2026-04-01
Yesenia Lopez Hernandez, Horatiu Vinerean, Saurabh Aggarwal
Isotonitazene is a highly potent benzimidazole-derived synthetic opioid increasingly implicated in fatal intoxications. Despite growing forensic detection, controlled in vivo toxicity data remain limited. We determined the median lethal dose (LD50) of isotonitazene in adult male and female Fischer 344 rats using the Dixon up-and-down procedure following single intraperitoneal administration. Mortality and clinical signs were monitored for 14 days. The estimated LD50 was 0.08 mg/kg (95% CI 0.02996–0.182 mg/kg) in males and 0.25 mg/kg (95% CI 0.04841–0.768 mg/kg) in females, indicating greater male sensitivity. Rapid respiratory depression and neuromuscular rigidity occurred within minutes of dosing and preceded lethality. The narrow separation between survival and death demonstrates a steep dose–response relationship and limited safety margin. These findings provide quantitative data for toxicological risk assessment of nitazene opioids and underscore the importance of sex as a biological variable in opioid toxicity studies.
DOI: 10.3389/ftox.2026.18186842026-04-01
Harm J. Heusinkveld, Ellen V. S. Hessel, Edwin P. Zwart, Eric R. Gremmer, Jeroen L. A. Pennings
IntroductionThe zebrafish is a well-known whole organism model to study developmental effects of chemical exposure, including developmental neurotoxicity (DNT). One method used to screen for DNT effects is the zebrafish light-dark transition test (LDT), which measures locomotory behavior under alternating light and dark conditions. Because the LDT is relatively new, experimental protocols and data analysis tools for this assay are still evolving. To advance our knowledge on potential applications of the LDT, we explored using artificial intelligence (AI) methods to distinguish between zebrafish exposed to vehicle controls or pharmaceuticals (fluoxetine, paroxetine, carbamazepine, phenytoin) on 5, 10 or 14 days post-fertilization (dpf).MethodsAI methods were trained to distinguish control versus exposed zebrafish behavior and then applied to assign fish to either group, using five-fold cross-validation. This was done with four different AI methods that differ in nature and complexity, namely Generalized Linear Model (GLM), Random Forest (RF), Gradient Boosting Machine (GBM), and Deep Learning (DL).ResultsAverage prediction accuracy increased from 67% at 5 dpf to 76% at 10 and 14 dpf upon continuous exposure. For fish analyzed at 14 dpf but exposed for shorter durations, we found DNT effects clearly persistent for the antidepressants fluoxetine and paroxetine, but less clearly for the anticonvulsants carbamazepine and phenytoin. The AI methods GLM, RF and DL showed comparable performance, whereas GBM accuracies were lower.DiscussionCompared to conventional univariate analysis, AI appears more sensitive in detecting DNT effects. Overall, this shows the potential of implementing AI methods in DNT screening of chemicals and further development of this approach.
DOI: 10.3389/ftox.2026.17896912026-03-26
Ji Hyun Lee, Hongku Lee, Kang Ho Ahn, Mi Seong Jo, Jin Kwon Kim, Youngshang Han, Yong Taek Kwon, Elaine M. Faustman, Il Je Yu
IntroductionVolatile organic compounds (VOCs) readily penetrate the respiratory tract and cross the air–liquid interface (ALI), yet their in vitro deposition behavior and dose uniformity remain poorly characterized.MethodsIn this study, we evaluated the delivered concentration in the basolateral compartment and inter-transwell variability of isopropyl alcohol (IPA), acetone, and benzyl alcohol (BA) in a six-transwell ALI exposure system. VOCs were generated by atomization at airflow rates of 0.2−3 L/min and delivered for 1–2 h. Delivered concentrations were quantified using photoionization detection or UV–Vis spectroscopy, and aerosol characteristics were monitored by scanning mobility particle sizing.ResultsDespite rapid evaporation and predominantly vapor-phase transport of IPA and acetone, and semi-volatile aerosol BA, VOC delivery to the ALI was reproducible and spatially uniform. IPA and acetone exposures produced coefficients of variation (CVs) below 10% and 16%, respectively, across transwells, whereas benzyl alcohol exhibited higher variability (CV ≤ 19%) due to its lower vapor pressure and higher viscosity. No statistically significant positional differences were observed under any exposure condition.DiscussionThese findings indicate that volatile organic compounds delivered to the ALI undergo diffusion-driven gas–liquid delivery, resulting in a relatively uniform spatial distribution. This study provides exposure-relevant delivery characterization for VOCs and semi-VOCs in ALI systems, supporting future NAM-based inhalation studies without implying biological dose equivalence.
