Table 2_Stevia rebaudiana extract (main components: chlorogenic acid and its analogues) as a new safe feed additive: evaluation of acute toxicity, sub chronic toxicity, genotoxicity, and teratogenicity.docx

IntroductionStevia rebaudiana extract (SREC), primarily composed of chlorogenic acid and its analogues, is a promising feed additive with potential benefits for livestock performance, gut health, and antioxidant capacity. However, its safety evaluation has not been comprehensively studied.
MethodsThe safety of SREC was assessed through a series of tests, including acute oral toxicity in mice and rats, a 90-day subchronic toxicity test in rats, genotoxicity assays (Ames test, mouse bone marrow micronucleus test, and mouse sperm abnormality test), and teratogenicity evaluation in pregnant rats.
ResultsThe acute oral toxicity test indicated that the LD₅₀ of SREC in mice and rats was greater than 5,000 mg/kg body weight. In the 90-day subchronic toxicity test, SREC was non-toxic at doses up to 50,000 mg/kg in feed. The Ames test showed no mutagenic effects on Salmonella typhimurium strains TA97, TA98, TA100, and TA102. No genotoxicity was observed in the mouse bone marrow micronucleus test or the sperm abnormality test, with no significant differences compared to controls (p > 0.05). Similarly, no teratogenic effects were found in pregnant rats, with normal embryonic development across treatment and control groups.
DiscussionSREC exhibited low toxicity in both acute and subchronic tests, and no evidence of genotoxicity or teratogenicity was observed. These findings suggest that SREC is safe as a potential feed additive and provide valuable reference data for its safety evaluation.

引言
甜叶菊提取物（Stevia rebaudiana extract, SREC）主要由绿原酸及其类似物构成，是一种颇具应用前景的饲料添加剂，对畜禽生产性能、肠道健康及抗氧化能力均具有潜在益处。然而目前尚未对其开展全面的安全性评价研究。

方法
本研究通过一系列试验对SREC的安全性进行评估，包括小鼠与大鼠的急性经口毒性试验、大鼠90天亚慢性经口毒性试验、遗传毒性试验（Ames试验（Ames test）、小鼠骨髓细胞微核试验以及小鼠精子畸形试验），以及妊娠大鼠致畸试验。

结果
急性经口毒性试验结果显示，SREC对小鼠和大鼠的半数致死量（LD₅₀）均大于5000 mg/kg体重。在90天亚慢性毒性试验中，饲料中添加剂量高达50000 mg/kg的SREC未表现出毒性作用。Ames试验（Ames test）结果表明，SREC对鼠伤寒沙门氏菌TA97、TA98、TA100及TA102菌株均无致突变效应。小鼠骨髓细胞微核试验与精子畸形试验均未观察到遗传毒性，与对照组相比无显著差异（p > 0.05）。同样，妊娠大鼠致畸试验未发现致畸效应，各给药组与对照组的胚胎发育均处于正常水平。

讨论
SREC在急性及亚慢性毒性试验中均表现出低毒性，且未观察到遗传毒性或致畸性相关证据。上述研究结果表明，SREC作为潜在饲料添加剂具有安全性，可为其安全性评价提供有价值的参考数据。