Safety Evaluation of Modified Si Jun Zi Tang, Assessment of Controlled and Randomized, Acute and Subchronic Mouse Oral Toxicity Studies

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1 Safety Evaluation of Modified Si Jun Zi Tang, Assessment of Controlled and Randomized, Acute and Subchronic Mouse Oral Toxicity Studies Shuang Ma DVM, MS, Aituan Ma DVM, MS, PhD ABSTRACT In order to evaluate the clinical safety of a modification of the classical formula Si Jun Zi Tang (Wei Qi Booster a (WQB)), acute and subchronic mouse oral toxicity tests were conducted. An acute oral 7-day mouse study using 80 Kunming (KM) mice randomly divided into a saline control and 7 dosages of WQB (range g/kg BW) was conducted to determine LD 50. No mortality or adverse effects were observed in any parameter tested which included food consumption, clinical toxicity, body weights, organ/body weight ratios (liver and kidney) and histologic morphology of the liver, spleen, lung, kidney and duodenum. To determine the maximum tolerated dose (MTD) of WQB, g/ kg BW was given twice daily for 7 days. No mortality or adverse effects were observed in any parameter tested in this study which included all parameters evaluated in the LD 50 study along with hematology. In the subchronic oral study, 15 mice were divided into 3 groups with the control group fed normal chow diet and each of the other two groups either receiving normal chow with 1% WQB or levamisole hydrochloride 0.01 g/kg BW (positive control) for 90 days. No mortality or adverse effects were observed in the 1% WQB in any parameter tested which included all parameters evaluated in the acute studies and with clinical chemistry replacing hematology. Results of these preclinical safety studies conducted with WQB at the highest dose tested which is g/kg BW for 7 days and 1% of food consumed for 90 days demonstrated no toxicity. Key words: Si Jun Zi Tang, Wei Qi Booster, LD 50, MTD, pre-clinical safety testing, hematology; biological indexes; immunity indexes, mouse, Chinese herbal medicine ABBREVIATIONS ALT Alanine aminotransferase ALB Albumin AST Aspartate aminotransferase BUN Blood urea nitrogen CHOL Cholesterol Crea Creatinine GLU Glucose HGB Hemoglobin LD 50 Median Lethal Dose 50 LYM Lymphocyte MID Leukocytes (neutrophil, eosinophil, basophil) MTD Maximum tolerated dose RBC Red blood cell TG Triglyceride UA Uric acid WBC White Blood Cell WQB Wei Qi Booster From: College of Traditional Chinese Veterinary Medicine, Agricultural University of Hebei, Baoding China (S. Ma, A, Ma) Herbal medicinal products and supplements have increased exponentially over the past three decades and are now used by more than 80% of the worldwide population. 1 With the increased use of these therapeutic agents, the safety of Chinese herbal medicine has drawn more attention recently from both domestic and abroad. 2 Establishing the safety of therapeutic compounds through toxicity studies is an active stage of research for western pharmaceuticals and is a requirement for approval under government regulations that approve the release of new drugs. In these studies, safety data are collected with the main goal to assess a product s safety profile. Studies of a therapeutic agent s toxicity include identification of organs that may be targeted by the compound; as well as any long term adverse effects. These studies should mimic the planned route, duration and schedule of drug administration as closely as possible. 3 These non-clinical studies may include acute toxicity (7 or 14 day studies), subacute (28-day study), subchronic (90 days or greater) and chronic (1 year) studies usually in a rodent species. Body weights, clinical signs of toxicity, food consumption, clinical pathology, histopathology, organ weights and 29

2 behavioral changes are usually evaluated in these studies along with mortality. A brief review of the literature reveals there is some safety testing of Chinese herbal medicines similar to pharmaceuticals being performed with both single herbs and herbal formulas. Safety evaluation of Xiang Gu (Shitake) was performed by Gao et al in a 14-day study in mice containing 3 dose groups along with a saline control. There were no adverse events recorded and a complete blood count and serum biochemical tests of liver and kidney were all within normal range. 