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bioRxiv2026

Sequence-dependent modulation of hepatorenal biochemical markers following Artemether-Lumefantrine and Sulfadoxine-Pyrimethamine exposure in Wistar rats

Malaria remains one of the most pressing health problems, especially in Sub-Saharan Africa. Various antimalarial drugs, used to combat this debilitating illness, may directly or indirectly affect blood indices in humans. This study aims to evaluate the toxicological effects of sequential administration of Artemether-lumefantrine and sulfadoxine-pyrimethamine in male Wistar rats. Thirty (30) mature male Albino Wistar rats weighing between 190-280g were randomly divided into five groups comprising six (6) rats each. Group 1 served as control, Group 2 received Artemether-lumefantrine (8 mg/kg/bw) for 3 days, Group 3 received sulfadoxine-pyrimethamine (0.079 mg/kg/bw) for 1 day, Group 4 received a sequential dose of Artemether-Lumefantrine for 3days and sulfadoxine-pyrimethamine for 1 day, while Group 5 received a sequential dose of SP for 1 day and AL for 3 days. Sequential administration of AL and SP resulted in a significant (p < 0.05) elevation of ALT, AST, ALP, serum total and direct bilirubin levels, urea, creatinine, and HDL. There was a significant (pL0.05) decrease in the serum total protein and albumin. Notably, HDL levels increased significantly in the SP [->] AL group (p < 0.05), while other lipid parameters showed sequence-specific significant changes compared to the control. Sequential administration, particularly the SP [->] AL sequence, was observed to have more pronounced effects on hepatorenal biomarkers compared to independent administration. These findings are relevant, especially in malaria-endemic regions where unregulated self-medication and drug switching are rampant.

pharmacology and toxicology↗

bioRxiv2024

Cell circuits underlying nanomaterial specific respiratory toxicology

Nanomaterials emerged as boundless resource of innovation, but their shape and biopersistence related to respiratory toxicology raise longstanding concerns. The development of predictive safety tests for inhaled nanomaterials, however, is hampered by limited understanding of cell type-specific responses. To advance this knowledge, we used single-cell RNA-sequencing to longitudinally analyze cellular perturbations in mice, caused by three carbonaceous nanomaterials of different shape and toxicity upon pulmonary delivery. Focusing on nanomaterial-specific dynamics of lung inflammation, we found persistent depletion of alveolar macrophages by fiber-shaped nanotubes. While only little involvement was observed for alveolar macrophages during the initiation phase, they emerged, together with infiltrating monocyte-derived macrophages, as decisive factors in shifting inflammation towards resolution for spherical nanomaterials, or chronic inflammation for fibers. Fibroblasts, central for fibrosis, sensed macrophage and epithelial signals and emerged as orchestrators of nanomaterial-induced inflammation. Thus, the mode of actions identified in this study will significantly inspire the precision of future in vitro testing. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/579746v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1d6f061org.highwire.dtl.DTLVardef@3fabb1org.highwire.dtl.DTLVardef@21040eorg.highwire.dtl.DTLVardef@1915c3b_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2024

Leveraging zebrafish embryo phenotypic observations to advance data-driven analyses in toxicology

Zebrafish have emerged as a central model organism in toxicological research. Zebrafish embryos are exempt from certain animal testing regulations which facilitates their use in toxicological testing. Next to the zebrafish embryo acute toxicity test (ZFET) according to the OECD TG 236, fish embryos are used in mechanistic investigations, chemical screenings, in ecotoxicology, and drug development. However, inconsistencies in the applied test protocols and the monitored endpoints in addition to a lack of standardized data formats, impede comprehensive meta-analyses and cross-study comparisons. To address these challenges, we developed the Integrated Effect Database for Toxicological Observations (INTOB), a comprehensive data management tool that standardizes collection of metadata and phenotypic observations using a controlled vocabulary. By incorporating data from more than 600 experiments into the database and subsequent comprehensive data analyses, we demonstrate its utility in improving the comparability and interoperability of toxicity data. Our results show that the ZFET can detect toxicity spanning seven orders of magnitude at the scale of effect concentrations. We also highlight the potential of read-across analyses based on morphological fingerprints and their connection to chemical modes of action, provide information on control variability of the ZFET, and highlight the importance of time for mechanistic understanding in chemical exposure-effect assessments. We provide the full FAIR dataset as well as the analysis workflow and demonstrate how professional data management, as enabled with INTOB, marks a significant advancement by offering a comprehensive framework for the systematic use of zebrafish embryo toxicity data, thus paving the way for more reliable, data-driven chemical risk assessment. SynopsisThis article shows how a novel data management tool for zebrafish embryo toxicity data advances comparative chemical risk assessment by enhancing data and metadata standardization, comparability, and cross-study analyses.

