International Journal of Toxicology

Many antiviral agents are known to induce off-target mitochondrial toxicity due to the prokaryotic origin of mitochondria. Mitochondrial dysfunction is frequently linked to cardiotoxicity. We aimed to elucidate the mitochondrial toxicity profile of molnupiravir via focusing on mitochondrial dynamics, biogenesis, and oxidative stress in cardiac cells. Mitochondrial function was evaluated by luminometric measurement of ATP (adenosine triphosphate) content, and flow cytometric analysis of mitochondrial membrane potential, and mitochondrial mass. The expression levels of genes involved in mitochondrial fusion-fission were assessed by RT-PCR. In addition, molecular docking analysis was performed to evaluate the interaction between molnupiravir and the dynamin related-protein DRP1. Protein carbonylation was determined as an oxidative stress parameter. Toxicity evaluation was further investigated in Caenorhabditis elegans to support the in vitro findings at the organismal level. Molnupiravir exposure led to a significant dose-dependent reduction in intracellular ATP level and mitochondrial mass, accompanied by increased protein carbonylation. Mitochondrial membrane potential remained slightly increased. Alterations in the expression of genes regulating mitochondrial dynamics suggested an imbalance between fusion and fission processes, while mitochondrial biogenesis-related signaling was progressively suppressed. C. elegans exposed to higher concentrations of the drug (20-500 µM) exhibited significant lifespan reduction at all doses. Molecular docking analysis demonstrated a moderate binding affinity of molnupiravir to DRP1, supporting a potential direct interaction with mitochondrial fission machinery. In conclusion, our results demonstrate that molnupiravir induces mitochondrial stress through oxidative damage, impaired biogenesis, and altered dynamics, emphasizing the need for careful evaluation of mitochondrial safety of molnupiravir in cardiac tissue.

5-Methoxy-N,N-dimethyltryptamine (5-MeO-DMT) is a serotonin receptor agonist, in clinical development for the treatment of major depression and other psychiatric disorders. A critical safety concern for serotonergic compounds is their potential to induce seizures, as a severe manifestation of serotonin toxicity. The objective of this study was to evaluate the seizure liability of 5-MeO-DMT in a canine model. Beagle dogs (n = 8) were surgically instrumented for continuous telemetric recording of electroencephalography (EEG) and electromyography (EMG), coupled with behavioral video monitoring. Following a baseline period, animals were intranasally administered with control vehicle or escalating doses of 5-MeO-DMT (0.5, 1.0, and 1.5 mg/kg/day), once daily for nine consecutive days. Plasma samples for determination of pharmacokinetic parameters were collected from a satellite group (n = 3). Intranasal administration of 5-MeO-DMT resulted in centrally mediated and dose-dependent behavioral signs, such as head shaking, salivation, repetitive movements, dilated pupils, increased muscle tone, and tremors. A majority of these signs had a rapid onset and offset, which correlated with the peak plasma levels and resolved within 1 hour post-dose, and they were consistent with serotonergic agonism of 5-MeO-DMT. The continuous EEG recording revealed no evidence of seizures or epileptiform discharges at any dose levels throughout the study. In a canine model, considered sensitive to serotonergic drug-induced seizures, daily intranasal administration of escalating doses of 5-MeO-DMT did not induce seizures or epileptiform activity, even at doses causing significant physiological signs of serotonergic stimulation. These results provide robust critical safety information for a low seizure liability of 5-MeO-DMT.