DOI: 10.3389/ftox.2026.17838712026-03-26
Tyler Nicholas, Eric Morinello, Chandrika Moudgal, Alexandre Beaulieu, Jessica Graham
IntroductionProtein kinases regulate various cell cycle functions, and small molecule kinase inhibitors (SMKIs) represent a growing class of therapeutics. In the pharmaceutical industry, occupational toxicologists enable research and development (R&D) and manufacturing activities for SMKIs by deriving health-based exposure limits (HBELs), including occupational exposure limits (OELs) and occupational exposure bands (OEBs), often in data-limited R&D settings.MethodsThe objective of this study was to evaluate health hazard data for FDA-approved SMKIs and apply a quantitative framework to estimate OELs and map them to OEBs to guide occupational health and safety practices. We compiled health hazard data for SMKIs (n = 86) from literature sources and drug labels including data on genotoxicity, carcinogenicity, developmental and reproductive toxicity (DART), minimum daily therapeutic dose, and sensitive subpopulations. For orally administered SMKIs (n = 83), we estimated OELs using a standardized dose-based approach that incorporates adjustment factors and bioavailability. Estimated OELs were then mapped to OEBs and evaluated by target family.ResultsEstimated OELs ranged from 0.05 to 96 µg/m3, with 82% falling between 1 and <100 µg/3, spanning OEB 2 and OEB 3A. Developmental toxicity was regarded a class effect, while genotoxicity and carcinogenicity were less frequent and varied by target family.DiscussionSeveral target families, including JAK, FGFR, MEK, mTOR, PI3K, and VEGFR inhibitors, exhibited lower estimated OELs and may warrant more protective OEBs in data-limited R&D settings. This analysis demonstrates that a default OEB of 1 to <10 µg/m3 (3A) is likely protective for most SMKIs. These findings provide a quantitative, mechanism-informed framework to guide occupational risk management for SMKIs.
DOI: 10.3389/ftox.2026.17435582026-03-26
Kamilah Na’imah Muhammad, Tracey Woodlief, Xian Wu
Neuroinflammatory contributions play a critical role in Parkinson’s disease onset and progression. Key drivers of neuroinflammation include glial cell reactivity, cytokine signaling, protein aggregation, and mitochondrial dysfunction. Although animal models have been extensively used to investigate the mechanisms, their translational relevance is limited because neuroinflammation in humans is typically chronic, heterogeneous, and sustained over years, whereas in rodents is often acute, transient, and resolves within days to weeks. This paper highlights the utility of human stem cell–derived models in studying Parkinson’s disease by recapitulating patient-specific genetic mutations, neuroinflammatory microglia–neuron interactions, α-synuclein aggregation, and dopaminergic dysfunction, thereby enabling mechanistic studies in the human-relevant models. In addition, we examine how micro- and nanoplastics may exacerbate neuroinflammation in PD. This review concludes by highlighting how human-relevant stem cell-based approaches advance mechanistic understanding of Parkinson’s disease.
DOI: 10.3389/ftox.2026.17230922026-03-24
D. Gil, S. José, A. Ascenso, M. Novak Babič, E. Segal, J. Meletiadis, J. P. Gangneux, C. J. Weiskerger, H. M. Solo-Gabriele, E. Valério, J. Brandão
Antifungal resistance is an on-growing public health concern due to the difficulty in managing or treating medical conditions that often favour fatal fungal infections. The changing climate and globalisation, which increase fungal persistence and propagation, adds to that concern. Wastewater disposal is one potential source to the environment as antifungals are released into it. Considering that most fungal infections originate from the environment and considering the One Health principle, introducing antifungals through wastewater effluents has the potential to promote the emergence and dissemination of antifungal resistance. The objective of this study was to generate knowledge that can assist regulating the release of antifungals in the environment by quantifying predicted no-effect concentrations (PNECs) that would not promote antifungal resistance. For this purpose, a systematic review was performed to consolidate information on antifungals released to the environment and respective concentrations. The systematic literature review followed Preferred Reporting Items for Systematic literature reviews and Meta Analyses extension for Scoping Reviews (PRISMA-SLR). The analysis of 122 reviewed articles using this approach showed high concentrations and dispersion of antifungals in water, wastewater or soil. This highlights their potential dispersion in the environment, thus increasing the potential of fungal antimicrobial resistance. Due to the lack of PNEC values using fungi as model organisms in this review, PNECs for 17 antifungals were calculated using Candida albicans as model, as it is done for clinical purposes. We consider that the antifungal PNECs calculated and consolidated from the literature can be used to prioritise them for regulation and to determine acceptable levels in wastewater effluents.
