vMost-DILI-concern" drugs were detected with a sensitivity of 72% and a specificity of 89%, and "vMost-DILI-concern" drugs affecting the nervous system were detected with a sensitivity of 92% and a specificity of 91%. The robustness and relevance of this evaluation were assessed using a 5-fold cross-validation. The good predictivity, together with the in vivo-like morphology of the liver microtissues and scalability to a 384-well microplate, makes this method a promising and practical in vitro alternative to 2D cell line cultures for the early hepatotoxicity screening of drug candidates., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society of Toxicology.)

Currently, the OECD has adopted three defined approaches (DAs) for eye hazard identification of non-surfactant liquids and solids (TG 467) according to the three UN GHS categories. We are now expanding the applicability domain with a new DA for chemicals having surfactant properties (DASF). It is based on a combination of recombinant human cornea-like epithelium test methods (TG 492: EpiOcular™ EIT or SkinEthic™ HCE EIT) and a modification of the Short Time Exposure (TG 491) method. The aim of the current study was to compare the performance of the DASF with the performance of other NAMs currently included in the OECD TGs and with the classification based on the Draize eye test to identify potential additional DAs. The minimum performance criteria (75% Cat. 1, 50% Cat. 2, 70% No Cat.) used for the adoption of the DAs currently included in TG 467 were used for this purpose. The DASF identified 90.9% of Cat. 1 (N = 23), 77.8% of Cat. 2 (N = 9), and 76.0% of No Cat. (N = 17) surfactants, meeting the minimum performance criteria. Some of the NAMs that are currently included in the TGs seem promising methods to become part of a DA to identify Cat. 1 or No Cat. for eye hazard assessment of surfactants. However, the number of surfactants that have been tested to evaluate their reliability and relevance was often too low. To date, the DASF is the only DA that has evaluated a sufficiently large number of surfactants and whose performance meets the minimum performance criteria.

Bisphenol A (BPA) is a widely used industrial compound found in polycarbonate plastics, epoxy resin, and various polymer materials, leading to its ubiquitous presence in the environment. The toxicity of BPA to aquatic organisms has been well documented following in vivo exposure scenarios, with known cytotoxic and endocrine-disrupting effects. As such, BPA was used in this study as a well-characterized chemical to implement more ethical and resource-efficient scientific practices in toxicity testing through new approach methods (NAMs). Due to the frequent use of Daphnia spp. as a model organism in toxicology, we developed an in vitro cell culture system from Daphnia magna embryos, with optimized medium to support cell longevity. The cultures were maintained for up to two months, demonstrating their stability and suitability for cytotoxicity studies. Using this novel system, lethal concentration 50 (LC 50 ) values were determined at the 24 and 48 h time points following BPA exposure. Subsequently, oxidative stress, endocrine disruption, and DNA damage were assessed through gene expression, activity assays, and a comet assay in BPA-exposed cells. LC 50 values of 52 µM and 20 µM BPA were calculated after 24 and 48 h exposures, respectively. BPA cells exposed to 20 and 52 µM had significantly increased GSH, GPx, and GST activity levels. mRNA expression analysis revealed significant upregulations in the expression of hsp70, hsp90, gst, gpx, vtg1, and cyp4, with downregulations of sod, cat, and ecr following BPA exposure. Furthermore, comet assays showed a significantly higher level of DNA damage induced by BPA compared to controls, with greater comet and tail lengths. This study established a novel in vitro Daphnia model, using BPA as a case study for determining toxic effects, further highlighting the importance and applicability of utilizing alternative methods in ecotoxicological research through reducing animal use., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier B.V.)