Excessive advanced glycation end products (AGEs) can lead to cardiovascular diseases such as myocardial fibrosis (MF). Although studies have found that AGEs induce cuproptosis, and cuproptosis promotes fibrosis, it has not been confirmed whether AGEs promote MF through cuproptosis. AGEs increased intracellular copper levels, promoted the expression of solute carrier family 31 member 1 (SLC31A1), and downregulated the expression of ferredoxin 1 (FDX1), lipoic acid synthetase (LIAS), lipoylated (Lip)-dihydrolipoamide S-succinyltransferase (DLST), and Lip-dihydrolipoamide S-acetyltransferase (DLAT) in cardiac fibroblasts (CFs). All of these key cuproptosis regulatory gene expression abnormalities indicate that cuproptosis is induced. AGEs also decreased adenosine triphosphate content and inhibited the activities of mitochondrial complexes I and III, but these regulatory effects were significantly weakened after SLC31A1 downregulation. Meanwhile, AGEs significantly promoted the expression of alpha smooth muscle actin, collagen I, collagen III, and transforming growth factor-β1, while SLC31A1 siRNA or copper chelator ammonium tetrathiomolybdate (TTM) blocked these promoting effects. Similarly, CuCl₂ also induced fibrosis gene expression, while SLC31A1 overexpression (SLC31A1-O) further enhanced these effects, but TTM reduced CF activation induced by CuCl₂ plus SLC31A1-O. In addition, AGEs significantly promoted cell migration and enhanced the expression and secretion of matrix metalloproteinase (MMP)-2 and MMP-9, while SLC31A1 siRNA or TTM weakened these effects. SLC31A1-O plus copper treatment also had similar effects to AGEs, and these effects could also be blocked by TTM. Therefore, AGEs enhance copper transport by promoting SLC31A1 expression, which leads to intracellular copper overload and then induces cuproptosis, and finally promotes CF activation and migration.

Quercetagetin (QG) is a naturally occurring flavonoid found in marigold (Tagetes erecta). It has garnered attention for its potential to support immune health, regulate blood sugar levels, and enhance metabolism. Yet, unlike the closely related compound quercetin (QC), QG has an extra hydroxyl (-OH) group at the C-6 position of the flavonoid A-ring, whereas quercetin has only a hydrogen atom at that position, which has raised safety concerns. QG has not been thoroughly studied for long-term safety. To fill this gap, we conducted a 90-day study in rats to assess the response to repeated oral doses of QG. The QG-administered group of animals received 500, 1000, or 3000 mg/kg bw per day, and we monitored a wide range of health indicators, from body weight and food intake to blood chemistry, hormone levels, and indicators of organ health. The results were reassuring as there were no changes in the urine parameters, deaths, clinical signs, or changes in growth or eye health. Changes were non-adverse and/or of no significant toxicological concern in blood, and organ weight differences were minor and not associated with any tissue damage. Based on these findings, the No Observed Adverse Effect Level (NOAEL) was established at 3000 mg/kg bw/day, the highest dose tested.

We also tested QG for mutagenicity/clastogenicity using standard genotoxic assays, including bacterial mutation tests and micronucleus assays in CHO-k1 cells (in vitro) and Swiss albino mice (in vivo). Across all systems, QG showed no evidence of mutagenicity and/or clastogenicity.

Nalmefene is an opioid antagonist used in OPVEE^® nasal spray approved for use in patients 12 years of age and older. To support future clinical studies in younger pediatric populations, 2 GLP juvenile rat studies were conducted to assess the safety of nalmefene and its associated systemic exposure. Nalmefene was administered daily via intranasal instillation from postnatal day (PND) 7 to PND 28 (corresponding to birth through approximately 3 years) with recovery to PND 91, or from PND 22 to PND 70 (2 years to adolescent) with recovery to PND 114. Exposures on PND 7 were 28-40 times higher than those on PND 28, and exposures on PND 22 were 3-5 times higher than those on PND 70. Exposures on PND 28 were comparable to those on PND 70, attaining adult levels. Considering the extensive metabolism and urinary clearance of nalmefene, these findings are consistent with the maturation of metabolic and urinary clearance pathways during early development. No effects were observed on clinical signs, ophthalmology, food consumption, femur length, sperm parameters, immune responses, or neurodevelopment as assessed by neurohistopathology and neurobehavioral testing for locomotor activity, sensory function, or learning and memory. There were no macroscopic or microscopic findings indicative of target organ, systemic, or neurotoxicity. The NOAELs were 4.25 mg/dose, the highest doses tested. On PND 28, C_max (224 ng/mL) and AUC (311 ng·hr/mL) were 21- and 8-fold higher than those reported in human adults, respectively. The AUC-based safety margins in pediatric patients were estimated to range from 1.5 to 12, depending on the age group (birth-12 years; 3-40 kg).

