2.3Experimental design
The oral chronic toxicity study was evaluated accordance with to Organization for Economic Cooperation and Development (OECD) guideline 408 and Central Council for Research in Ayurvedic Sciences (CCRAS) guideline (6, 7). Wistar albino Rats of both sexes (over night fasting free access to water), aged 8–12 weeks old were used. In 80 rats (40Male and 40females) were randomized into groups separately based on bodyweight. After the randomization process, each study animal was assigned a unique number and identified by a picric acid mark. Group-I served with vehicle as normal control, Group-II, Group-III and Group-IV administered with test drug orally at a dose of 10 mg/kg, 50 mg/kg and 100 mg/kg respectively for 90 days. Before drug administration, the body weight of each animal was determined and the dose was calculated according to the body weight for every week consecutively for 90 days (8). Rat general health, and signs of toxicity, body weight, mortality, food and water intake was monitored. At 90th day 50% of the experimental animals (40 animals form both sex) in each group were subjected to euthanasia. The necropsy was carried out on all euthanized animals and the organs were isolated and observed macroscopically for abnormalities. After 30days of post treatment the remaining half animals (40animals) were euthanized and organs were collected and observed macroscopically for abnormalities. The collected organs during the treatment and post treatment were preserved in 10% formaldehyde solution for histopathological examination.