4 The median lethal dose (LD 50 ) and the maximum tolerated dose (MTD) of a formula containing Huang Lian (Rhizoma coptidis) and Ku Shen (Sophora flavescens) was tested in a mouse model. These animals were given 40 g/kg BW of the test herbs three times a day for 7 days without mortality or adverse effects on body weight, food and water consumption or clinical signs. The LD 50 was determined to be greater than 40g/kg BW and the MTD greater than 120 g/kg BW. The authors concluded that the traditional Chinese medicines had very low toxicity or no toxicity and were safe to use in the clinic. 5 Kim and his group investigated the safety of Ren Shen Bai Du San (Insampaedok-san water extract, ISSE) in mice, when receiving 5g/kg BW once daily for 15 days. Again, no mortality or adverse effects occurred and ISSE was considered safe for consumption. 6 The acute toxicity of the herbal formula Jin Weng Zhi Li Ke Li which contains several herbs including Huang Qi (Astragalus membranaceus) and Jin Yin Hua (Lonicera japanica thunb) was evaluated in mice in a 7-day study. The results showed the LD 50 was greater than 20 g/ kg BW with no adverse effects in study parameters evaluated. 7 In a 13- week subchronic oral toxicity study of Liu Wei Di Huang Tang, the results showed that all data measured which included mortality, clinical signs, body weight changes, food and water consumption, ophthalmologic findings, urinalysis, hematological and biochemical parameters, gross necropsy findings, organ weights, and histological examination of organs, were within normal limits. 8 A modification of the classical formula Si Jun Zi Tang (Wei Qi Booster a ) has been used in multiple species of domestic animals with success to tonify Wei Qi, Qi and Blood, and clear Heat-Toxins. 9,10 This Chinese herbal medicine consists of Dang Shen (Codonopsis pilosula), Huang Qi (Astragalus membranaceus), Wu Yao (Lindera aggregata), Chen Pi (Citrus reticulata), Dang Gui (Angelica sinensis), Xuan Shen (Scrophularia ningpoensis), Bai Hua She She Cao (Hedyotis diffusa) and Ban Zhi Lian (Scutellaria barbata). Numerous pharmacological studies have shown that this group of herbs can enhance body immunity and provide antitumor effects (references?) Liu and his colleagues demonstrated that Huang Qi saponins, one of the main ingredients of Huang Qi (Astragalus membranaceus), inhibits tumor growth in the hepatoma 22 (H 22 ) model and sarcoma 180 (S 180 ) implants in mice. 11 Bai Hua She She Cao (Oldenlandia) has also demonstrated anti-neoplastic effects as well as antiinflammatory properties. 12,13 In addition, the combination of Huang Qi (Astragalus) and Dang Gui (Angelica) has been shown to stimulate erythropoetin production in cultured kidney cells which therapeutically benefits the treatment of anemia. 14 Although WQB has demonstrated numerous benefits when administered to animals both in clinical and pharmacological studies, its safety in non-clinical toxicity testing remains unknown. The objective of the present study was to evaluate the safety of WQB in acute and subchronic oral rodent studies by the collection of data on body and organ weight changes, histologic changes of selected organs and clinical pathology changes (hematology, clinical chemistry) in order to provide guidance for safe use of this Chinese herbal medicine in clinical applications. The aim of these studies was to set an MTD (maximum tolerated dose), and LD 50 (median lethal dose) for WQB and identify any long term adverse effects (subchronic exposure) from oral administration of the compound. The hypothesis for this group of experimental studies was that WQB would demonstrate very low or no toxicity in these studies. MATERIALS AND METHODS Safety assessment of WQB was conducted through a series of 3 safety tests which included 2 acute toxicity tests (LD 50 and MTD) and 1 subchronic assay. The dosage and experimental procedures used in the LD 50 and MTD studies were consistent with the government legislation Evaluation Procedures and Methods on Health Food Safety Toxicology and Preclinical Safety Evaluation and Experiment issued by the Chinese Ministry of Health. 15,16,17 Consistent with the regulatory requirements, calculation of doses for evaluating compound safety in the LD 50 study included multiples of 1.4X for 7 dosages. If no adverse effects occurred at the highest dose level in this study, then the MTD study dose would be set at double the highest LD 50 dose. Dose determination for the subchronic study was based on a previous study conducted with chickens and was set at 1% WQB added into the normal chow for 90 days. 10 All animals for these studies were sourced from the Laboratory Animal Center of Hebel Medical University. The mice were housed in polypropylene cages with cellulose fiber chip bedding at a temperature of 25ºC and 30-40% humidity along with a 14-hour light and 10-hour dark cycle. All animals were given free access to rodent feed and water in glass bottles with rubber stoppers for an adaptive period of 7 days. All animal care and use was in accordance with the guidelines of the Chinese Council for Animal Care. The first acute toxicity test performed was to establish an LD 50 for WQB. This study contained 80, 8-week-old adult mice with an average weight of 30±2 g. After the adaptive period, the mice were randomly 30

