Rodent Toxicity Archives Reveal Candidate Longevity Agents

Researchers have identified a set of chemical compounds that increased survival rates in long-term rodent toxicity studies, suggesting that existing regulatory safety archives can be repurposed to discover candidate agents for healthy aging.

The motivation for this research stems from the difficulty of finding interventions that extend both lifespan and healthspan in mammals. While efforts to combat age-related chronic disease are intensifying, translation to humans requires rigorous demonstration of efficacy and safety in mammalian models. Large, controlled lifespan studies in genetically heterogeneous mice are considered the preclinical gold standard, but they are slow and costly, limiting the number of interventions that can be tested at scale. The NIA Interventions Testing Program has provided a standardized framework since 2004, yet reproducible extensions of mouse lifespan remain uncommon and are typically modest and sex-specific.

Hormesis, the adaptive activation of protective stress-response pathways by low-level toxic or metabolic challenges, offers a mechanistic rationale for many longevity interventions. This concept is well-documented in short-lived models like C. elegans and Drosophila, where low-dose stressors often produce robust increases in lifespan. However, demonstrating hormesis in mammals is substantially more challenging because it requires long-term dose-response studies with large cohorts. Regulatory long-term toxicity studies submitted to agencies like the European Food Safety Authority (EFSA) and the US Environmental Protection Agency (EPA) constitute an underused repository of high-quality rodent survival data, routinely testing multiple dose groups in large cohorts under stringent quality-control frameworks.

The researchers mined the BfR electronic Archive of Studies and Assessments for long-term rodent studies of pesticide active substances tested at multiple doses, typically three, using at least 50 rats and mice per dose and sex. Typically, studies in rats lasted over two years, yielding median survival at the endpoint of 52% in males and 48% in females. In mice, only 33% of studies were conducted over 22 months, with similar median survival rates, while shorter studies resulted in higher survival but reduced statistical power to detect changes. From a random sample of 545 database entries, 154 contained long-term toxicity and survival data.

Among these 154 entries, 27 substances, representing 18% of the sampled data, increased survival by at least 10% at study termination with statistical significance (p < 0.05) in at least one species, sex, or dose. Only one of these, ascorbic acid, had previously been shown to extend lifespan in male mice. Most of the other substances also exhibited non-significant survival increases across other concentrations and cohorts, with survival decreases being rare. Out of the 27 substances, 15 can be publicly disclosed for further investigation, including alanycarb, ascorbic acid, bifenazate, bromuconazole, bromoxynil, chlorfenapyr, chlortoluron, clothianidin, cyantraniliprole, cyproconazole, cyprodinil, dimethenamid, etoxazole, fenpyroximate, and lenacil.

The survival effects were often sex-biased. Four substances increased survival exclusively in males, while two did so only in females. Sex biases for the remaining nine substances were typically concentration and/or species-dependent. Statistical analyses using Bonferroni and Benjamini-Hochberg corrections indicated that the most robust survival effects remained significant under stringent correction, while others fell below formal false discovery rate thresholds.

Investigations into the mechanisms behind these survival benefits revealed that several substances affected food intake and body weight. Chlorfenapyr, clothianidin, and etoxazole tended to reduce both food intake and body weight at the doses that improved survival, suggesting a potential dietary restriction effect. In contrast, bromoxynil, cyproconazole, cyprodinil, and lenacil reduced body weight without reducing food intake, implying they might have acted as exercise mimetics or produced organ toxicity. The most common chronic toxic effect, typically at the maximum dose tested, was increased liver weight, often with centrilobular hypertrophy, indicating a detoxification response. No substance was regarded as genotoxic, but indications for reproductive toxicity, neurotoxicity, and carcinogenicity were summarized in supplementary tables.

To understand how these substances promote survival, the researchers examined their effects on electron transfer systems, which are frequently reported in non-mammalian systems. In plants, some of these substances inhibit Photosystem II, while in insects or plants, they inhibit mitochondria. Given that perturbations of mitochondria can activate stress response pathways and extend lifespan in C. elegans and mice, the effects of the 15 disclosed substances on cellular oxygen consumption rates (OCR) in mammalian cells were examined at both low (LD20) and high (LD80) doses using a Seahorse XF Analyser. This approach allowed the researchers to assess the direct impact of these compounds on mitochondrial function in a controlled cellular environment.