Ketamine, an N-methyl-D-aspartate (NMDA) receptor antagonist, was first approved by the US FDA as KETALAR® for induction and maintenance of general anesthesia. Ketamine has been used off-label for depression treatment, while its S-enantiomer, esketamine (SPRAVATO®), is approved for treatment-resistant depression and major depressive disorder with acute suicidal ideation or behavior. High doses of ketamine have been linked to neurotoxicity. The effects of acute ketamine exposure on brain development during childhood and adolescence in humans remain poorly understood. To better characterize the safety of ketamine, we treated juvenile (postnatal day (PND) 21, 30, 35) and adult rats (PND 90) with a single subcutaneous dose of ketamine (50, 75, and 100 mg/kg). Seventy-two hours later, tissue samples were collected. Tissue samples were processed, mounted as 5-µm-thick sections, and stained with H&E for neurohistopathological evaluation. Additionally, key pharmacokinetic parameters of ketamine were determined. Neuronal necrosis was detected in the retrosplenial cortex in adult female rats treated with ketamine (100 mg/kg), while no histological changes were observed in any other groups. These data are the first to demonstrate that acute ketamine can cause neuronal necrosis in adult animals and do not support juvenile rats as having an increased susceptibility to ketamine-induced neuronal death. Adult female rats showed a marked increase in norketamine, the primary metabolite of ketamine in serum. We hypothesize that the elevated levels of norketamine may contribute to these sex- and age-specific histopathological changes.

The Expert Panel for Cosmetic Safety (Panel) assessed the safety of 8 naturally-sourced clay ingredients, of which 6 were previously reviewed, as used in cosmetic formulations. All of these ingredients are reported to function in cosmetics as absorbents and bulking agents; other cosmetic functions are also reported. The Panel reviewed all relevant data and concluded that Kaolin is safe in cosmetics in the present practices of use and concentration described in this safety assessment. The remaining 7 naturally-sourced clay ingredients are safe in cosmetics in the present practices of use and concentration, with the exception that the available data are insufficient to make a determination that these ingredients are safe in products that may be incidentally inhaled.

Siamenoside I, a cucurbitane glycoside 300 times sweeter than sucrose, is found in monk fruit (Siraitia grosvenorii). The triterpene glycosides in monk fruit, such as mogroside V, siamenoside I, and mogroside III, comprise around 1% of the fruit flesh. These mogrosides share a core structure, mogrol, with varying numbers of glucose units attached to carbon-3 of the cucurbitane backbone and/or carbon-24 of the triterpene side chain. Previous in vitro fecal homogenate metabolism and in vivo pharmacokinetic research indicated that mogrosides are deglycosylated to a common metabolite, the aglycone mogrol, by intestinal flora prior to absorption. This study was conducted using a purified siamenoside I produced via fermentation and a monk fruit extract in an in vitro human fecal homogenate system to confirm the deglycosylation of siamenoside I by intestinal microflora to the common metabolic intermediate, mogrol. The rates of deglycosylation of siamenoside I and monk fruit extract showed essentially complete metabolism to mogrol within 8 h at 0.2 mg/mL and 2 mg/mL for siamenoside I, and 8 h at 0.2 mg/mL and 16 h at 2 mg/mL for monk fruit extract, in both male and female pooled fecal homogenates with no apparent sex differences. Overall, no difference was observed in the deglycosylation rates of purified siamenoside I or siamenoside I present in monk fruit extract to the mogrol metabolite further confirming the deglycosylation of mogrosides in the lower gastrointestinal tract.