3 divided via a random number table into 8 groups with 10 mice in each group. Mice in the control group were gavaged with 0.4 ml normal saline, while mice in groups 1-7 received the same amount of saline mixed with 2.0 g, 2.80 g, 3.92 g, 5.49 g, 7.68 g, g and g WQB/ kg BW twice daily for 7 days respectively. The animals received daily evaluation of food consumption, clinical signs of toxicity and monitoring for mortality. After 1 week of treatment, the mice were humanely euthanized by ketamine/xylazine overdose and blood was collected for hematology. Parameters evaluated included intermediate cells (eosinophil, neutrophil, basophil leukocytes) (MID), lymphocytes (LYM), red blood cells (RBC) and hemoglobin (HGB) with a commercial hematology analyzer b. Final body weights were recorded along with immediate collection of liver, spleen, lung, kidney and duodenum from the control and high dose groups. The organs were weighed and then placed in Bouin s solution for histopathological evaluation. The second acute toxicity test was to establish the MTD for WQB. This is the highest dose of an agent that can be administered without causing excessive toxicity or decreased survival. This study contained ten 8-week-old mice, with an average weight of 30±2 g. It was conducted as part of the LD 50 study, therefore, the control animal group was shared. The 10 study animals received g/kg BW of WQB twice daily for 7 days. All animals were given free access to food and water after gastric infusion. The animals received daily evaluation of food consumption, clinical signs of toxicity and monitoring for mortality. After 1 week of treatment, the mice were humanely euthanized as previously described and blood was collected for hematology (MID, LYM, RBC, HGB) with evaluation performed by a commercial hematology analyzer b. In addition, final body weight and organ weights (liver, kidney) were recorded c. Protocol organs which included liver, spleen, lung, kidney and duodenum were immediately collected and fixed in Bouin s solution for histological evaluation d. Both acute studies were repeated twice to verify results obtained. Subchronic toxicity studies (90-day or 13 weeks) are conducted to evaluate the safety of long-term administration of drugs or other therapeutic products. To evaluate the long-term safety of WQB, a 90-day study was conducted in mice. The study contained 45 healthy 8-week-old mice randomly divided into 3 groups of 15 animals. The control group was fed normal chow diet with each of the other two groups either receiving normal chow combined with 1% WQB or levamisole hydrochloride e 0.01 g/kg BW. Levamisole hydrochloride was used as a positive control due to its confirmed ability to enhance body immunity and used as comparison of immune enhancement that may occur subsequent to dosing with WQB. Mice were weighed every 2 weeks and received daily evaluation for signs of clinical toxicity, food consumption and mortality. After 90 days, the mice were humanely euthanized as previously described and blood was collected for clinical chemistry evaluation. Serum was obtained from the blood samples by centrifugation at 3,000 rpm for 10 min. The level of triglyceride (TG), cholesterol (CHOL), glucose (GLU), uric acid (UA), creatinine (Crea), alanine aminotransferase (ALT), blood urea nitrogen (BUN), total protein (TP), albumin (ALB) and aspartate transaminase (AST) were measured f. Final body weight and organ weights (liver, kidney) were recorded along with collection of the liver, spleen, lung, kidney and duodenum for histopathologic evaluation. The tissues were fixed in Bouin s solution and processed for histology by dehydration in a graded ethanol series. They were then embedded in paraffin and serial sections (4-5 um in thickness) were stained with a standard hematoxylineosin (H&E) stain for evaluation by light microscopy. Data were expressed as means plus