*2.2. Care and Handling of Experimental Animal*

The three oral toxicity studies used a total of 208 Sprague Dawley rats purchased from BioLASCO Taiwan Co. Ltd. (Taipei City, Taiwan). The rats, hereafter referred to as the test system (TS), arrived at four weeks of age and were quarantined for two weeks under the care of the veterinarian. Following quarantine, the TS were housed individually in the Individual Ventilated Cage (IVC) and acclimatized to the experimental environment for seven days. The room had a 12 h light and dark cycle where the lights were switched on from 7 a.m. to 7 p.m.; the temperature was kept at 22 ± 3 ◦C and relative humidity was maintained at 57.5 ± 7.5%. The cages and bedding were changed every two weeks. Food (200 g) was placed in each cage weekly and replenished if inadequate. The amount of food left weekly was measured and used to calculate the week's food consumption. Reverse osmosis water was provided ad libitum and the water intake was also measured in a similar approach. This study followed the Principle and Guide to Ethical Use of Laboratory Animals prepared by the Ministry of Health Malaysia (2000) [25]. Approval from the Animal Care and Use Committee (ACUC) at the Institute for Medical Research, Malaysia was obtained; ACUC number ACUC/KKM/02(5/2008).

#### *2.3. Single Dose Acute Oral Toxicity Study*

The acute oral toxicity was done following the OECD Guideline No. 420 [22], where the sighting study was done using single dosages of 0.005, 0.05, 0.30 and 2 g/kg BW of test item and administered orally to female TS. First, one of the TS was orally administered with the lowest dose and observed for any signs of acute toxicity 24 h post-dosing. As the first TS showed no signs of toxicity, a second TS was administered the second lowest dose (0.05 g/kg BW) and observed in the same manner. This procedure was repeated until the highest dose (2 g/kg BW) was administered. The highest dose did not trigger any acute effects; thus 2 g/kg BW was used in the main study. Four new TS were given 2 g/kg BW of the test item and monitored for 14 days. The TS were monitored for any signs of acute toxicity, including morbidity and mortality, at 0.5, 1, 2 and 4 h after treatment, then twice daily until day 14. The BW, food and water consumption were measured weekly. The TS were sacrificed following the 14-day observation period and gross observations of all organs were recorded.

#### *2.4. Subacute 28-Day and Chronic 6-Month Oral Toxicity Studies*

#### 2.4.1. Subacute 28-Day Experimental Design

The subacute oral toxicity study was based on the OECD Guideline No. 407 [23], with modifications. Male and female TS were grouped into four groups of five TS, where each group received doses of 0.14, 0.29 and 1 g/kg BW of test item, orally administered in 2 mL volumes. The control group received distilled water. The TS were observed twice daily for any clinical signs, morbidity and mortality. Individual TS's dosage was corrected each week to correspond with the TS's body weight. Detailed clinical observations and food intake measurements were performed, while water was given ad libitum. The TS were anaesthetized and blood samples were collected by cardiac puncture. Blood samples were sent for hematology analysis (2.5 mL) in EDTA tubes. The TS were then sacrificed with an overdose of diethyl ether and necropsies were performed. At necropsy, gross pathology was conducted and the internal organs; lung, heart, liver, kidneys, adrenals, ovaries, testes, spleen, stomach and intestinal tract were collected. All organs were cleaned of excess fat and absolute weights were taken immediately. The organs were then placed in 10% formalin for histopathology evaluations.

### 2.4.2. Chronic 6-Month Experimental Design

The chronic 6-month oral toxicity study was carried out in accordance with OECD Guideline No. 452 [24], with modifications, where 20 rats per group per sex were used in the same dosage group (0.14 g/kg BW, 0.29 g/kg BW, 1 g/kg BW and Control) as in the subacute study. The test item (2 mL) was orally administered daily. During the in-life phase, the TS were observed daily for mortality, morbidity and clinical observations. Their food and water intake were monitored weekly, while detailed clinical observations by the attending veterinarian was conducted monthly. At necropsy, gross observation was conducted and blood was collected for hematology and serum clinical biochemistry analysis. The absolute weights of the internal organs were recorded, then treated with 10% formalin for histopathology investigation. The list of organs was as in the subacute study.

#### 2.4.3. Hematology Analysis

Blood was analyzed using Medonic CA620 Vet Analyzer (Boule Diagnostics AB, Stockholm, Sweden) for white blood cells (WBC), red blood cells (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular hemoglobin concentration (MCHC) and platelets (PLT) for the subacute study. In the chronic study, the RBC, HGB, HCT and PLT levels were determined.

#### 2.4.4. Clinical Biochemistry Analysis

Serum in the chronic study was analyzed using the biochemistry analyzer (Vitalab Selectra E-series, Vital Scientific N.V., Spankeren/Dieren, Netherlands). Liver function profile—total protein, albumin, enzymes; alkaline phosphatase (ALP), alanine amino-transferase (ALT) and aspartate amino-transferase (AST), lactate dehydrogenase (LDH), creatine kinase (CK); renal profile—creatinine, urea and uric acid; lipid profile—cholesterol and triglycerides; and glucose and calcium were determined.