War and terrorism demonstrate the need for an improved antidote against nerve agents that cause brain damage following inhibition of acetylcholinesterase (AChE). Our laboratories invented substituted phenoxyalkyl pyridinium oxime AChE reactivators (US patent 9,227,937) that demonstrate the ability to cross the blood–brain barrier in in vivo rat tests with a sarin surrogate by reducing time to cessation of seizure‐like behaviors, glial fibrillary acidic protein levels, and neuropathology, compared to the FDA-approved oxime, 2‐pyridine aldoxime methyl chloride (2‐PAM). This study investigated the safety profile of novel oximes through evaluation of their cytotoxicity and genotoxicity. Cytotoxicity was evaluated in mouse areolar fibroblasts using a lactate dehydrogenase (LDH) assay and an MTS assay. All novel oximes showed no toxicity in the LDH assay from 1.25 nM to 5 mM, whereas 2-PAM did; novel oximes were significantly different from 2-PAM (P < .00001). The MTS assay demonstrated that novel oximes at 2 µM to 500 µM significantly reduced the metabolic activity of fibroblasts compared to 2-PAM (P = .00001). A modified Ames assay using 5 bacterial strains with and without S9 assessed the genotoxicity of the lead compound, Oxime 20, at concentrations from 100 µM to 10 mM. No concentration, whether with S9 or without, showed any genotoxicity. The LDH and Ames results indicated no cytotoxicity or genotoxicity was caused by the novel oximes.

The extensive application of bisphenol A (BPA), an endocrine-disrupting substance in polymer-based products and consumer commodities, has sparked concerns over its carcinogenic potential. However, the molecular mechanisms underlying BPA-induced gastric carcinogenesis remain poorly understood. We used network toxicology and molecular docking analysis to uncover the pivotal targets and mechanisms underlying BPA-associated gastric carcinogenesis. Potential BPA-related targets and differentially expressed genes in gastric cancer (GC) were collected from publicly available databases to identify hub genes. A prognostic risk assessment model utilizing hub genes, with molecular docking simulations, was performed to confirm the binding interactions between BPA and target proteins. A total of 27 potential targets associated with both BPA exposure and GC were identified. The protein-protein interaction network analysis revealed 19 hub genes, with core targets including MMP9, ADRB2, PTGS1, SLC6A4, ERBB2, MAPT, AR, SLC6A3, CNR1, PDE5A, DRD2, KCNH2, MC4R, CALCR, CHRM1, ADRB3, CXCR2, MMP1, and SHBG. Enrichment analysis demonstrated these hub genes were significantly involved in neuroactive ligand-receptor interaction, calcium signaling pathway, cGMP-PKG signaling pathway, and chemical carcinogenesis-receptor activation pathways. Prognostic analysis identified six hub genes (CALCR, ADRB3, CNR1, HRH2, KCNH2, and AR) significantly associated with patient survival. Molecular docking simulations demonstrated robust interaction affinities of BPA with the identified core target proteins. This study reveals that BPA may influence GC development and progression through multiple targets and signaling pathways, particularly involving G protein-coupled receptor signaling, hormone regulation, and neurological pathways.

The Expert Panel for Cosmetic Ingredient Safety (Panel) assessed the safety of Basic Yellow 87, which is reported to function as a hair dye in cosmetic products. The Panel reviewed the available data to determine the safety of this ingredient. The Panel concluded that Basic Yellow 87 is safe for use as a hair dye ingredient in the present practices of use and concentration described in this safety assessment.