or minus the standard deviation (mean ± SD). Differences between groups were evaluated by one-way analysis of variance (ANOVA) using SPSS software package for Windows g. In all tests, statistical significance was considered p<0.05 (*) significant, and p<0.01 (**) very significant. RESULTS There was no mortality in the acute studies (MTD, LD 50 ) in any study group over the 7-day observation period. In addition, no meaningful differences were seen in food consumption and clinical signs of toxicity when treated groups were compared to the control group. The ratio of liver and kidney organ weights to final body weight did not show any statistically significant effect in the WQB dose groups of the LD 50 study, including the 30.12g group in the MTD study (p>0.05) (Table 1). In addition, there were no statistically significant findings in any hematology parameter evaluated in either acute study (Table 2). In both acute studies, histological examination of liver sections from the g WQB group in the LD 50 study (Figure 1 B) and the 30.12g WQB group in the MTD study (Figure 1 C) demonstrated normal hepatocellular morphology with no evidence of hepatocyte hypertrophy or cytoplasmic vacuolation (lipidosis) when compared with their control groups (Figure 1 A). Examination of white pulp in the splenic sections demonstrated normal size and normal lymphocyte populations and volume in both the g and g WQB groups (Figure.1 E, Figure.1 F) when compared to the control group (Figure 1 D). Both WQB groups (Figure 1 H, Figure 1 I) had normal lung morphology when compared to controls (Figure 1 G) characterized by normal alveoli with no interstitial fibrosis or hemorrhage. Kidney sections in the treated groups (Figure 1 K/L) were examined for inflammatory infiltrates, glomerular changes, fibrosis, renal tubule cell injury and urothelium cell loss and were not different from the control group (Figure 1 J). Examination of duodenal sections in the g WQB group from the LD 50 study (Figure 1 N) 31

4 Table 1: Acute toxicity studies; Effect of WQB on organ to final body weight ratio of liver and kidney (n=10) Group WQB Dose (g per Liver weight to body Kidney weight to P-value kg BW) weight ratio body weight ratio P-value Control ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± MTD ± ± In all Tables, statistically significant (p<0.05), is denoted with (*) and very significant (p<0.01), with (**); statistics calculated by ANOVA (Tables 1-5). Table 2: Acute toxicity studies; Effect of WQB on hematological parameters (n=10) Group Dose (g per kg BW) MID(%) P-value LYM(%) P-value RBC (10 12 /L) P-value HGB(g/L) P-value Control Group ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± MTD Group ± ± ± ± *(p<0.05) significant; **(p<0.01)very significant and the MTD study (Figure 1 O) when compared with the control group (Figure 1 M) were normal with no villus blunting or crypt hyperplasia. In the subchronic 90-day study, all mice grew well with normal food consumption along with no mortality or signs of clinical toxicity. There was no statistically significant body weight effect (p>0.05) in any of the treatment groups at any study time point. All animals gained weight equally throughout the study (Table 3) when body weights at various study time points were compared to starting weights during week 1. In addition, there was no statistically significant difference in the kidney/body weight ratio when the treatment groups were compared to the control group. A statistically significant difference occurred in the liver/body weight ratios both in the levamisole group (P-value=0.02) and 1% WQB group (P-value=0.05) (Table 4). There were minor increases and decreases in various clinical chemistry parameters when the 1% WQB group was compared to the control group, however, none of the differences were statistically significant (Table 5) and all chemistry values were within the study reference range. Histopathology of protocol organs in the subchronic study included liver, spleen, lung, kidney and duodenum. Liver sections were examined for toxicologic changes such as abnormalities of the artery, vein and bile ducts in the portal triad, hepatocyte hypertrophy, areas of necrosis and numbers of stellate cells and reticulum fibers in the perisinusoidal spaces (Figures 2 A/B). The white and red pulp were evaluated in the splenic sections for volume of 32