DOI: 10.3389/ftox.2026.17679252026-03-24
Debora Curci, Martina Franzin, Enrico Pobega, Stefania Braidotti, Anna Flamigni, Gilda Paternuosto, Giulia Schillani, Riccardo Addobbati, Stefania Norbedo, Natalia Maximova
IntroductionThe adverse effect of metals and metalloids on neural development is a significant health concern, especially in vulnerable populations, such as pediatric patients. Excessive accumulation of these elements in the developing central nervous system (CNS) can induce oxidative stress, trigger cell death, and promote neuroinflammation. However, few studies have quantified metal and metalloid concentrations in cerebrospinal fluid (CSF) in pediatric patients, and, to our knowledge, none have compared these levels between children with and without neurological disorders.MethodsIn this exploratory study, we compared pediatric patients with neurological diseases or CNS infections to control pediatric patients to assess differences in metal and metalloid concentrations and in CSF inflammatory cytokine profiles.ResultsWe observed higher levels of most metals and metalloids in neurological and infection patients, while group-specific differences were observed in cytokines. Notably, the cytokines Pentraxin-3 and IL-8 showed positive correlations with calcium, copper, iron, antimony, and chromium, suggesting a possible functional relationship in neuroinflammation.DiscussionIn conclusion, this study presents a comparative analysis of CSF metal and metalloid levels in conjunction with inflammatory cytokine profiles in a pediatric population, providing a basis for further research into their roles in neuroinflammation and neurological disease.
DOI: 10.3389/ftox.2026.17669042026-03-24
Eneko Madorran
The persistent gap between preclinical findings and clinical outcomes highlights the limitations of current in vitro models in regulatory toxicology. While New Approach Methodologies (NAMs) promise mechanistic insight, reduced reliance on animal testing, and enhanced human relevance, their translational accuracy remains constrained by oversimplified assumptions. This manuscript identifies foundational aspects of human physiology—degeneracy, interconnected pathways, variability among individuals, biological rhythms, and context dependency—that are often underrepresented in existing systems. I propose reframing biological effects as probabilistic outcomes rather than deterministic events, recognizing that cells and organisms operate through overlapping networks where redundancy, variability, and timing shape the likelihood of specific responses. Building on this framework, I outline a probabilistic and context-dependent cell culture model that integrates viability, functional fidelity, pathway mapping, temporal resolution, and Bayesian inference. Translational relevance is further strengthened by anchoring in vitro measurements to clinically meaningful benchmarks, incorporating patient perspectives, and aligning with regulatory oversight. Although challenges remain—including replicating cell–cell communication, multi-organ synchronization, and harmonizing probabilistic outputs with deterministic regulatory frameworks—embedding these principles into NAMs offers a pathway to overcome the translational bottleneck. By embracing complexity rather than reducing it, NAMs can evolve into tools that are not only mechanistically informative but also predictive, acceptable, and implementable in real-world milieu, ultimately advancing safer and more ethical toxicological assessment.
DOI: 10.3389/ftox.2026.17657532026-03-20
Zahra Miri, Johanna Laakkonen, Emilia Toivonen, Niina Väljä, Susanna Miettinen, Hanna Vuorenpää
The use of animal-derived reagents in biomedical research poses challenges for reproducibility due to batch-to-batch variability and inter-species differences, along with ethical concerns related to their origin. In pursuing a human-relevant in vitro model, an animal-free and defined cell culture process is preferred to improve relevance and reproducibility. We investigated the use of serum replacement (SR) consisting of human hepatocyte-derived proteins in cell culture and recombinant antibodies with a plant-derived blocking solution (animal-free blocker, AFB) in immunocytochemical staining of cells. Human serum (HS) instead of animal-derived serum was used in this study for comparison with SR. We showed that bone marrow stem/stromal cells (BMSCs) maintain their proliferation capacity and cell-specific morphology in SR-supplemented medium, whereas human umbilical vein endothelial cells (HUVECs) show compromised growth under similar conditions. In a more complex co-culture, BMSCs + HUVECs formed a stable vascular network in SR-supplemented medium. In immunocytochemical staining, we compared the performance of recombinant antibodies with animal-derived antibodies and an AFB solution with a bovine serum albumin (BSA)-based blocking solution. Adipose stem/stromal cells (ASCs) showed their typical spindle-shaped morphology when stained with recombinant antibodies against alpha-smooth muscle actin (αSMA) in both AFB and BSA-based blocking solutions. We detected partial non-specific binding of recombinant antibodies and animal-derived antibodies against β-tubulin III in ASC. In contrast, we did not observe non-specific binding on these neuronal antibodies in HUVECs in any tested condition. While protocol optimization depends on the cell type used, our findings indicate that animal-derived materials can reliably be replaced.
DOI: 10.3389/ftox.2026.1741716