An essential aspect of the EU’s Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) regulation is the European Chemicals Agency’s (ECHA) evaluation of testing proposals submitted by registrants to address data gaps. Registrants may propose adaptations, such as read-across, to waive standard testing; however, it is widely believed that ECHA often finds justifications for read-across hypotheses inadequate. From 2008 to August 2023, 2,630 testing proposals were submitted to ECHA; of these, 1,538 had published decisions that were systematically evaluated in this study. Each document was manually reviewed and information extracted for further analyses, focusing on 17 assessment elements (AEs) from the Read-Across Assessment Framework (RAAF) and testing proposal evaluations (TPE). Each submission was classified as to the AEs relied upon by the registrants and by ECHA. Data was analyzed for patterns and associations. Adaptations were included in 23% (350) of proposals, with analogue (168) and group (136) read-across being most common. Of the 304 read-across hypotheses, 49% were accepted, with group read-across showing significantly higher odds of acceptance. Data analysis examined factors such as tonnage band (Annex), test guidelines, hypothesis AEs, and structural similarities of target and source sub­stances. While decisions were often context-specific, several significant associations influencing acceptance emerged. Overall, this analysis provides a comprehensive overview of 15 years of experience with testing proposal-specific read-across adaptations by both registrants and ECHA. These data will inform future submissions as they identify most critical AEs to increase the odds of read-across acceptance.

The rapid increase in the production and global use of chemicals and their mixtures has raised concerns about their potential impact on human and environmental health. With advances in analytical techniques, in particular, high-resolution mass spectrometry (HRMS), thousands of compounds and transformation products with potential adverse effects can now be detected in environmental samples. However, identifying and prioritizing the toxicity drivers among these compounds remain a significant challenge. Effect-directed analysis (EDA) emerged as an important tool to address this challenge, combining biotesting, sample fractionation, and chemical analysis to unravel toxicity drivers in complex mixtures. Traditional EDA workflows are labor-intensive and time-consuming, hindering large-scale applications. The concept of high-throughput (HT) EDA has recently gained traction as a means of accelerating these workflows. Key features of HT-EDA include the combination of microfractionation and downscaled bioassays, automation of sample preparation and biotesting, and efficient data processing workflows supported by novel computational tools. In addition to microplate-based fractionation, high-performance thin-layer chromatography (HPTLC) offers an interesting alternative to HPLC in HT-EDA. This review provides an updated perspective on the state-of-the-art in HT-EDA, and novel methods/tools that can be incorporated into HT-EDA workflows. It also discusses recent studies on HT-EDA, HT bioassays, and computational prioritization tools, along with considerations regarding HPTLC. By identifying current gaps in HT-EDA and proposing new approaches to overcome them, this review aims to bring HT-EDA a step closer to monitoring applications., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Toxic emission from industrial activity is a serious problem, particularly with regard to the quality of water. Thus, the ISO 11348-3 standard for assessing water quality has been established. This method is used to determine solution toxicity from the bioluminescence inhibition of Aliivibrio fischeri. However, the accuracy of measurements is influenced by the selection of individual reaction time points. This study explores the utility of the area under the curve (AUC) method in water quality detection and evaluates how A. fischeri responds to three toxicants, namely ethanol, acetone, and zinc sulfate, over time. The half-maximal effective concentrations of these three substances were found to be 10.13 %, 5.02 %, and 19.49 mg/L, respectively. Compared with the results from individual reaction time point assessments, the results of AUC comprehensively captured the effects of the toxicants, including time-dependent effects and hormetic effects, by capturing dynamic changes under different toxicant concentrations and reaction times. Therefore, AUC analysis mitigates the pitfalls associated with individual reaction times and provides a more accurate and reliable assessment method for water quality detection, contributing to a better understanding of the impact of toxic substances on aquatic environments., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Yung-Sheng Lin reports financial support was provided by National Science and Technology Council, Taiwan. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Standard information reporting helps to ensure that assay conditions and data are consistently reported and to facilitate inter-laboratory comparisons. Here, we present recommendations on minimum information for reporting on the TEER (trans-epithelial/endothelial electrical resistance) assay (MIRTA). The TEER assay is extensively used to evaluate the health of an epithelial/endothelial cell culture model and as an indicator of the potential toxicity of a test substance. This publication is the result of an international collaboration─called the RespTox (Respiratory Toxicity) Collaborative─through which twelve laboratories shared their protocols for assessing the barrier function of respiratory epithelial cells using the TEER assay following exposure to substances. The protocols from each laboratory were reviewed to identify general steps for performing the TEER assay, interlaboratory differences between steps, the rationale for differences, whether these differences impact results or cross-laboratory comparisons between TEER measurements. While the MIRTA recommendations are focused on respiratory epithelial cell systems, these recommendations can be adapted for other cell systems that form barriers. The use of these recommendations will support data transparency and reproducibility, reduce challenges in data interpretation, enable cross-laboratory comparisons, help assess study quality, and facilitate the incorporation of the TEER assay into national and international testing guidance., Competing Interests: Declarations. Conflict of interest: The authors declare no conflict of interest., (© 2024. The Author(s).)