5 copyright 2017 by AJTCVM All Rights Reserved Figure 1: Effect of WQB on liver, spleen, lung, kidney and duodenum histologic morphology in acute toxicity studies (n=10) Photomicrographs of liver: A-C; spleen: D-F; lung: G-I; kidney: J-L; duodenum: M-O Control Group: A, D, G, J, M; Dose Group (15.06 g/kg BW): B, E, H, K, N; MTD Group (30.12 g/ kg BW): C, F, I, L, O; Hepatocyte morphology is normal (arrow) in all WQB dosed groups with no evidence of hepatocellular cytoplasmic vacuolation or lipidosis. There is no difference between the volume of white pulp with lymphocyte numbers and germinal centers in the spleen (arrow) when g/kg BW WQB group and MTD group is compared with the control group. Lung histologic morphology in the g/kg BW WQB group and in the MTD group demonstrates normal lung alveoli (arrow), no interstitial fibrosis or hemorrhage when compared to control mice. Kidney glomerular morphology (arrow) along with renal tubular epithelium in g/kg BW WQB group and in MTD group is normal and shows no evidence of hyperplasia, vascular changes or cellular injury when compared to the control group. The duodenum in the g/kg BW WQB group and in MTD group demonstrates normal villous height and cellular morphology (arrow) with no evidence of villus blunting, crypt hyperplasia or dilatation when compared with the control group. Black bar in right corner of photomicrographs represents 100 μm. 33

6 Table 3: Subchronic study; Effect of WQB on body weight (n=15) Time Control group Levamisole group P-value 1%WQB group P-value 1 week 24.14± ± ± weeks 32.58± ± ± weeks 36.06± ± ± weeks 43.85± ± ± weeks 42.90± ± ± weeks 44.57± ± ± weeks 44.21± ± ± *(p<0.05) significant; ** (p<0.01) very significant Table 4: Subchronic Toxicity Study; Effect of WQB on organ to final body weight ratio (n=15) Indexes Control group Levamisole group P-value 1%WQB group P-value Liver weight to body weight ratio 3.17± ±0.04 * ±0.04* 0.05 Kidney weight to body weight ratio 1.24± ± ± *(p<0.05) significant; ** (p<0.01) very significant Table 5: Subchronic Toxicity Study; Effect of WQB on clinical chemistry parameters in mice (n=15) Indexes Control group Levamisole group P-value 1% WQB group P-value CHOL (mmol/l) 3.54± ± ± TG (mmol/l) Crea (umol/l) Glu (mmol/l) AST (U/L) ALT (U/L) TP (g/l) ALB (g/l) BUN (mmol/l) UA (mg/dl) *(p<0.05) significant; ** (p<0.01) very significant 2.10± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

7 copyright 2017 by AJTCVM All Rights Reserved Figure 2: Effect of 1% WQB in diet on liver, spleen, lung, kidney and duodenum histologic morphology in subchronic toxicity study (n=15) Photomicrographs of: Liver A-B: Spleen C-D; Lung E-F; Kidney G-H; Duodenum I-J. Control Group A, C, E, G, I; 1% WQB Group B, D, F, H, J. The hepatocytes and portal triads (arrow) in 1% WQB and controls demonstrate normal morphology along with rare observation of stellate cells and reticulum fibers in perisinusoidal spaces. Splenic red and white pulp (arrow) in 1% WQB and controls was filled with appropriate numbers of red blood cells and lymphocytes, respectively, with normal germinal centers in the white pulp and no inflammatory infiltrate. The histologic morphology of lung in both 1% WQB and controls was normal with clear alveolar lumens (arrow), no congestion, edema, inflammation or fibrosis noted in any sections. The renal stroma, including glomeruli (arrow) in the 1% WQB group was normal and no inflammatory cells, fibrosis or renal tubule cell loss was noted. The morphology of the intestinal mucosa in the 1% WQB group was normal with no pathological changes of congestion, edema, inflammatory cell infiltration, erosion or ulcers noted. Black bar in right corner of photomicrographs represents 100 μm. 35

8 erythrocytes, size of the germinal centers and increased inflammatory infiltrate of the interstitium (Figures 2 C/D). Lung section morphology included evaluation for edema, inflammation and fibrosis (Figure 2 E/F) while kidney sections were examined for inflammatory infiltrates, fibrosis, renal tubule cell injury and urothelium cell loss (Figure 2 G/H). The villous morphology of duodenal mucosa was assessed for histologic changes such as edema, inflammatory cell infiltrates, erosions and ulcers (Figure 2 I/J). All were found to be within normal limits. DISCUSSION It is a scientific and regulatory requirement that before any potential new human and some veterinary drugs can be administered to patients in a clinic situation, the toxicity of the compounds should be investigated in order to define safe doses. Safety tests, also referred to as preclinical safety testing, are intended to reveal potential toxicity associated