# Toxicological Evaluation and Antimicrobial Activity of a Natural Thymol–Eucalyptol-Based Mixture

## TL;DR

This study evaluates a natural mixture of thymol and eucalyptol for low toxicity and strong antimicrobial properties, showing promise as a safe alternative to harmful chemicals.

## Contribution

The study introduces a low-toxicity, natural antimicrobial mixture with potential for safe use in food and animal health.

## Key findings

- The mixture showed low toxicity in chickens and Artemia salina, with no systemic adverse effects observed.
- The extract improved intestinal barrier integrity and exhibited antimicrobial activity against several pathogens.
- It demonstrated antimicrobial effects comparable to organic acids used as preservatives.

## Abstract

Currently, safe alternatives with very low toxicity and good antimicrobial activity are being sought to replace chemical compounds that can be harmful to animal and human health. For this reason, this study evaluated the safety and biofunctional microbiocidal potential of an extract composed of thymol and eucalyptol. Toxicity tests showed low toxicity in both chickens (2000 mg/kg bw) and Artemia salina (EC50 = 2003 mg/L) and Daphnia magna (EC50 = 87 mg/L), indicating a safe usage profile. Oxidative stress biomarkers (nitrite and MDA) and antioxidant enzymes (SOD and catalase) improved in treated chickens at 20 days of age. The hematological and biochemical parameters of the treated birds showed normal values similar to those of the control group chickens, with better protein levels and lower AST levels. Histology of the kidney, intestine, and liver showed no changes in any group, confirming the absence of systemic adverse effects. At the molecular level, an improvement in the expression of tight junction proteins (claudin and occludin) was observed, suggesting a strengthening of the intestinal barrier integrity. Finally, the extract demonstrated an antimicrobial effect (E. coli, C. perfringens, Salmonella sp. and Pseudomonas sp.) comparable to that of organic acids commonly used as food preservatives, positioning it as a promising alternative in applications.