The manual counting of juveniles in enchytraeid soil toxicity tests is time-consuming, labour-intensive, repetitive, prone to subjectivity, but can potentially be automated through deep learning methods using convolutional neural networks. This study investigated if RootPainter can be used as a tool to automatically quantify Enchytraeus crypticus juveniles in toxicity tests using different soil types. Toxicity tests were performed following OECD guideline 220 using five different pesticides (two fungicides and three insecticides) and four different soil types (three OECD artificial soils and one natural LUFA 2.2 soil). Manual counts were done by three different operators, with each operator counting images for one pesticide. Correlations between automated and manual counts were strong and significant in all four soils for all operators, with Pearson's correlation coefficients ≥ 0.955 and intraclass comparability coefficients ≥ 0.936. Toxicity values (EC 50 and EC 10 ) calculated from the manual and automated counts were within a factor of 0.85 - 1.30. Overall, the results show that RootPainter is a suitable tool for a reliable, repeatable and accurate quantification of enchytraeid juveniles, and can eliminate the time-consuming manual counting process., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Drug toxicity is an important cause of chronic liver damage, which in the long term can lead to impaired bone homeostasis through an imbalance in the liver-bone axis. For instance, non-steroidal anti-inflammatory drugs (e.g., diclofenac), which are commonly used to control pain during orthopaedic interventions, are known to reduce bone quality and are the most prevalent causes of drug-induced liver damage. Therefore, we used human cell lines to produce a stable, reproducible, and reliable in vitro liver-bone co-culture model, which mimics the impaired bone homeostasis seen after diclofenac intake in vivo. To provide the best cell culture conditions for the two systems, we tested the effects of supplements contained in liver and bone cell culture medium on liver and bone cell lines, respectively. Additionally, different ratios of culture medium combinations on bone cell scaffolds and liver spheroids' viability and function were also analysed. Then, liver spheroids and bone scaffolds were daily exposed to 3-6 µM diclofenac alone or in co-culture to compare and evaluate its effect on the liver and bone system. Our results demonstrated that a 50:50 liver:bone medium combination maintains the function of liver spheroids and bone scaffolds for up to 21 days. Osteoclast-like cell activity was significantly upregulated after chronic exposure to diclofenac only in bone scaffolds co-cultured with liver spheroids. Consequently, the mineral content and stiffness of bone scaffolds treated with diclofenac in co-culture with liver spheroids were significantly reduced. Interestingly, our results show that the increase in osteoclastic activity in the system is not related to the main product of diclofenac metabolism. However, osteoclast activation correlated with the increase in oxidative stress and inflammation associated with chronic diclofenac exposure. In summary, we established a long-term stable liver-bone system that represents the interaction between the two organs, meanwhile, it is also an outstanding model for studying the toxicity of drugs on bone homeostasis., Competing Interests: Declarations. Conflict of interest: The authors declare that they have no conflict of interest. Ethical approval: This study does not contain any studies with human or animal subjects performed by any of the authors., (© 2024. The Author(s).)