pharmacology and toxicology↗

bioRxiv2022

CSL-Tox: An open-source analytical framework for the comparison of short-term and long-term toxicity end points and exploring the opportunities for decreasing in-vivo studies conducted for drug development programs.

In-vivo toxicity assessment is an important step prior to clinical development and is still the main source of data for overall risk assessment of a new molecular entity (NCE). All in-vivo studies are performed according to regulatory requirements and many efforts have been exerted to minimize these studies in accordance with the (Replacement, Reduction and Refinement) 3Rs principle. Many aspects of in-vivo toxicology packages can be optimized to reduce animal use, including the number of studies performed as well as study durations, which is the main focus of this analysis. We performed a statistical comparison of adverse findings observed in 116 short-term versus 78 long-term studies in order to explore the possibility of using only short-term studies as a prediction tool for the longer-term effects. Annotation of treatment related findings was one of the challenges faced during this work. A specific focus was therefore put on the summary and conclusion sections of the reports since they contain expert assessments on whether the findings were considered adverse or were attributed to other reasons. Our analysis showed a general good concordance between short-term and long-term toxicity findings for large molecules and the majority of small molecules. Less concordance was seen for certain "target organ systems findings. While this work supports the minimization of in-vivo study durations, a larger-scale effort would be needed to provide more evidence. We therefore present the steps performed in this study as an open-source R workflow (CSL-Tox) and we provide the dataset used in the work to allow researchers to reproduce such analysis and to promote large-scale application of this study.

pharmacology and toxicology↗

bioRxiv2024

Effect Of Ayahuasca on Experimental Models of Ethanol Addiction and Immunohistochemistry Analysis in Rodents

BackgroundAyahuasca, a psychoactive Amazonian preparation, is increasingly studied for substance-use disorders. ObjectivesInvestigate whether oral lyophilised-ayahuasca attenuates ethanol-induced conditioned place preference (CPP) in mice and alters {Delta}FosB expression in nucleus accumbens (NAc). MethodsMale Swiss mice received water or ayahuasca (130-1950 mg/kg, p.o.) 30 min before each of eight ethanol pairings (2g/kg i.p.) in a CPP paradigm. A separate cohort underwent acute toxicology (650-5000mg/kg) with behavioural-observation and rotarod. Alkaloids were quantified by LC-MS/MS. {Delta}FosB-immunoreactive nuclei were counted in NAc 24h after the CPP post-test. ResultsAlkaloids levels were within traditional ranges. High-dose ayahuasca(5000 mg/kg) produced transient-serotonergic-syndrome-like signs and rotarod locomotor-deficit; lower doses did not express toxicity. Ethanol produced a moderate-CPP in controls({Delta}Time{approx}+60s), whereas ayahuasca-pretreatment abolished preference at all doses({Delta}Time within{+/-}7s). One-way ANOVA on {Delta}Time showed a robust-Treatment effect(F(3,36)=8.83, p=0.00016); Tukey tests: control differed from each ayahuasca group (all p<0.05), with no differences among ayahuasca doses. {Delta}FosB density did not differ among groups(p>0.05). ConclusionsAyahuasca was well tolerated at ceremonies-equivalent doses and blocked ethanol-induced-CPP across all doses, while {Delta}FosB levels in NAc were unchanged at 24h. Limitations on the CPP baseline and {Delta}FosB results may limit sensitivity, generalisability and interpretation. Findings provide preliminary evidence that ayahuasca-pretreatment may blunt ethanol-context preference, reinforcing the need of replication with stronger reward baselines, naive controls and complementary molecular markers.

