pharmacopuncture is a new form of acupuncture treatment combining acupuncture and herbal medicine . with acupuncture based on the meridian theory and herbal medicine based on qi and flavor theory pharmacopuncture treatment requires differential diagnosis by a traditional oriental medicine practitioner to select a matching herbal extract , and the injections are given at several acupoints to attain benefits of both acupuncture and herbal medicine . thus , a single procedure , can achieve both the effects of acupuncture and herbal medicine . as the pharmacopuncture does not pass through the digestive system , it works fast and has effects that oral administration does not have . for this reason , the constituents of the eun - bi san pharmacopuncture are angelicae gigantis radix , strychni semen and glycyrrhizae radix . these were extracted at low temperature and low pressure in an aseptic room at the korean pharmacopuncture institute ( kpi ) . eun - bi san pharmacopuncture is known to be beneficial to pain control and to have an anti - inflammatory effect clinically . studies on the toxicities of the components eun - bi san pharmacopuncture have been published . seo and roh conducted a philological study on the poisoning and side effects of angelicae sinensis radix , and kang conducted a study on the antimicrobial effect and the cytotoxicity of glycyrrhizae radix in vitro . nevertheless , objective single - dose toxicity testing of the eun - bi san pharmacopuncture , which is a complex combination of herbs , has not been conducted yet . therefore , this study was performed to analyze the single - dose toxicity and to determine the lethal dose of eun - bi san pharmacopuncture in rats . studying the acute and the subacute toxicities through good laboratory practice ( glp ) regulations is the current research trend for single - dose toxicity testing of extracts . the eun - bi san pharmacopuncture was made in a clean room at the kpi ( k - gmp ) . after the mixing process with sterile distilled water , the ph was controlled to between 7.0 and 7.5 . then , nacl was added to make a 0.9% isotonic solution by using sterilized equipment . the completed extract was stored in a refrigerator ( 2.1  5.5c ) , until it was used . the date of manufacture on this extract was 2013 - 4 - 21 , and its valid date was 2013 - 10 - 21 . in this study , six - week - old sprague - dawley ( sd ) rats reared by orientbio were used . the reason sd rats were chosen is that they have been widely used in safety tests in the field of medicine , so the results can be easily compared with many other data - bases . the weights of the male rats ranged from 177.6 to 193.2 g , and those of female rats ranged from 150.3 to 176.8 g at the time of injection . upon import , for all animals , a visual inspection was conducted ; all animals were weighed using a cp3202s system ( sartorius , germany ) . during 7 days of acclimatization the temperature of the breeding environment was 21.1  22.7c , the humidity was 44.3%  56.6% , and the illumination was 150  300 lux . feedstuff ( teklad certified irradiated global 18% protein rodent diet 2918c ) and uv filtered water were provided . after 7 days of acclimatization , animals were selected and grouped by using the criteria of their weights being close to the mean weight . in total , 20 male rats and 20 female rats were selected . the animals were randomly distributed into 4 groups ( 5 male and 5 female mice per group ) , as shown in table 1 . because the usual dose for eun - bi san pharmacopuncture is 1.0 ml per treatment in clinical applications , 1.0 ml of eun - bi san pharmacopuncture was injected into each male and female rat in a pilot test . in this study , 1.0 ml/ animal was set as a high dose , and 0.5 ml / animal and 0.25 ml / animal were set as the mid dose and the low doses , respectively . in the control group , disposable syringes were used to inject a single dose , 0.25 and 0.5 ml / animal , of eun - bi san pharmacopuncture into the left thigh muscle of the rats in the low dose and the mid dose groups , respectively , 0.5 ml of eun - bi san pharmacopuncture was injected into each thigh muscles of the rats in the high dose group , for a total of 1.0 ml / animal . this study was performed under the approval of the institutional animal ethics committee of biotoxtech co. , ltd . the general symptoms were observed once a day from the 1 day to 14 day after