The use of rare earth elements has increased in recent years, leading to a rise in environmental concentrations. Despite the growth in number of studies regarding toxicity, knowledge gaps remain. For Daphnia magna, standardized test methods involve exposure periods of either 48 h or 21 days to assess toxicological effects. In this study, the exposure period was adjusted to 7 days to evaluate sublethal endpoints not measurable in 48-h tests. Additionally, this approach enabled us to obtain results within a shorter time frame than that required for 21-day tests. This study focused on the individual toxicity of lanthanum (La) and gadolinium (Gd) to Daphnia magna. We assessed mortality, feeding rate, somatic growth, and maturity under static conditions, modifying the media by adding MOPS buffer to maintain an initial pH of 6.8 and providing Raphidocelis subcapitata as a daily food source. Results showed that the solubility of La decreased considerably, with the highest recovery rate dropping from 133.33% at the start to 32.73% by the end of the 7-day exposure period. In contrast, Gd solubility remained stable, with a recovery rate of 86.88% at the start and 81.30% at the end of the test. Daily lethal concentrations (LC x ) were calculated, revealing LC 10 values on the first day, LC 20 on the second day, and LC 50 by the third day for La and the second day for Gd. By the test's end, the LC 10 , LC 20 , and LC 50 values were 30.40, 78.96, and 403.67 µg L -1 for La, and 10.67, 33.73, and 241.28 µg L -1 for Gd. For the sublethal endpoints, maturity was the most sensitive endpoint with the EC 20 and EC 10 corresponding to 0.79 and 0.26 µg L -1 for La and 0.39 and 0.14 µg L -1 for Gd. Gd had a higher toxicity in all endpoints assessed. While a thorough comparison to existing literature remains challenging due to variations in endpoints assessed, the methodology employed in this study yielded a range of informative results. This approach provides a useful range-finding test for Daphnia magna toxicity assessments, particularly for preliminary screening, and may complement standardized methodologies., Competing Interests: Declarations. Ethical approval: Not applicable. Consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Concentration-dependent cytotoxicity experiments are frequently used in toxicology. Although it has been reported that an adequate choice of concentrations improves the quality of the statistical inference substantially, a recent literature review of three major toxicological journals has shown that the corresponding methods are rarely used in toxicological practice. In this study the performance of different sets of concentrations, also called designs, are analyzed, while the overall goal is to promote the advantages of optimal design procedures and to present a user-friendly guideline for planning new cytotoxicity concentration-response experiments. We compare the frequently used log-equidistant design to a Bayesian design, which is constructed by methods of optimum design theory. Using both a dense data set of concentration-cytotoxicity data of valproic acid (VPA) and regular assay data of 104 substances, the performance of the different designs is analyzed in two scenarios, where detailed previous knowledge on VPA is available or not. The results show that it is critical to apply a specific design strategy to determine optimal concentrations for cytotoxicity testing. In particular, the Bayesian design technique with and without incorporating pre-existing knowledge of a specific test substance resulted in a more precise statistical inference than the other used designs. Finally, we present a guideline for upcoming experiments and an accessible user-friendly Shiny app (see http://shiny.statistik.tu-dortmund.de:8080/app/occe )., Competing Interests: Declarations. Conflict of interest: The authors declare that they have no conflict of interest. Ethical approval: Not applicable. Consent to participate: Not applicable. Consent for publication: Not applicable. Code availability: The corresponding Shiny app can be found online openly accessible at http://shiny.statistik.tu-dortmund.de:8080/app/occe ., (© 2024. The Author(s).)