pharmacology and toxicology↗

bioRxiv2023

Impact of gene selection criteria on transcriptomic ontology-based point of departure estimates

Apical effects are typically associated with changes in gene expression, which allows for the use of short- term in life transcriptomic studies to derive biologically relevant points of departure (POD). These methods offer cost savings over conventional toxicology assessments and can derive data from very short-term studies where apical effects may not yet be present. When there is limited or insufficient data for a conventional POD assessment, a transcriptomic screen could provide valuable data for deriving a cellular bioactivity POD for chemical screening and hazard assessment. We used existing transcriptomic data from published 5-day rat in vivo kidney and liver exposures to examine the effect of differential gene expression metrics for the selection of genes used for ontology pathway-based POD derivation. Williams Trend Test (WTT) indicate no gene expression dose-response in 6 instances and ANOVA in one, while DESeq2 detected differentially expressed genes in all instances. The three statistical metrics produced consistent POD values. One chemical (PFOA in liver) showed ontology enrichment indicative of a cytotoxic response at the highest dose, emphasizing the effect which too high a dose can have on the derivation of POD values if such response is not accounted for. Whether the choice of a gene selection metric combining both a statistical significance criterion as well as a minimum magnitude of change threshold affects the sensitivity of POD values depends on the specifics of the dose- response. Existing alternative and complementary analyses could be utilized with existing analyses pipelines to better inform analytical decisions when using transcriptomics and BMD for point of departure determinations.

pharmacology and toxicology↗

bioRxiv2018

Development of a large-scale computer-controlled ozone inhalation exposure system for rodents

Complete systems for laboratory-based inhalation toxicology studies are typically not commercially available; therefore, inhalation toxicologists utilize custom-made exposure systems. Here we report on the design, construction, testing, operation, and maintenance of a newly developed in vivo rodent ozone inhalation exposure system. Key design requirements for the system included large-capacity exposure chambers to facilitate studies with large sample sizes, automatic and precise control of chamber ozone concentrations, as well as automated data collection on airflow and environmental conditions. The exposure system contains two Hazelton H-1000 stainless steel and glass exposure chambers, each providing capacity for up to 180 mice or 96 rats. We developed an empirically tuned proportional-integral-derivative (PID) control loop that provides stable ozone concentrations throughout the exposure period (typically 3h), after a short ramp time ([~]8 minutes), and across a tested concentration range of 0.2 to 2 ppm. Specific details on the combination analog and digital input/output system for environmental data acquisition, control, and safety systems are provided, and we outline the steps involved in maintenance and calibration of the system. We show that the exposure system produces consistent ozone exposures both within and across experiments, as evidenced by low coefficients of variation in chamber ozone concentration and consistent biological responses (airway inflammation) in mice, respectively. Thus, we have created a large and robust ozone exposure system, facilitating future studies on the health effects of ozone in rodents.

pharmacology and toxicology↗

bioRxiv2022

2,4-Di-tert-butylphenol Induces Adipogenesis in Human Mesenchymal Stem Cells by Activating Retinoid X Receptors