with a prospective medicine. Careful consideration of the doses used in preclinical studies is necessary to fulfill the need to identify target organs that may experience excessive toxicity and usually are identified by setting low, intermediate and high doses, plus a control group. This span of doses will usually cover from no effect to adverse effects or until limited by volume or limit dose (highest dose that should be used in the absence of demonstrable toxicity). Establishing the safety of Chinese herbal medicines as they experience increased use in clinic settings is becoming more common. Two examples of Chinese herbal medicines used as veterinary therapeutics after preclinical safety testing demonstrated their oral safety include Di Weng Ke Li composed of Bai Tou Weng (Pulsatillae Radixand) and Huang Lian (Coptidis Rhizoma) for piglets and Hu Pi Su (Quercetin) for use as a feed additive in animal production. These medicines, similar to the present study, had no deleterious effects, including no reduction in body weights when tested at the highest dose level possible. 18,19,20 In the current experimental study, a modification of the classical formula Si Jun Zi Tang (Wei Qi Booster a ), which is a veterinary specific Chinese herbal medicine, was evaluated for oral safety by using both acute and subchronic mouse studies modeled after preclinical safety studies used to evaluate new pharmaceuticals. Safety assessment of WQB was conducted through a series of 3 safety tests which included 2 acute toxicity tests (LD 50 and MTD) and 1 subchronic assay. The dosage and experimental procedures used in the design of these studies were consistent with government legislation Evaluation Procedures and Methods on Health Food Safety Toxicology and Preclinical Safety Evaluation and Experiment issued by the Chinese Ministry of Health. 15,16,17 There was no mortality in the acute studies (LD 50, MTD) over the 7-day observation period or in the subchronic study over its 90-day observation period. In all studies, no meaningful differences were seen in food consumption and clinical signs of toxicity when treated groups were compared to the control group. The ratio of liver and kidney organ weights to body weight did not show any statistically significant effects in any acute study WQB dose group including the very high dose of 30.12g/ kg BW group in the MTD study (p>0.05). Histopathology of all other organs collected in the WQB groups in the acute and subchronic studies were within normal limits for that organ. In addition, both hematology and clinical chemistry parameters were unaffected in animals dosed with WQB in all safety studies. A statistically significant increase occurred when comparing control liver/final body weight ratio and both treatment groups in the subchronic toxicity study. This was considered a non-adverse finding due to the following: there was no histologic abnormalities including hepatocellular hypertrophy, clinical chemistry values were normal and there was no dose response increase in the liver/body weight ratios in the acute studies. Most likely this finding is secondary to one control group animal with a low liver weight (outlier) which caused an artefactual lowering of the group s liver organ weight ratio results. According to the experimental design and statistical basis of drug evaluation, a Chinese herbal medicine can be considered safe if there is no mortality or adverse clinical signs in animals by gavage at a maximum tolerated oral dose of 10 g/kg BW. 21 There was no adverse effect observed at the highest dose level tested (30.12 g/kg BW) in the acute studies to determine LD 50 and MTD. Since the MTD of WQB was 30.12g/kg body weight (3 times higher than 10 g/kg BW), 0.14 g of WQB is considered acutely nontoxic to a 50 g mouse. When considering the recommended therapeutic dose of WQB for different species, 1.0 mg/kg for humans, 1.87 mg/kg for dogs or 9.1 mg/kg for mice given twice daily; the maximum dose tested (30.12g/kg BW) indicates a very large acute margin of safety (approximately 300X depending on species) when using this Chinese herbal medicine. In the subchronic study, when evaluating long term exposure, 1% WQB was added into the normal feed of experimental animals. With the study mice consuming approximately 6-8g of feed per 30-40g BW/day, the study mice received an approximate dose of 2g/kg or 2000 mg/kg BW of WQB per day for 90 days. When considering the recommended therapeutic dose of WQB for different species, the maximum dose found to have no adverse effects (2000 mg/kg BW) again indicates a large chronic exposure margin of safety of approximately 15X (depending on species). Results of this group of safety studies in mice with modified Si Jun Zi Tang (Wei Qi Booster a ) clearly demonstrated no acute or subchronic toxicity to experimental animals in any study. It may be concluded that WQB is a safe Chinese herbal medicine, even at 36

