This study was approved by Institutional Animal Care and Use Committee of Biotoxtech Co. (Approval No: 130365). The tests were performed according to Good Laboratory Practice (GLP) regulation and toxicity test guidelines of the Ministry of Food and Drug Safety (MFDS).
To process the tissues of deer antlers (DA), we chopped 35 g of DA into small pieces and put them into 500 mL of 95% ethanol. The DA mixture was stirred 4 hours a day during cold storage for one week to extract all DA components; then, the stirred DA mixture was filtered with a Whatman™ nylon membrane filter (pore size: 0.45 μm), and 12 g of the filtered mixture was added to 1000 cc of safflower oil in a round flask. This was followed by decompressed concentration to volatilize ethanol completely. Ethanol vaporized at 55°C and was liquefied through the cooling tube; the DA components were dissolved into the safflower oil. The liquid ethanol was discarded, and the DA extract solution was transferred to the preparation tank. The transferred DA and safflower oil solution (CFC solution) was mixed by using nitrogen gas (N2, 99.99% purity) agitation for over an hour. The CFC solution was filtered with 0.2 μm pore filter paper (KA2DFLP02, Pall Corporation, U.S.A.) to remove endotoxin, and the filtered solution was added to water for injection (WFI) at an adequate rate in the reaction container. The CFC solution and an emulsifier were mixed with high speed agitator to make a WCFC solution. The pH of the WCFC solution was adjusted to pH 7.3 by using NaOH. The WCFC solution was passed through a filter (0.1 μm Sartorius) between the preparation and the filling tanks and was transferred to the filling tank by using N2 gas. The WCFC solution was subdivided, placed into vials, and sterilized at high pressure (high pressure sterilizer, fine FA, Korea) at 121°C for 30 minutes. The vials containing the WCFC solution were kept refrigerated at 4°C.
Five-week-old male (n = 24) and female (n = 24) Sprague-Dawley (SD) rats with weights ranging from 112.2 to 125.6 g in males and from 110.5 ─ 131.0 g in females were purchased from Orientbio Inc., Korea, for use in this study. When the animals were received, they underwent basic visual examinations, and their weights were measured by using an electronic scale (CP3202S, Sartorius, Germany). General symptoms were observed daily during the 7-day stabilization period. On the last day of the stabilization period, weights, general symptoms and weight changes were measured to confirm the normal conditions for all animals.
Animals were kept in stainless-steel wire cages with dimensions of 260 W × 350 D × 210 H (mm) under the following conditions: temperature in the range of 22.0 ─ 23.9°C, relative humidity in the range of 50.3% ─ 77.3%, a air change rate of 10 ─ 15 times/hour, a illumination time of 12 hours/day (7:00 am ─ 7:00 pm), and an intensity of illumination of 150 ─ 300 Lux. The breeding cage was changed once every two weeks. The breeding apparatuses were cleaned by using an automatic washer and were sterilized by using an autoclave. Solid feed (Teklad Certified Irradiated Global 18% Protein Rodent Diet 2918C, Harlan Laboratories, Inc., U.S.A.) was put into the feeding system for free intake. Drinking water was sterilized by using an ultraviolet water sterilizer.
All animals were put into grouped on the last day of stabilization (grouping day). Twenty male and twenty female rats whose weights were approximately equal to the mean weight were selected. Selected animals were randomized into 4 groups with 5 male rats and 5 female rats for each group (male weights: 179.8 ─ 197.0 g, female weights: 145.7 ─ 177.0 g) so that average weights per group were about the same. The remaining animals were excluded from the study.
An intramuscular injection route using a method applicable to Korean medicine clinics was selected because many kinds of pharmacopunctures have been injected into muscle or subcutaneous tissue to treat diseases at Korean medicine clinics. Dosages for the control and the high dose groups were 0.5 mL/animal of saline and of WCFC, respectively, and those for the middle dose group and the low dose group were 0.25 and 0.125 mL/animal, respectively. WCFC was injected into the muscle of the left femoral region by using a disposable syringe (1 mL, 26 gauge) (Table 1).