2,4-di-tert-butylphenol (2,4-DTBP) is an important commercial antioxidant and a toxic natural secondary metabolite that has been detected in humans. However, there is scant information regarding its toxicological effects. Here we asked whether 2,4-DTBP is a potential obesogen. Using a human mesenchymal stem cell (MSC) adipogenesis assay, we found that exposure to 2,4-DTBP led to increased lipid accumulation and expression of adipogenic marker genes. Antagonist assays revealed that 2,4-DTBP increased lipid accumulation by activating the peroxisome proliferator-activated receptor {gamma} (PPAR{gamma})-retinoid X receptor (RXR) heterodimer. 2,4-DTBP likely activated the PPAR{gamma}/RXR heterodimer by activating RXR but not directly binding to PPAR{gamma}. We confirmed that 2,4-DTBP directly bound to RXR by solving the crystal structure of this complex, then predicted and demonstrated that related compounds could also activate RXR. Our study demonstrated that 2,4-DTBP and related chemicals could act as obesogens and endocrine disruptors via RXR. These data showed that 2,4-DTBP belongs to a family of compounds whose endocrine-disrupting and obesogenic effects can be strongly modulated by their chemical composition and that structure-activity studies such as the present one could help guide the rational development of safer antioxidants. SYNOPSISLittle research exists on the effects of commercially valuable antioxidants on biological systems. This study reports that di- and tri-tert-butylphenols can act as endocrine disruptors and potential obesogens by activating nuclear hormone receptors.

pharmacology and toxicology↗

bioRxiv2025

Elucidating the potential carcinogenic molecular mechanisms of parabens in head and neck squamous cell carcinoma through network toxicology and molecular docking

ObjectiveThis study aims to systematically investigate the molecular mechanisms through which parabens may contribute to head and neck squamous cell carcinoma (HNSCC) carcinogenesis using integrated network toxicology and molecular docking. Materials and MethodsSix commonly used parabens (ethyl-, propyl-, methyl-, heptyl-, butyl-, and benzylparaben) were selected for toxicity prediction via ProTox 3.0 and ADMETlab2.0. Their potential targets were retrieved from Swiss Target Prediction, ChEMBL, and the Similarity Ensemble Approach (SEA). HNSCC-related targets were collected from GeneCards, OMIM, and CTD, while differentially expressed genes (DEGs) were identified using TCGA-HNSC data. Functional enrichment, protein-protein interaction (PPI) network construction, hub gene identification, molecular docking, and immune infiltration analyses were performed using DAVID, STRING, Cytoscape, CB-DOCK2, and TIMER2.0. ResultsWe identified 80 common targets through which parabens may exert toxic effects in HNSCC. Among these, 12 hub genes (CCNB1, CDK1, CCNA2, CDK2, CDK4, TYMS, AURKA, CCNA1, CHEK1, CCNB2, PLK1, CDC25A) were significantly overexpressed in HNSCC tissues and were primarily enriched in cell cycle regulation, p53 signaling, and viral carcinogenesis pathways. CCNA1 was further validated as an independent prognostic factor associated with poor survival. Molecular docking revealed strong binding affinities between parabens and hub proteins. Immune infiltration analysis indicated a negative correlation between CCNA1 expression and CD8+ T cell and B cell infiltration. ConclusionsParabens may promote HNSCC progression by disrupting cell cycle regulation and immune responses via direct interactions with key hub genes. These findings provide a novel mechanistic basis for the carcinogenic potential of parabens in HNSCC and underscore the need for further experimental and epidemiological validation.

pharmacology and toxicology↗

bioRxiv2022

Adverse PFAS effects on mouse oocyte in vitro maturation are associated with carbon-chain length and inclusion of a sulfonate group