9 the highest dosage (30.12g/kg BW) for one week and also long term usage at 1% of feed or 2000 mg/kg BW. The hypothesis was therefore accepted that WQB would demonstrate no toxicity in experimental animals under the conditions of these studies. In conclusion, these studies provide evidence of the safety of this Chinese herbal medicine and support the basis of its safe use in clinical practice. ACKNOWLEDGEMENTS This work was financially supported by the Science and Technology Development Fund of Agricultural University of Hebei (NO ) Footnotes a. Wei Qi Booster, Dr. Xie s Jing Tang Herbal Inc, Reddick, FL USA b. KM-23vet, Komeic company, Shanghai, China c. Sartorius, Germany d. Huangxing Science and Technology Co. LTD, Beijing, China e. Levamisole hydrochloride, Jinpai Pharmaceutical, Xuzhou, China f. Bio Sino, Beijing, China g. SPSS 13.0 software, IBM Corporation, Armonk, NY, USA REFERENCES 1. Rong WH, Zhan ZS, Pu CY et al. Developing current situation of Chinese herbal and some thought. Journal of Bethune Military Medical College 2011; 9: Keysser CH. Preclinical safety testing of new drugs. Ann Clin Lab Sci. 1976; 6(2): Ye ZG, Zhang GP. Progress of safety evaluation of traditional Chinese medicines and its strategy in further studies. Chinese Journal of Experimental Traditional Medical Formulae. 2014;20(16): Gao ZZ, Liu RR, Hu YL et al. A randomized and controlled experimental study of the acute and subacute toxicity of lentinan extracted from Xiang Gu (Shitake) in mice. AJTCVM 2014; 9(2): Liu YW, Wang DM, Zhao Y et al. Detection of the toxicity of traditional Chinese medicines used to treat diseases caused by candida albicans. Journal of Pathogen Biology 2010; 5: Kim YJ, Lee MY, Kim OS et al. Acute oral toxicity of Insampaedok-san, a traditional herbal formula in rats and its protective effects against ovalbumin-induced asthma via anti-inflammatory and antioxidant properties. BMC Complementary and Alternative Medicine 2014; 14: Yi Q, Li Sk, Zhu ZR et al. Experimental studies of the actue and repeated administration toxicity of the jin weng zhi li granules. Heilongjiang Animal Science and Veterinary Medicine 2015; 2: Ha HY, Lee JK, Lee HY et al. Safety evaluation of yukmijihwang-tang: assessment of acute and subchronic toxicity in rats. Evid Based Complement Alternat Med 2011; doi: /2011/ Patricia B. Acupuncture and Chinese Herbal Therapy for Acute Upper Respiratory Disease in a Geriatric Quarter Horse. AJTCVM 2009;4: Li S. Influence of WQB on the immune response to Newcastle disease vaccine in chickens [D]. Hebei, China, Agricultural University of Hebei 2013; Liu MH, Ren MP, Chen JP et al. Study on antitumor activity of Astragalus saponins. Pharmacology and Clinics of Chinese Materia Medica 2009; 25: Wang Q. Studies on anti-tumor and anti-inflammatory experiment of fujian hedyotis diffusa willd [D]. Fujian, China, Fujian University of Traditional Chinese Medicine 2013; Mao Y, Xu F, Xu XJ et al. Research progress on chemic constituent and anti-tumor activity of oldenlandia diffusa willd. Modern Preventive Medicine 2015;42: Zuo ZC, Gan M, Cui HM et al. Effects of Chinese herbal compound on the blood physiological parameters and cytokines of myelosuppressive mice. Chinese Veterinary Science 2013;43: Chinese ministry of health. The Evaluation Procedures and Methods on Health Food Safety Toxicology [S] Yuan BJ, Wang ZQ. The preclinical safety evaluation of new drug. Military Medical Science Press [M]1997, Veterinary drug test specification assembly. The Ministry of Agriculture Veterinary Drug Evaluation Committee Office [S] Teo S, Stirling D, Thomas S et al. A 90-day oral gavage toxicity study of D-methylphenidate and D, L-methylphenidate in Sprague-Dawley rats. Toxicology 2002;179, Fang X. Toxicity test and therapeutic effect of diweng keli on piglet white scour [D]. Jilin, China, Jilin University 2015, Feng XA. Safety Evaluation of Quercetin as a Feed Additive [D]. Heilongjiang, China, Northeast Agricultural Uuiversity, 2013; Liu CX, Sun RY. Experimental design and statistical basis of drug evaluation. Military Medical Science Press [M] 1998;