The consumption of dietary supplements to enhance physical performance has increased significantly in the last century, especially thermogenic pre-workout supplements. Nevertheless, this industry has faced criticism for inadequate safety measures surveillance in regulatory issues regarding their products. The aims of our study were to investigate two pre-workout supplements with respect to (1) mutagenicity utilizing Salmonella/microsome assay; (2) genotoxicity employing cytokinesis-block micronucleus (CBMN) assay protocols; and (3) hepatocytoxicity using WST cell proliferation, activities of lactate dehydrogenase (LDH) and alkaline phosphatase using human liver carcinoma (HepG2) and mouse fibroblast (F C3H) cells. Oxidative stress was determined through glutathione (GSH) measurement and in silico for predictions of pharmacokinetics and toxicity for the most abundant isolated substances present in these supplements. Both supplements induced mutagenicity in all examined bacterial strains, especially in the presence of exogenous metabolism. Further, tested supplements significantly elevated the formation of micronuclei (MN) as well as other cellular phenomena. Concentration- and time-dependent curves were observed for hepatotoxicity in both studied cell lines. In addition, both supplements decreased levels of intracellular and extracellular GSH. In silico predictions showed that the isolated individual compounds failed to induce the observed outcomes. Our findings provide contributions to the molecular mechanisms underlying two pre-workout supplement-induced toxicity and the need for surveillance. [GRAPHICS]

Popular ergogenic supplements for weight loss raise safety concerns due to potential cardiovascular, liver, and kidney toxicity. Their genotoxicity remains insufficiently studied, highlighting the need for more research on their long-term effects. The Brazilian health surveillance and regulatory system does not include dietary supplements under OECD genotoxicity regulatory guidelines. This study evaluates the mutagenic, cytotoxic, and genotoxic effects of a multi-herbal ergogenic supplement using the Ames test, WST-1 cell viability assay, and cytokinesis-block micronucleus tests in HepG2 and HEK293 cell lines. The Ames test revealed dose-dependent mutagenicity, particularly with metabolic activation, while the WST-1 assay showed hepatocytotoxic and nephrocytotoxic potential with dose- and time-dependent response. The cytokinesis-block micronucleus assay further confirmed genotoxicity, with significant formation of micronuclei and other cellular damage indicators. Our study emphasizes the need for careful safety monitoring of ergogenic supplements, highlighting the toxicity risks associated with their mutagenic, cytotoxic, and genotoxic potential.

Octodrine (2-amino-6-methylheptane; DMHA) is a sympathomimetic stimulant detected in sports-performance and weight-loss supplements despite limited toxicological characterization and regulatory concerns. Here, we used 7-day-post-fertilization zebrafish larvae to characterize the acute cardiac, neurobehavioral, and transcriptional effects of octodrine after 2 h of exposure. Cardiac responses were biphasic: 5 µM octodrine significantly increased atrial and ventricular rates, whereas concentrations ≥ 150 µM produced progressive cardiac depression and higher concentrations disrupted atrioventricular conduction. At 450 µM, all larvae showed complete absence of detectable cardiac activity; mortality was not independently assessed. Modelling of the inhibitory phase yielded IC₅₀ values of 195.6 µM for atrial rate and 188.8 µM for ventricular rate. At substantially lower concentrations, octodrine significantly reduced spontaneous locomotor activity. After Holm correction across all 24 planned time-by-treatment comparisons, a transient effect remained detectable at 50 nM, with more sustained hypoactivity at 0.5 and 5 µM across most of the 2 h exposure period. At the transcriptional level, 3.5 µM octodrine significantly reduced fosab and egr1 expression, whereas fkbp5 was induced at selected concentrations; npas4a showed a significant overall treatment effect but no dose-specific comparison remained significant after blocked analysis and multiplicity correction. nr4a1 was significantly downregulated at 350 µM, whereas th1 and kcnh2a expression was not significantly altered. Together, these findings show that neurobehavioral/transcriptional responses and pronounced cardiac dysfunction occurred over different concentration ranges under the conditions tested. The results provide an initial in vivo hazard characterization of octodrine and support further investigation of its neuropharmacological mechanisms, internal exposure, and cardiovascular safety.

Octodrine (2-amino-6-methylheptane; DMHA) is a sympathomimetic stimulant detected in sports-performance and weight-loss supplements despite limited toxicological characterization and regulatory concerns. Here, we used 7-day-post-fertilization zebrafish larvae to characterize the acute cardiac, neurobehavioral, and transcriptional effects of octodrine after 2 h of exposure. Cardiac responses were biphasic: 5 µM octodrine significantly increased atrial and ventricular rates, whereas concentrations ≥150 µM produced progressive cardiac depression and higher concentrations disrupted atrioventricular conduction. Exposure to 450 µM resulted in 100% lethality. Nonlinear concentration–response analysis yielded EC50 values of 194.8 µM for atrial rate and 187.9 µM for ventricular rate. At substantially lower concentrations, octodrine significantly reduced spontaneous locomotor activity, with effects detectable at 50 nM and more sustained hypoactivity at 0.5 and 5 µM across most of the 2 h exposure period. At the transcriptional level, exposure to 3.5 µM suppressed several neuronal activity–responsive genes, including fosab, egr1, and npas4a, while nr4a1 was downregulated at higher concentrations. The stress-responsive glucocorticoid target fkbp5 was induced at selected concentrations, whereas th1 and kcnh2a expression was not significantly altered. Together, these findings reveal a marked separation between low-concentration neurobehavioral and transcriptional effects and overt cardiac toxicity at substantially higher concentrations. The results provide an initial in vivo toxicological profile of octodrine and support further investigation of its neuropharmacological mechanisms, internal exposure, and cardiovascular safety.