Per- and polyfluoroalkyl substances (PFAS) are man-made chemicals that are used in products such as non-stick cookware, stain-resistant coating, and food packaging. PFAS are characterized by their fluorinated carbon chains that make them hard to degrade and bioaccumulate in human and animals. Toxicological studies have shown PFAS toxic effects: cytotoxicity, immunotoxicity, neurotoxicity, and reproductive toxicity. Two major categories of PFAS are perfluoroalkyl carboxylic acid (PFCA) and perfluoroalkyl sulfonic acid (PFSA). In this study, we used a mouse-oocyte-in-vitro-maturation (IVM) system to study how the structures of PFAS, such as carbon-chain length and functional groups, determine their reproductive toxicity. We found the toxicity of PFAS is elevated with increasing carbon-chain length and the inclusion of the sulfonate group. Specifically, at 600 {micro}M, perfluorohexanesulfonic acid (PFHxS) and perfluorooctanesulfonic acid (PFOS) reduced the rates of both germinal vesicle breakdown (GVBD) and polar body extrusion (PBE) as well as induced the formation of relatively large polar bodies. However, the shorter PFSA, perfluorobutanesulfonic acid (PFBS), and all PFCA did not show similar adverse cytotoxicity. We further examined mitochondria and cytoskeleton, two essential factors for cell division, in PFOS- and PFHxS-treated oocytes. We found that 600 {micro}M PFHxS and PFOS exposure induced excess reactive oxygen species (ROS) and decreased mitochondrial membrane potential (MMP). Cytoskeleton analysis revealed that PFHxS and PFOS exposure induced chromosome misalignment, abnormal F-actin organization, elongated the spindle formation, and symmetric division in the treated oocytes. Together, our study provides new information on the structure-toxicity relationship of PFAS. SynopsisReproductive toxicity of PFAS, a group of persistent organic pollutants, is determined by their chemical structures.

pharmacology and toxicology↗

bioRxiv2020

Rearing medium dictates variability across replicates in untreated and arsenic challenged zebrafish larvae

Reproducibility and consistency are hallmarks of scientific integrity. Biological systems are inherently noisy, posing a challenge to reproducibility. This is particularly relevant to the field of environmental toxicology, where many unaccounted experimental parameters can have a marked influence on the biological response to exposure. Here, we extend the use of zebrafish as a robust toxicological model for studying the effects of inorganic arsenic (iAs) on liver biology. We observed that iAs toxicity in this system is not influenced by important parameters including genetic background, rearing container material or rearing volume but the dose response to iAs is influenced by the rearing medium. We compared mortality as a measure of iAs toxicity to embryos cultured in two standard rearing media: egg water made from dehydrated ocean salts dissolved in water and a defined embryo medium which is a pH adjusted, buffered salt solution. Larvae reared in egg water were more susceptible to iAs compared to those reared in embryo medium. This effect was independent of the pH differences between these solutions. These culture conditions did not cause any difference in the global hepatic transcriptome of control zebrafish. Further, no difference in the expression of genes involved in the unfolded protein response (UPR) in larvae exposed to iAs treatment or in a stress independent system to activate UPR genes by transgenic overexpression of activating transcription factor 6 (nAtf6) in hepatocytes was observed. However, the clutch-to-clutch variation in gene expression was significantly greater in larvae reared in egg water compared to those in embryo medium. These data demonstrate that egg water affects reproducibility across replicates in terms of gene expression and exacerbates iAs mediated toxic response. This highlights the importance of rigorous evaluation of experimental conditions to assure reproducibility.

pharmacology and toxicology↗

bioRxiv2025

AmesNet: A Deep Learning Model Enhancing Generalization in Ames Mutagenicity Prediction

Regulatory agencies require comprehensive genotoxicity assessments for all novel small-molecule therapeutics prior to human trials. Developers often delay these studies until a candidate is nearing regulatory submission because they are expensive and secondary to bioactivity. This timing creates a bottleneck where late-stage failures can jeopardize >$10 million in capital and multiple years of developmental progress per candidate. The Ames assay is used to detect a molecules mutagenic potential. Regulators now explicitly support the use of in silico Ames mutagenicity models through enabling legislation, dedicated FDA AI toxicology programs, internationally harmonized guidelines, and benchmark challenges. However, current Ames models suffer from a dramatic sensitivity drop-off when they evaluate molecules outside their training domain. Sensitivity is the most important metric in Ames prediction because false negatives allow mutagenic compounds to advance undetected and trigger the most costly late-stage failures. Attempts to fix this sensitivity drop-off often reduce overall model performance, which can be represented by balanced accuracy. For example, DeepAmes reports high levels of sensitivity only by sacrificing its balanced accuracy. We introduce AmesNet, a novel Task-Conditioned modeling paradigm that achieves both class-leading sensitivity and balanced accuracy in novel chemical spaces. AmesNet utilizes a dual branch architecture containing a molecular encoder and a dedicated channel to condition Ames assay context such as metabolic activation and bacterial strain type. In comparative benchmarks, AmesNet reached a sensitivity of 0.73 (95% confidence interval: 0.68-0.77) and a simultaneous balanced accuracy of 0.81 (95% confidence interval: 0.79-0.83) on the out-of-domain test data. This represents an improvement in sensitivity of up to 46% over existing approaches without a trade-off in balanced accuracy. Structural analysis demonstrates that AmesNet recovers difficult-to-detect mutagenic compounds missed by existing models. This framework provides a high-confidence filtering mechanism that enables drug developers to turn a costly late-stage safety bottleneck into a proactive decision-making edge.