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., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

The increasing emphasis on animal welfare and ethics, as well as the considerable time and cost involved with animal testing, have prompted the replacement of many aspects of animal testing with alternative methods. In the area of developmental toxicity, the embryonic stem cell test (EST) has played a significant role. The EST evaluates toxicity using mouse embryonic stem cells and somatic cells and observes the changes in heartbeat after cardiac differentiation. Nevertheless, the EST is a relatively complex testing process, and an in vitro test requires a long duration. Several attempts have been made to develop a more straightforward testing method than the EST, with improved reproducibility and accuracy, leading to the development of the embryoid body test (EBT) . Unlike the EST, which involves cardiac differentiation stages, the EBT verifies toxicity by measuring the changes in the area of the embryoid body. Despite its short testing period and simple procedure, the EBT offers high accuracy and reproducibility and is fully validated through two rounds of validation, making it ready for practical application. The EBT is expected to play a crucial role in the rapidly increasing demand for alternative methods to animal testing, particularly for screening early developmental toxicity., Competing Interests: The authors declare no conflicts of interest.

Hemocytes are the circulating immune-competent cells in bivalve mollusks and play a key role in several important functions of cell-mediated innate immunity. During the early stages of the immune response, hemocytes actively migrate to the site of infection. This inherent motility is a fundamental characteristic of these cells. It represents a key cellular function that integrates multiple processes, such as cell adhesion, cell signaling, cytoskeletal dynamics, and changes in cell volume. Therefore, alterations in cell motility following exposure to drugs or pollutants can serve as a useful toxicological endpoint. Despite the fundamental role of cell motility in cellular physiology, it has been poorly investigated from a toxicological perspective. This work proposes a novel in vitro method for the rapid and sensitive assessment of the toxicity and ecotoxicity of pollutants, based on evaluating the hemocyte motility of Mytilus galloprovincialis. We developed a cell motility assay on hemocytes adhering to the bottom of a 96-well polystyrene microplate. Following exposure to increasing concentrations of drugs, cell trajectories, and velocities were quantified by cell tracking under time-lapse microscopy, allowing us to measure the effects on hemocyte motility. Due to the ease of hemocyte collection from the animals in a relatively non-invasive manner, the proposed method offers an alternative test for screening the effects and mechanisms of action of pollutants and drugs. It aligns with the 3Rs (Replacement, Reduction, and Refinement) criteria, addressing ethical concerns and contributing to the reduction of vertebrate in vivo animal testing.

For many years overnight fasting of rats before the collection of clinical pathology blood samples and necropsy has been a common procedure in toxicological studies for regulatory purposes. The fasting was thought to minimize the intragroup variability for clinical pathology and organ weights. However, depriving rats of food overnight will impact animal welfare by interfering with the general metabolism and may result in physiological and behavioural changes. The effects of overnight fasting in comparison to rats that were not fasted prior to necropsy was investigated in lactating rats based on an evaluation of organ weights, haematological, and clinical biochemical parameters. The results of 92 OECD 422 studies were analysed (i.e., Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test) of which approximately half of the studies incorporated fasting prior to necropsy and the other half did not. Terminal body and organ weights from all 92 studies were evaluated. Clinical pathology was included in 78 of these 92 studies. Decreased glucose levels following fasting had been reported in the literature but were not observed when comparing 39 studies with fasted conditions versus 39 studies with non-fasted conditions. Both literature and the analysed database exhibited a reduction in liver weight, alanine aminotransferase, and alkaline phosphatase levels in overnight fasted groups. These differences between fasted and non-fasted states are considered of little account as study results are always interpreted based on the differences in parameter values between treated animals compared to control animals within a study. Contrarily to previously suggested, intragroup variability was lower in the majority of parameters in non-fasted animals. According to laboratory historical data, clinical pathology and organ weight parameters are found to be very similar in male and female rats, indicating that the results of this study may be extrapolatable to non-lactating female and male rats. Based on these comparisons, it is recommended not to fast rats prior to necropsy but to continue feeding all rats ad libitum, to minimize physiological changes in these animals, to reduce variability, improve animal welfare and thereby improve the scientific value of study results., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)