**2.6.2 Two weeks sub-chronic toxicity test**

Twenty eight Wistar rats divided into four weight-matched groups, of seven rats each, (both sexes) were used for the study. Three test groups received 500, 1000, 2000 mg/kg *V. amygdalina* by gavage with biomedical needle (G 16, Length 76.2mm, diameter 3mm, Straight Harvard Apparatus) for 14 days. The control group received normal saline.

Food and water intake were measured daily while the animal's body weights were taken every other day. All animals were observed at least once daily for clinical signs (behavior such as lethargy, hyperactivity, depression and diarrhea). On day 14, immediately prior to euthanasia, all animals were anesthetized with chloroform and bled via the descending aorta for hematology and clinical chemistry determination. Organs were dissected and weighed to determine absolute and relative weight. The blood for clinical chemistry was allowed to clot in microtainer separator tubes, centrifuged and sera collected and stored at - 200C till ready for biochemical analyses. Commercial kits for Biosystem BTS-310, (Biosystem S.A Costa Brava 30, Barcelona, Spain) and Vitrous DT systems, Orthoclinical Diagnostics Johnson Company (US17) were used to analyze liver function, renal function and the electrolyte test.

The hematological tests were carried out in an ethylene diamine tetra-acetic acid (EDTA) – anticoagulated blood. Hemoglobin (Hb) concentration was analysed by the cyanmethaemoglobin method, packed cell volume (PCV) by the micro-method, and white blood cell (WBC, total and differential) and platelet counts by visual methods Dacia et al, 1991). The mean cell hemoglobin concentration (MCHC) was calculated by dividing Hb by PCV ( Dioka et al, 2002).

#### **2.6.3 Six weeks sub-chronic toxicity test**

For the six-week exposure studies, twenty eight male Wistar rats were divided into four weight-matched groups of seven rats each. Three of the four test groups received 750 1500, 3000 mg/kg *V. amygdalina* by gavage with biomedical needle (G 16, Length 76.2mm, diameter 3mm, Straight Harvard Apparatus) consecutively for 43 days. The control group received distilled water vehicle only, via gavage.

Food and water intake were measured daily while the animal's body weights were taken preexposure and weekly during exposure. All animals were observed at least once daily for clinical signs (behavior such as lethargy, hyperactivity, depression and diarrhea), and once/week clinical observations were performed on each rat by removing it from its cage and examining it for changes in general health. On day 44, immediately prior to euthanasia, all animals were anesthetized with chloroform and bled via the descending aorta for hematology and clinical chemistry determination. Organs were dissected and weighed to determine absolute and relative weight. The blood for clinical chemistry was allowed to clot in microtainer separator tubes, centrifuged and sera collected and stored at -70 0C until performing the biochemical analyses. The biochemical and hematological parameter were analyzed in the (New Italian Laboratory). Mbarara University of Science and Technology (MUST).

The collected plasma samples were analyzed using a (HumanStar 180 and Humalyze 2000, Germany) autoanalyzer. Sixteen biochemical parameters were studied; plasma sodium, potassium and chloride were also assayed with (Humalyzer 17410, Germany) autoanalyzer with appropriate Human Kits.

Hematological parameters were analyzed using (Beckman and Coulter, USA) with the appropriate kit (Coulter ACT 5 diff Diluent's).