pharmacology and toxicology↗

bioRxiv2025

The Lipidome Landscape of Amiodarone Toxicity: An in vivo Lipid-centric Multi-Omics Study

Amiodarone is an effective therapy for arrhythmias, its prolonged management may lead to significant adverse drug reactions. Amiodarone-induced hepatoxicity is described by phospholipidosis, hepatic steatosis, cholestatic hepatitis, and cirrhosis. However, the systemic and hepatic lipidome disturbances and underlying toxicological mechanisms remain comprehensively elucidated. Untargeted lipidomics were utilized to analyze serum and liver samples from the rats orally administered a daily dose of amiodarone of either 100 or 300 mg/kg for one week. Changes in the expression of hepatic lipid-related genes were also examined utilizing transcriptomics. We found a higher magnitude of lipidome alterations in the 300 mg/kg than those in the 100 mg/kg groups. Treated animals showed elevated abundances of phosphatidylcholines, ether-linked phosphatidylcholines, sphingomyelins, and ceramides, and decreased levels of triacylglycerols, ether-linked triacylglycerols, and fatty acids. We also found 199 lipid-related differentially expressed hepatic genes between the 300 mg/kg group versus controls, implying lipid metabolism and signaling pathways disturbances. Specifically, elevation of serum phosphatidylcholines and ether-linked phosphatidylcholines, as well as hepatic bismonoacylglycerophosphates were associated with reduced expression of phospholipase genes and elevated expression of glycerophospholipid biosynthesis genes, possibly driving phospholipidosis. Perturbations of sphingolipid metabolism might also be the key events for amiodarone-induced toxicity. Alterations in gene expression levels related to lipid storage and metabolism, mitochondria functions, and energy homeostasis were also found. Collectively, our study characterized the sophisticated perturbations in the lipidome and transcriptome of amiodarone-treated rats and suggested potential mechanisms responsible for amiodarone-induced hepatotoxicity.

pharmacology and toxicology↗

bioRxiv2017

Early afterdepolarisation tendency as a simulated pro-arrhythmic risk indicator

Drug-induced Torsades de Pointes (TdP) arrhythmia is of major interest in predictive toxicology. Drugs which cause TdP block the hERG cardiac potassium channel. However, not all drugs that block hERG cause TdP. As such, further understanding of the mechanistic route to TdP is needed. Early afterde-polarisations (EADs) are a cell-level phenomenon in which the membrane of a cardiac cell depolarises a second time before repolarisation, and EADs are seen in hearts during TdP. Therefore, we propose a method of predicting TdP using induced EADs combined with multiple ion channel block in simulations using biophysically-based mathematical models of human ventricular cell electrophysiology. EADs were induced in cardiac action potential models using interventions based on diseases that are known to cause EADs, including: increasing the conduction of the L-type calcium channel, decreasing the conduction of the hERG channel, and shifting the inactivation curve of the fast sodium channel. The threshold of intervention that was required to cause an EAD was used to classify drugs into clinical risk categories. The metric that used L-type calcium induced EADs was the most accurate of the EAD metrics at classifying drugs into the correct risk categories, and increased in accuracy when combined with action potential duration measurements. The EAD metrics were all more accurate than hERG block alone, but not as predictive as simpler measures such as simulated action potential duration. This may be because different routes to EADs represent risk well for different patient subgroups, something that is difficult to assess at present.

