2.1. Preparation of TA extract and control materials
TA consists of seven herbs (Table 1). All materials were purchased from an herbal materials company (Jayeondameun, Yangju, Korea). The TA extract was manufactured as the finished product (60.5 mg/mL in a sealed vial) using water and alcohol (v/v = 1:1) at an extramural facility, meeting Korean Good Manufacturing Practice standards (Namsangcheon Herbal Medicine Dispensary, Yongin, Korea). The TA extract was stored in a refrigerator at 4.2–5.4°C. Normal saline was used as a control, in the single-dose intramuscular toxicity test.
2.2. Experimental animals
For the single-dose intramuscular toxicity test, Sprague–Dawley (SD) rats (6-weeks-old, male, and female, n = 5, per group) were purchased from Orient Bio Inc (Seongnam, Korea). The body weight ranged from 190.1–206.2 g for the male and 147.4–163.4 g for the female rats, respectively. When the animals first arrived, a visual inspection was performed and their weight was recorded with electronic scales (CP3202S, Sartorius, Gottingen, Germany).
The general condition and changes in the animals were checked, once daily, during 1 week of acclimatization, and all the animals were judged to be normal. SD rats were housed under controlled environmental conditions with an ambient temperature of 22.5 ± 2°C, relative humidity of 54 ± 6%, 12-h/12-h light/dark cycle, and free access to food (Teklad Certified Irradiated Global 18% Protein Rodent Diet 2918C, Envigo, Huntingdon, UK) and water. The mice were divided into two groups after the acclimatization period.
The animal were cared for and treated according to the Korean National Institute of Health and the Korean Academy of Medical Sciences guidelines. This study was conducted at Biotoxtech (Cheongwon, Korea) under the Good Laboratory Practice regulations , with approval of the Institutional Animal Ethics Committee of Biotoxtech (No. 170574, 170549).
2.3. Single-dose intramuscular toxicity test in Sprague–Dawley rats
2.3.1. Administration and group designation
Since the muscles are the target tissues for pharmacopuncture, the rats were administered TA extract was via intramuscular injection. The expected clinical dose of the test extract, TA, is 0.1 to 1.0 mL (0.1 mL/time) in humans. In a preliminary test (Biotoxtech Study No.: B17513P1), no mortalities were observed after intramuscular injection with a single dose of 0.25, 0.5, and 1.0 mL of TA in male and female rats. Based on the preliminary test, the dose of TA was set to 1.0 mL in this study (G2) and the same amount of saline was administered in the control group (G1) (Table 2). Ten male and 10 female rats, with body weights closest to the mean weight were selected for this experiment. The selected animals were divided into two groups (5 rats of each sex per group) and were administered intramuscular injections into the muscles of the left and the right femur, with a total dose of 1.0 mL per animal.
2.3.2. Observation and inspection
On the day of injection, day 0, the general conditions, including toxicity indication type, toxicity expression and recovery times, and any deaths, were observed at 30 minutes, and 1, 2, 4, and 6 hours after injection. Their general conditions were observed once daily, from day 1 to day 14 after injection. The animals’ body weights were measured before injection on day 0, and on days 3, 7, and 14 after injection.
After the observation period, all animals were anesthetized with CO2 and exsanguinated through the abdominal aorta. Complete postmortem examinations were performed for all animals. During the autopsy, the injection sites were harvested and preserved in 10% neutral buffered formalin. For the histopathological evaluation, preserved tissues were trimmed, dehydrated, paraffin-embedded, and then sectioned and stained with hematoxylin and eosin. All residual tissues were preserved in 10% neutral buffered formalin.
2.4. Statistical analysis
Statistical evaluation of the single-dose intramuscular toxicity test was performed by analyzing the body weights. After testing for equipotentiality using the Folded-F test and finding equal variance, Student’s t-test was performed (P < 0.05, P < 0.01). SAS version 9. 3 (SAS Institute Inc., Cary, NC, USA) was used for all analyses.