In a pilot test, no deaths were observed for intramuscular injections of 0.5 mL/animal of WCFC into male and female rats (Biotoxtech Study No.: B13477P). Based on the pilot study and clinical experience, the injection doses selected for this study were 0.5 mL/animal for the high dose, 0.25 mL/animal for the middle dose, and 0.125 mL/animal for the low dose, the dose being reduced by a factor of 2 from group to group. Normal saline (Choongwae Pharma Corp., Korea) at a dose of 0.5 mL/animal was injected into the control group.
General clinical sign, such as types of toxicity, onset time, recovery time, etc., and deaths were observed 30 minutes, 1, 2, 4, and 6 hours after the first injection on the day of the first injection (day 0). General symptoms were observed daily for 14 days after injection. The body weights of the SD rats were measured on the day of the injection (before injection) and on the third, seventh, and fourteenth after the injection. Before necropsy, all animals were fasted for more than 18 hours. On the necropsy day (fifteenth day after injection), animals were anesthetized with isoflurane, and blood was taken from the abdominal aorta. Blood was kept in a 1-mL ethylenediaminetetraacetic acid (EDTA) tube, and hematologic tests were done by using a blood corpuscle analyzer (ADVIA 2120i, Siemens, Germany). The red blood cell count (RBC), hemoglobin concentration (HGB), hematocrits (HCT), mean corpuscular cell volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular cell hemoglobin concentration (MCHC), platelet count (PLT), white blood cell count (WBC), WBC differential counts (neutrophils, lymphocytes, monocytes, eosinophils, and basophils), and reticulocyte (Reti) count were measured. For blood coagulation tests, blood was drawn in 2-mL tubes that contained 3.2% sodium citrate. The blood was centrifuged at 3,000 rpm for 10 minutes to collect the plasma. The prothrombin time (PT) and the active partial thromboplastin time (APTT) were measured with a coagulation time analyzer (Coapresta 2000, Sekisui, Japan). The blood that remained after the hematologic test was used for the serum biochemical analysis. The blood was centrifuged at 3,000 rpm for 10 minutes to collect the serum. Blood urea nitrogen (BUN), creatinine (Cr), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), gamma glutamyl transpeptidase (GGT), total protein (TP), albumin (Alb) and albumin/ globulin ratio (A/G ratio), total bilirubin (T-bili), total cholesterol (T-Chol), triglycerides (TGs), phosphorus (P), glucose (Glu), and calcium (Ca) were measured with a blood chemical test analyzer (7180, Hitachi, Japan). Sodium (Na+), potassium (K+) and chloride (Cl-) were measured with an electrolyte analyzer (AVL9181, Roche, Germany).
Detailed visual examinations of entire organs were done for all animals after necropsy had been conducted. From the necropsied animals, injection sites were removed and fixed with 10% neutral buffered formalin (NBF). Fixed tissues underwent a general tissue processing process such as dehydration, paraffin embedding, and sectioning to make tissue slices, which was followed by hematoxylin and eosin (H&E) staining. Residual tissues were preserved with 10% NBF. Microscopic examinations were conducted for all injection sites on all test group rats, and histopathologic examinations were performed for all injection sites of the rats in all groups.
The data for weights, hematology and serum biochemistry were analyzed by using a statistical analysis system (SAS, version 9.3, SAS Institute Inc., U.S.A.). The continuous data are presented as means ± standard deviations. The test for equality of variance was done with the Bartlett test (P < 0.05). If the variance data were normally distributed, the one way analysis of variance (ANOVA) was performed, followed by Dunnett's t-test for a post-hoc analysis. If those were not homogeneous, the Kruskall-Wallis test was done. A significance level of 0.05 for the two-sided test was considered statistically significant.