Complement-dependent cytotoxicity (CDC) is critical for tumor cell proliferation. Previous work has revealed that epalrestat (EPA), an inhibitor of aldose reductase, plays a crucial role in tumor cell proliferation. However, whether CDC is involved in EPA-mediated inhibition of cancer cell proliferation remains unclear. This study evaluates reactive oxygen species (ROS) levels, 5-ethynyl-2'-deoxyuridine (EdU) incorporation, and Cell Counting Kit-8 assay to determine the effect of EPA on cell proliferation in colorectal cancer cells. Quantitative real-time PCR, immunoblotting, rescue experiments combined with luciferase reporter assay, and chromatin immunoprecipitation (ChIP) assay were performed to explore the mechanism of EPA on CDC. In the present study, we observed that EPA significantly downregulates cell proliferation characterized by enhanced ROS levels and reduced EdU⁺ positive cells, which was attributed to the decreased CD46 expression. Ectopic CD46 expression reversed the above EPA-induced phenomenon. Mechanistically, EPA treatment suppressed protein kinase D (PKD)/signal transducer and activator of transcription 3 (STAT3) phosphorylation, leading to reduce the binding ability of STAT3 to the CD46 promoter, further attenuating CD46 transactivation. Activation of PKD by phorbol 12-myristate 13-acetate largely blocked the effect of EPA-mediated CD46 transactivation. Together, the current findings revealed a novel mechanism whereby EPA modulates CDC to inhibit the proliferation of cancer cells, broadening the pharmacological functions of EPA in anti-tumor therapy.

In a 4-week rat study, a nucleotide prodrug antiviral agent containing a synthetic non-natural amino acid (C-331) induced a dose-related vacuolation in the brain’s white matter tracts without concurrent neuronal loss, gliosis, or neurological deficits. Routine safety pharmacology studies showed no effects. Penetration of the drug through the blood-brain barrier was negligible. Rat-specific metabolites were not evident. A follow-up 13-week rat study with a 4-week interim assessment was performed using oral doses of 0, 100, 300, or 1000 mg/kg/day for 4 or 13 weeks. Intermittent, transient clonic convulsions (<1 min) occurred in 1 male rat at 100 mg/kg/day (3 observations) and 5 animals at 1000 mg/kg/day during the dosing phase but were not observed during recovery; their relationship to brain vacuolation remains uncertain. No test article-related neurobehavioral or open-field changes, or ophthalmic abnormalities were noted. In all treated groups, widespread vacuolation predominantly affecting white matter was observed in the cerebellum, thalamus, and striatum. The severity of vacuolation increased over time and persisted after the 4-week recovery period. Based on ultrastructural evaluation, the vacuolation resulted from myelin sheath splitting (intramyelinic edema) without axonal degeneration. The vacuolation was considered to be an adverse effect based on the severity and extent of the changes and the potential for causing oligodendrocyte dysfunction. An NOAEL in the rat was not identified. Because of multiple unknowns, including the lack of consistent neurobehavioral signs, species specificity, low exposure multiples, and the potential human significance, further development of C-331 was terminated. The study describes the unusual presentation of species-specific, drug-induced splitting of myelin sheaths/intramyelinic edema without clear correlative neurofunctional effects.

Traditional food production systems are constrained by limited arable land, climate change, and environmental pressures, necessitating the exploration of alternative protein sources. Mycoprotein from Fusarium compactum (F. compactum) MM-135 has shown promise due to its balanced amino acid profile, high dietary fiber, and low-fat content. Previous studies, including a 14-day dietary exposure study, have shown safety at high doses up to 150,000 ppm. This study further evaluated the subchronic toxicity and teratogenicity of mycoprotein from F. compactum MM-135, testing higher doses than previously reported. In the 90-day oral toxicity study, rats were fed diets containing 50,000, 100,000, or 200,000 ppm of mycoprotein with no adverse effects. The NOAEL was 200,000 ppm (15.26 g/kg/day in males and 16.20 g/kg/day in females). In the teratogenic study, pregnant rats were exposed to the same doses from gestation days 6 to 15. No adverse effects on maternal pregnancy or fetal development were observed, with an NOAEL of 200,000 ppm (16.53 g/kg/day). These results confirm that mycoprotein from F. compactum MM-135 is safe at the tested dose of 200,000 ppm, exceeding the safety threshold established in prior studies, and support its potential as a sustainable alternative protein source.

