Subacute oral toxicity study

Male Sprague Dawley (SD) rats at 6 week-old with an average weight 170 grams were used in this study. For the toxicological or toxicokinetic properties of structurally related chemicals, male rats are likely to be more sensitive compare to female rats, thus male rats are more compatible to be selected (5). A total of six rats were systematically selected out of a population of 30 rats by systematic randomization techniques and kept in their cages for 5 days prior to dosing to allow for acclimatization to the laboratory conditions. The rats were acclimatised to the housing conditions in polycarbonate plastic cages with temperature within the range of 22~27°C, humidity at the range 40~70% and balance of 12 hr light/12 hr dark cycle. The rats were supplied by Alchemy Supplies Sdn. Bhd, Seri Kembangan, Malaysia. Clean sterile straw bedding was provided to the animals. The animals were fed with commercially available standard pellet chow and unlimited supply of filtered drinking water.

Subacute toxicity study was carried out in vivo. The subacute oral toxicity study was conducted using the dose test of up and down procedure according to OECD/OCDE Test Guidelines (5). In control group, the rats received normal saline and in vehicle group the rats received 5% DMSO by oral gavages.

Wellness parameters of animals were observed continuously during the first 30 min after dosing and observed periodically (with special attention given during the first 4 hr) for the next 24 hr and then daily thereafter, for 28 days. Individual weekly body weights of rats were recorded before the administration of extract on 1st week until 4th week of the experiment. Changes in the weight of individual rat was calculated and compared with that of the control and vehicle animals. The rats were observed daily for any mortality or signs of toxicity. The rats were fasted overnight before humanely euthanized with complete exsanguination under general anaesthesia with inhalation of carbon dioxide (CO₂) gas. Organs taken out after dissection, washed thrice in normal saline and dried with folds of filter paper.

Blood samples were collected once at the end of study using 23 gauge needle and 3 mL syringe via cardiac puncture for haematology and clinical biochemistry analyses. Liver and kidneys were collected for histopathology examination.

Blood samples were collected into ethylenediamine tetraacetic acid (EDTA) tube. Total numbers of white blood cells (WBCs) and red blood cells (RBCs), and haemoglobin (HB) concentration were analysed using an automated haematology analyzer (Cell Dyn^® 3700, Abbott Diagnostics, NJ, USA). Blood smear was prepared and stained with Wright stain. The differential WBC count was performed manually by counting 100 WBCs on the blood smears. Packed cell volume (PCV), icterus index, and plasma protein concentration were determined manually using standard methods. Mean corpuscular volume (MCV) and mean corpuscular haemoglobin concentration (MCHC) were calculated manually by using standard formula, MCV = (PCV [L/L] × 1000)/RBC (× 10¹²/L) and MCHC = Hb (g/L)/PCV (L/L).

Blood samples were collected into plain tubes and were centrifuged (Centrifuge S417R, Eppendorf, CA, USA) for 15 min at 3,000 rpm to obtain the serum. The serum samples were further analysed for creatinine, urea, total bilirubin, total protein (TP), albumin (ALB), globulin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and creatine kinase (CK) using an automated clinical chemistry analyser (Dimension Xpand Plus, Siemens, NY, USA).

At the end of the experimental period, all rats were sacrificed with inhalation of CO₂. Selected organs of each rat such as were collected in cold [pH 4] normal saline. The organs were cleaned from blood and weighed. The organs were then fixed in 10% neutral buffered formalin solution. The process of tissue fixation was in 48 hr duration in clean 10% formalin solution. The organs samples were processed at the Histopathology Laboratory, Faculty of Veterinary Medicine, Universiti Putra Malaysia (UPM), Serdang, Malaysia. All of the samples were trimmed about 0.5 cm thickness and were placed in cassettes. Later the cassettes were placed into 10% formalin solution overnight, before they were placed and undergone series of dehydrated process for about 16 hr in an automated processor (ASP300, Leica Biosystems, Nussloch, Germany). The samples then were embedded with paraffin to form a block by a processor machine (EG1160, Leica Biosystems). The samples were trimmed about 3~5 μm thickness using a sectioning rotary microtome RM2155, Leica Biosystems), and directly placed the tissue sectioning in 45°C water bath before mounting on slides. All the glass slides were labelled with a diamond pen and continue mounted on a hot plate (54°C) overnight. Later all slides were stained with hematoxylin and eosin (H&E), and examined microscopically at 10 ×, 20 ×, 40 ×, 60 × and 100 × magnifications. The microscopic analysis of all tissue samples were carried out as a blind study. Any histopathological changes deviant from the norm were carefully recorded.

Toxicological lesions such as inflammation, activated kupffer cells, hydropic degeneration, regeneration and necrosis in the liver were examined and were scored. Meanwhile in kidney tissue, toxicological lesions such as cellular cast, granular cast, protein cast, inflammation, hydropic degeneration and necrosis were examined and were scored. Lesions in both liver and kidney tissues were scored as 0 (normal), 0.5 (very mild), 1.0 (mild), 1.5 (mild to moderate), 2 (moderate), 2.5 (moderate to severe) and 3.0 (severe). The detail descriptions for lesion scoring method is presented in Table 1.

Lesion score and percentage of affected area (mm²)

The body weight, haematological and serum biochemical parameters, and relative organ weight results were expressed as mean ± SEM. The data were analysed using one-way ANOVA and Tukey HSD test using statistical analysis software, IBM SPSS Statistic 22.0 (IMB Corp., NY, USA). The histopathology results were expressed as mean ± SEM and analysed using Kruskal-Wallis test for global comparison of groups for all the parameters. Non-parametric Mann-Whitney-U test was used for comparison between two groups.