The study's central question is whether the CAPeo essential oil mixture exhibits any toxicity in rats. The context is the growing interest in herbal extracts for health benefits, driven by ethnopharmacological knowledge and recent research indicating CAPeo's antiviral properties and efficacy in treating upper respiratory tract infections. The importance of this research stems from the need to thoroughly assess the safety of herbal remedies, especially given that limited toxicity data exist for the individual components of CAPeo, particularly Cretan dittany. The study aimed to provide comprehensive acute and sub-chronic toxicity data for CAPeo in a rat model, addressing the lack of toxicity information for the mixture and for Cretan dittany specifically. The purpose was to evaluate the safety of CAPeo at various doses and assess potential hepatotoxicity, nephrotoxicity, or inflammatory responses. The study's significance lies in its potential to support the safe and effective use of CAPeo as a natural antiviral remedy, bridging the gap in toxicity information and reinforcing its potential as a therapeutic agent.

Existing literature highlights the role of oxidative stress in chronic diseases and the interest in natural antioxidants found in plants, including polyphenols. Herbal beverages, particularly in Mediterranean cultures like Crete, are recognized for their potential health benefits and association with longevity. Previous research had indicated that specific combinations of Cretan aromatic plants may be beneficial for cold and influenza prevention and treatment. The group had previously demonstrated CAPeo's antiviral activity in vitro and its effectiveness in reducing the duration and severity of upper respiratory tract infection symptoms in a clinical trial. However, toxicity data was limited for the individual components of CAPeo, especially Cretan dittany. While some studies existed for thyme and sage, they focused on different species, and no data were available for dittany. Studies on thyme often examined *Thymus vulgaris* instead of *Coridothymus capitatus*, and while *Thymus vulgaris* essential oil was less toxic than thymol, the latter had reported moderate acute oral toxicity. For Greek sage (*Salvia fruticosa*), no relevant data was available, while data for *Salvia officinalis* reported adverse effects only after prolonged use and overdose. The lack of toxicity data for the mixture motivated this comprehensive in vivo toxicity study.

The study used Sprague-Dawley rats in two experiments: an acute toxicity study and a sub-chronic toxicity study. **Acute Toxicity Study:** Male and female rats (n=4 per group) received a single oral dose of CAPeo (10x and 40x the human dose adjusted for rat metabolism) or olive oil (control) by gavage. Animals were monitored for 24 hours for visible signs of toxicity. Blood samples were collected at 2, 6, and 24 hours for hematological and biochemical analysis. A subset of animals (n=2 per sex) receiving the highest dose were monitored for 15 days and also underwent tissue analysis. Organ samples (kidney, liver, lungs, heart) were examined macroscopically and histologically (hematoxylin-eosin staining) for signs of injury or inflammation. **Sub-chronic Toxicity Study:** Male and female rats (n=10 per group) were divided into 5 groups: untreated, olive oil only (vehicle), and three CAPeo doses (1x, 5x, and 20x the human-equivalent dose). The study lasted 16 weeks, with weekly monitoring of body weight and food intake. Blood samples were collected at the start and end of the study for analysis of hematological and biochemical parameters (including liver and kidney function tests, and the neutrophil/lymphocyte ratio as a marker of inflammation). Organ samples underwent macroscopic and histological examination for signs of toxicity. **CAPeo Composition:** The CAPeo mixture was prepared via steam distillation and analyzed by Gas Chromatography-Mass Spectroscopy, yielding its chemical composition presented in Table 1. **Statistical analysis:** Appropriate statistical tests (t-test, ANOVA) were used to analyze the data.

In the acute toxicity study, there were no visible signs of toxicity (tremors, salivation, diarrhea) in any group within the 24 hours or 15 days observation periods. Biochemical markers of liver and kidney function (AST, ALT, creatinine, urea) remained unchanged compared to baseline and control groups, showing no hepatotoxicity or nephrotoxicity, even at the highest dose (40x the human equivalent). The neutrophil/lymphocyte ratio decreased in male rats at 24 hours, suggesting a possible anti-inflammatory effect. Histological examination revealed no signs of organ injury or inflammation. In the sub-chronic toxicity study, there was no mortality, and normal growth and food intake were observed. Biochemical markers of liver and kidney function remained stable across all groups, again demonstrating no organ toxicity. The neutrophil/lymphocyte ratio was significantly decreased in male rats after 4 months, a finding consistent with the acute study suggesting a sex-dependent anti-inflammatory effect. Histological analysis revealed no signs of toxicity, with minimal inflammation in kidneys observed across all groups (potentially attributable to housing conditions). These findings corroborate results from a previous clinical trial which reported no significant changes in liver and kidney function markers or neutrophil/lymphocyte ratio after 7 days of CAPeo administration to humans.

The study's findings directly address the research question by demonstrating the absence of acute and sub-chronic toxicity for the CAPeo mixture in rats at doses far exceeding those used in humans. The absence of hepatotoxicity, nephrotoxicity, or other adverse effects at doses up to 40 times the human equivalent in acute studies and 20 times the human equivalent in sub-chronic studies strongly supports the safety profile of CAPeo. The sex-dependent anti-inflammatory effect observed in male rats requires further investigation. The results significantly contribute to the field by providing crucial safety data for CAPeo, a plant-derived mixture lacking comprehensive toxicity information, and specifically for Cretan dittany. This data enhances the confidence in using CAPeo as a safe and effective antiviral agent.

The study conclusively demonstrates the absence of acute and sub-chronic toxicity for the CAPeo essential oil mixture in rats, supporting its safe use. This is particularly significant for Cretan dittany, for which no previous toxicity data existed. Future research could explore the sex-dependent anti-inflammatory mechanism observed and investigate the effects of CAPeo in other animal models or on specific cellular pathways. The findings reinforce the potential of CAPeo as a safe and effective natural remedy.

The study was conducted in rats, and results might not fully translate to humans. The highest dose tested was constrained by the administration volume limit in rats. The study focused on a specific composition of CAPeo; variations in composition may affect toxicity. Longer-term toxicity studies would provide further confirmation of long-term safety.