To evaluate the safety of Metabolyte™ (sodium dodecanedioate) for human consumption, results from in vitro and in vivo studies are reported, including a bacterial reverse mutation assay, an in vitro mammalian chromosome aberration test, an in vivo mammalian bone marrow micronucleus test, an in vivo mammalian bone marrow chromosome aberration test, and a 28-day repeat-dose oral toxicological evaluation. Metabolyte™ was not mutagenic in the in vitro and in vivo studies. In the 28-day study, Wistar rats were administered Metabolyte™ by gavage at doses of 0 (G1), 3250 (G2), 6500 (G3), and 13,100 (G4) mg test article/kg bw/day. Histological examination did not reveal any test item related lesions. In the G3 and G4 dose groups, significantly altered electrolyte levels and decrease in sodium and chloride levels in males, and increase in calcium and phosphorous in females, are indicative of possible kidney malfunction. The observed significant increase in triglycerides in G4 males and G3 and G4 females and bile acid in G3 and G4 males and G4 females, decrease in globulin and concurrent increase in albumin/globulin ratio in G4 males, and increase/decrease in ALT in G4 males and females indicate liver malfunction. Significant alterations in motor activity were observed, along with an increase in alkaline phosphatase, in G3 and G4 female animals. Significant changes in absolute relative weight of kidney and liver were detected in G4 females. The NOAEL was considered to be 3250 mg/kg bw/day, as at this dose there were no toxicologically relevant treatment-related findings in male or female rats.

Telmisartan, an angiotensin II receptor blocker with partial PPAR-γ agonist activity, exhibits pleiotropic effects beyond its antihypertensive action, including modulation of lipid metabolism, erythropoiesis, and renal function. However, its anorexigenic properties complicate interpretation of systemic outcomes in toxicology studies. This study employed a pair-feeding design to differentiate feed-dependent effects from direct pharmacodynamic actions in male Wistar rats treated with telmisartan (100 mg/kg/day) for 28 days. Both telmisartan and pair-fed groups showed reduced body weight and hepatic triglyceride levels, confirming feed-dependent metabolic effects. In contrast, telmisartan-specific changes included suppression of erythroid indices; reduced serum triglycerides; elevated urea, creatinine, potassium, and inorganic phosphate levels; and mild gastric submucosal inflammation, none of which were observed in pair-fed controls. These findings demonstrate that telmisartan’s hematological, renal, and lipid-lowering effects are feed-independent and mechanistically distinct from caloric restriction.

The Expert Panel for Cosmetic Ingredient Safety (Panel) assessed the safety of 8 Melaleuca alternifolia (tea tree)-derived ingredients as used in cosmetic formulations; 5 of these ingredients are reported to function in cosmetics as skin-conditioning agents. Because final product formulations may contain multiple botanicals, each containing the same constituents of concern, formulators are advised to be aware of these constituents and to avoid reaching levels that may be hazardous to consumers. Industry should use good manufacturing practices to minimize impurities that could be present in botanical ingredients. The Panel noted that oxidized tea tree oil could be a sensitizer, and stated that industry should employ methods to minimize oxidation of the oil in the final cosmetic product. The Panel considered all the data and concluded that these ingredients are safe in cosmetics in the present practices of use and concentration described in this safety assessment when formulated to be non-sensitizing.

Advances in the delivery and safety of nucleic acid-based therapeutics are enabling tissue-selective targeting and broadening the reach of genetic medicines. In addition to GalNAc-conjugated oligonucleotides, newer modalities—predominantly antibody- and peptide-conjugated oligonucleotides—combine innovative components with complex and often multimodal mechanisms of action. These constructs may require tailored nonclinical testing strategies to evaluate pharmacology, biodistribution, and component- and construct-driven toxicities. Here, we review the current state of these molecules, collectively termed here as bioconjugated oligonucleotide (BCO) therapeutics, summarize key safety liabilities across the protein scaffold, linker, and oligonucleotide payload, and highlight nonclinical approaches, including predictive safety assessments and in vivo toxicity studies that can support human clinical trials. We also discuss risk-based regulatory considerations that may differ from traditional biologics or small molecules.