5.3. Acute Oral Toxicity Tests

The study for acute oral toxicity has been performed according to the OECD guideline for the Testing of Chemicals No. 423 [21] at the Indian Institute of Toxicology, Pune, India. Healthy young adult (8–12 weeks of age) female (nulliparous and nonpregnant) Sprague-Dawley rats with uniform body weight (within ±20% of the mean) provided by National Institute of Biosciences, Thane, India, were used and acclimatized to laboratory conditions in single polycarbonate cages for a minimum of 5 days. They were maintained at a controlled temperature of 19.5–21.8 °C, relative humidity of 48.4–55.8%, and 12-h light/dark cycle. Animals were fed a rodent feed supplied by Pranav Agro Industries Ltd., Sangli, India, and allowed osmosis water from a local source, both ad libitum. A single dose of 2000 mg/kg bw of the freshly prepared extract I in distilled water (adjusted strength of 200 mg/mL, dose calculation 10 mL/kg of fasting body weight) was administered by oral gavage to three animals, followed by 14 days of observation (step 1). As there were no signs of any toxicity or mortality, three further female animals were added to the study and treated with the same dose (step 2). The applied dosage was selected based on data from the literature and from the knowledge of the phytochemical composition of the test item [1,6,7].

In each step, the animals have been controlled for clinical signs and mortality immediately after application at 5, 10, 30, and after 60 min as well as after 2, 4, and 6 h on the day of dosing, and followed daily for 14 days until sacrifice. Individual body weights were recorded prior to test item administration and thereafter weekly until study termination. All animals surviving the study period were sacrificed by carbon dioxide asphyxiation technique on day 15, followed by necropsy. OECD/EU-GLP approval code: Registration No: I01 by WIV-ISP, Brussels, Belgium, Date of approval: 4 January 2010, and Certificate No.: GLP/C-022 by National (Indian) GLP Compliance Monitoring Authority, New Delhi, India, Date of approval: 16 April 2010.