pharmacology and toxicology↗

bioRxiv2026

Phytochemical profiling, antioxidant capacity, and in vivo safety assessment of Warrigal spinach (Tetragonia tetragonioides) and Kensington Pride mango (Mangifera indica) extracts using Zebrafish larvae

Plant-derived bioactive compounds are recognised for their antioxidant potential and benefit for human diseases, including age-related diseases caused by oxidative stress. However, their antioxidant composition and safety profiles remain insufficiently understood. This study integrates phytochemical profiling, antioxidant evaluation, and in vivo toxicological assessment of Warrigal spinach (Tetragonia tetragonioides) and Kensington Pride mango (Mangifera indica). Spinach exhibited greater antioxidant capacity, and higher total phenolic and flavonoid content than mango: TPC (14.2 {+/-} 0.6 mg GAE/g vs 1.30 {+/-} 0.07 mg GAE/g) and TFC (9.61 {+/-} 0.39 mg QE/g vs 0.08 {+/-} 0.0 mg QE/g). LC-ESI-QTOF-MS/MS identified 187 metabolites dominated by flavonoids (53.5%) and phenolic acids (16%), with spinach showing greater chemical diversity. Quantitative analysis revealed higher levels of hydroxycinnamic acids and flavonoid glycosides in spinach, whereas mango contained distinct metabolites, including mangiferin and pyrogallol. Zebrafish embryo / larval assays demonstrated high safety margins, with LC50 values of 478.8 mg/L (spinach) and >480 mg/L (mango). At 480 mg/L spinach displayed developmental abnormalities and malformations. These findings demonstrate that antioxidant capacity is linked to phenolic composition, but does not predict toxicity. Thus, integrated phytochemical and safety evaluation for extracts with complex compound mixtures are critical to identify botanicals suitable for future drug development. HighlightsO_LIWarrigal spinach exhibited >10-fold higher phenolic content and antioxidant capacity than Kensington Pride mango. C_LIO_LILC-ESI-QTOF-MS/MS identified 187 metabolites, with flavonoids as the dominant phytochemical class. C_LIO_LIZebrafish assays confirmed high safety margins, demonstrating no direct correlation between antioxidant capacity and toxicity. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=114 SRC="FIGDIR/small/720960v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@f82bbeorg.highwire.dtl.DTLVardef@ffd6d4org.highwire.dtl.DTLVardef@711481org.highwire.dtl.DTLVardef@1f94d7d_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG

pharmacology and toxicology↗

bioRxiv2024

Screening and characterization of 133 physiologically-relevant environmental chemicals for reproductive toxicity

Reproduction is a functional outcome that relies on complex cellular, tissue, and organ interactions that span the developmental period to adulthood. Thus, the assessment of its disruption by environmental chemicals is remarkably painstaking in conventional toxicological animal models and does not scale up to the number of chemicals present in our environment and requiring testing. We adapted a previously described low-throughput in vivo chromosome segregation assay using C. elegans predictive of reproductive toxicity and leveraged available public data sources (ToxCast, ICE) to screen and characterize 133 physiologically-relevant chemicals in a high-throughput manner. The screening outcome was further validated in a second, independent in vivo assay assessing embryonic viability. In total, 13 chemicals were classified as reproductive toxicants with the two most active chemicals belonging to the large family of Quaternary Ammonium Compounds (QACs) commonly used as disinfectants but with limited available reproductive toxicity data. We compared the results from the C. elegans assay with ToxCast in vitro data compiled from 700+ cell response assays and 300+ signaling pathways-based assays. We did not observe a difference in the bioactivity or in average potency (AC50) between the top and bottom chemicals. However, the intended target categories were significantly different between the classified chemicals with, in particular, an over-representation of steroid hormone targets for the high Z-score chemicals. Taken together, these results point to the value of in vivo models that scale to high-throughput level for reproductive toxicity assessment and to the need to prioritize the assessment of QACs impacts on reproduction.