In intact cells with high doses (LD80), multiple substances significantly inhibited cellular OCR, whereas bromoxynil and chlorfenapyr stimulated it. At lower doses (LD20), only bifenazate and fenpyroximate inhibited OCR, and bromoxynil stimulated it. To examine mitochondria-specific effects, OCR was measured in permeabilized cells using complex-specific substrates. Complex I-specific inhibition was observed for chlortoluron, cyprodinil, bromuconazole, and bifenazate, while etoxazole and fenpyroximate inhibited sites other than complex I. Bromoxynil and chlorfenapyr stimulated mitochondrial OCR during active respiration, with bromoxynil’s uncoupler activity confirmed in state 4 OCR, aligning with in vivo observations of body weight reduction without changes in food intake.

Chlorfenapyr is a pro-drug to the active uncoupler tralopyril, which typically requires activation by cytosolic oxidases, explaining why no mitochondrial uncoupling activity was observed in permeabilized cells. Collectively, the results suggest that perturbation of mitochondrial function is a frequent mechanism in inducing pro-survival stress response pathways among the 15 substances that promoted survival in mammals. This finding highlights the potential of mitochondrial stress responses as strong candidate mechanisms supporting enhanced survival, linking the observed survival benefits to specific biochemical pathways.

While anti-aging interventions on lifespan effects in short-lived organisms may not always be replicated in mammalian models, healthspan outcomes have translational value. Accordingly, C. elegans healthspan was examined using spontaneous motility parameters, chosen as sensitive readouts of neuromuscular and metabolic function. Bifenazate and bromoxynil were selected as proof-of-concept examples, with the former inhibiting and the latter uncoupling mitochondria at LD20. Using the highest concentrations that did not result in acute or developmental toxicity, bifenazate increased the fraction of moving worms at higher ages, with an optimal effect at an intermediate concentration, but did not increase movement speed.

Bifenazate altered movement patterns, producing slower but more sustained activity, although lifespan was not significantly increased. In contrast, bromoxynil at 100 μM resulted in more worms remaining active during the first seven days of adulthood, faster movement at later ages, and increased total movement time and distance, indicating improved functional healthspan. Despite these functional improvements, lifespan was decreased in C. elegans, suggesting that the hormetic window may change or narrow with age, whereby mitochondrial uncoupling stress may shift from beneficial to detrimental as animals age. Both substances activated oxidative stress responses, with survival upon paraquat exposure increasing for each agent.

The study demonstrates that long-term toxicity archives can be repurposed to identify candidate compounds, targets, and mechanisms relevant to mammalian healthy aging. Leveraging such datasets complements conventional screening pipelines and aligns with the 3Rs principle by maximizing information from existing studies. It also provides rich training material for AI- and in silico-driven drug-discovery approaches that require large, high-quality datasets to prioritize interventions for rigorous follow-up in mammalian models. This approach offers a novel pathway for discovering longevity agents by repurposing data originally intended for safety assessment.

Subject of Research: Repurposing regulatory rodent toxicity data to identify candidate longevity agents and mitochondrial mechanisms

Article Title: Repurposing Regulatory Rodent Toxicology Safety Archives to Identify Candidate Hormetic Longevity Agents Highlights Emergent Mitochondrial Mechanisms

Article References: Miwa, S., Kucheryavenko, O., Herrmann, K., Raimundo, A., Xue, F., Mohanraj, K., Saunter, C., Niemann, L., Weinkove, D., & von Zglinicki, T. (2026). Repurposing Regulatory Rodent Toxicology Safety Archives to Identify Candidate Hormetic Longevity Agents Highlights Emergent Mitochondrial Mechanisms. Aging Cell, 25(11), Article e70797. https://doi.org/10.1111/acel.70797

Image Credits: AI Generated

DOI: 10.1111/acel.70797

Keywords: longevity, hormesis, mitochondrial function, toxicology archives, rodent studies, healthy aging, pesticides, survival analysis, drug discovery, C. elegans, Repurposing, Regulatory