pharmacology and toxicology↗

bioRxiv2021

Toxicological profile of umbilical cord blood-derived small extracellular vesicles

The development and adoption of cell therapies has been largely limited by difficulties associated with their safety, handling and storage. Extracellular vesicles (EV) have recently emerged as a likely mediator for the therapeutic effect of cells, offering several advantages over cell therapies. Due to their small size and inability to expand and metastasize, EV are generally considered safer than cell transplantation. Nevertheless, few studies have scrutinized the toxicity profile of EV, particularly after repeated high dose administration. The present study aimed to evaluate a preparation of small EV obtained from umbilical cord blood mononuclear cells (UCB-MNC-sEV) for its cytotoxicity in different cell lines, as well as its differential accumulation, distribution and toxicity following repeated intravenous (IV) administrations in a rodent model. In vitro, repeated sEV exposure in concentrations up to 1x1011 particles/ml had no deleterious impact on the viability or metabolic activity of peripheral blood mononuclear cells, THP-1 monocytes, THP-1-derived macrophages, normal dermal human fibroblasts or human umbilical vein endothelial cells. DiR-labeled sEV, injected IV for four weeks in healthy rats, were detected in clearance organs, particularly kidneys, spleen and liver, similarly to control dye. Moreover, repeated administrations during six and twelve weeks of up to 1x1010 total particles of sEV-dye were well tolerated, with no changes in general hematological cell counts, or kidney and liver toxicity markers. Importantly, unlabeled sEV likewise did not induce significant alterations in cellular and biochemical blood parameters, nor any morphological changes in heart, kidney, lung, spleen, or liver tissue. In sum, our data shows that UCB-MNC-sEV have no significant toxicity in vitro or in vivo, even when administered repeatedly at high concentrations, therefore confirming their safety profile and potential suitability for future clinical use.

pharmacology and toxicology↗

bioRxiv2026

Mutagenic and carcinogenic potency determinations for NDMA support the cumulative dose assumption underpinning the less-than-lifetime Threshold of Toxicological Concern

The management of N-nitrosamine impurities challenges pharmaceutical development and regulation worldwide. Because most medicinal exposures are shorter than lifetime and absolute impurity exclusion is impossible, reliable approaches to define duration-specific intake limits are essential. On the premise that carcinogenic risk is proportional to cumulative dose, the Less-Than-Lifetime (LTL) Threshold of Toxicological Concern (TTC) framework defines progressively lower intake limits for mutagenic impurities over longer exposures. However, N-nitrosamines are currently treated as a cohort of concern, necessitating compound-specific evaluation placing reliance on in vivo mutagenicity assays for impurity qualifications. To better understand durational potency relationships and the application domain of the LTL-TTC, we apply benchmark dose (BMD) modelling to cumulative-dose-scaled transgenic rodent (TGR), error-corrected sequencing and rodent carcinogenicity datasets for N-nitrosodimethylamine (NDMA) obtained from the published literature. For TGR, cumulative-dose scaling better resolved liver as the most sensitive organ and reduced interstudy variability: liver BMDs spanned [~]80-fold in daily-dose units but only [~]20-fold when scaled to cumulative dose. Among closely-matched mouse liver gavage studies, cumulative-dose BMDs only varied by [~]2.5-fold across 1 to 28-day treatment regimens. Error-corrected sequencing also demonstrated parity, with acute-dose regimens producing mutation burdens near-identical (< 1.2-fold) to those cumulated from 28-day repeat-dose regimens. Comparable results were obtained from carcinogenicity datasets confirming proportionality-of-effect to cumulative dose. These findings empirically support the validity of the LTL-TTC concept. More broadly, they demonstrate that short-term in vivo mutagenicity assays can serve as reliable surrogates for lifetime carcinogenicity studies, strengthening the scientific and regulatory basis for duration-adjusted acceptable intakes for N-nitrosamine impurities.

pharmacology and toxicology↗