treatment . on the administration day ( day 0 ) , the general symptoms ( kinds of side effects , revealing time , recovery time , etc . ) , as well as mortality , were examined at 30 minutes and at 1 , 2 , 4 , and 6 hours after injection . the weights were measured immediately before treatment and at 3 , 7 and 14 days after treatment . after fasting for more than 18 hours before autopsy , the rats were anesthetized by using isoflurane . blood samples were taken from the abdominal aorta on the day of autopsy ( 15 days after injection ) . a blood sample of about 1 ml was analyzed by using an automatic hematology analyzer ( advia 120 , siemems , germany ) . a blood sample of about 2 ml was centrifuged for the blood coagulation test ( 3,000 rpm , 10 minutes ) . the results were measured by using an automated coagulation analyzer ( coapresta 2000 , sekisui , japan ) . the results were measured by using an automatic analyzer ( 7180 , hitachi , japan ) and electrolyte analyzer ( avl9181 , roche , germany ) . finally , for all animals , the organs and tissues of the body were visually inspected and microscopically observed . the weights and the hematologic examination and blood chemical test results obtained from the experiments were analyzed by using statistical analysis system ( sas , version 9.3 , sas institute inc . a bartlett test was conducted to assess the homogeneity of the variance and the significance . the one way analysis of variance ( anova ) test was carried out when the homogeneity of the variance was recognized , and the kruskal - wallis test was conducted post - hoc . in this study , no deaths or abnormalities occurred in any of the groups ( tables 2,3 ) . in addition , no changes in weight were observed in any of the groups ( table 4 ) . finally , no remarkable changes were observed on the hematological examination , the blood chemical test , the necropsy or the histopathological examination ( tables 5,6,7,8 ) . significantly different from the control by dunnett s t - test : p < 0.05 . , number of animals ; rbc , red blood cell ; hgb , hemoglobin ; hct , hematocrit ; mcv , mean corpuscular cell volume ; mch , mean corpuscular hemoglobin ; mchc , mean corpuscular cell hemoglobin concentration ; plt , platelet ; reti , reticulocytes ; wbc , white blood cell ; neu , neutrophils ; lym , lymphocytes ; mono , monocytes ; eos , eosinophils ; baso , basophils ; pt , prothrombin time ; aptt , active partial thromboplastin time . significantly different from control by dunnett s t - test : p < 0.05 . s.d . , standard deviation ; n , number of animals ; alt , alanine aminotransferase ; ast , aspartate aminotransferase ; alp , alkaline phosphatase ; ggt , gamma glutamyltranspeptidase ; glu , glucose ; bun , blood urea nitrogen ; crea , creatinine ; t - bili , total bilirubin ; t - chol , total cholesterol ; tg , triglycerides ; tp , total protein ; alb , albumin ; a / g ratio , albumin / globulin ratio ; p , phosphorus ; ca , calcium ; na , sodium ; k , potassium ; cl , chloride . eun - bi san pharmacopuncture has been widely utilized in clinics . in spite of this , no clinical reviews on the effects of eun - bi san pharmacopuncture have been published , but there have been many studies on the component herbs of this pharmacopuncture have been published . pharmacopuncture of angelicae gigantis radix , which is a component of eun - bi san pharmacopuncture , has an anti - inflammatory property related to its inhibition of nitric oxide ( no ) and prostaglandin e2 ( pge2 ) production in lipopolysaccharide ( lps ) induced macrophages , so it may have a therapeutic potential for the treatment of various inflammatory diseases . angelicae gigantis radix pharmacopuncture also has inhibitory effect on elastase activity as well as an antioxidant effect on no and 1.1-diphenyl- 2-picrylhydrazyl ( dpph ) . angelicae gigantis radix pharmacopuncture has an anti - inflammatory effect related with its inhibition of pro - inflammatory cytokines such as interleukin 6 ( il-6 ) , tumor necrosis factor - alpha ( tnf- ) , granulocytecolony stimulating factor ( g - csf ) , granulocyte macrophage - colony stimulating factor ( gm - csf ) , and macrophage inflammatory protein-1alpha ( mip-1 ) in lps induced macrophages . strychni semen pharmacopuncture inserted into st36 is thought to have an effect on adjuvant arthritis in rats . finally , glycyrrhizae radix pharmacopuncture is beneficial in the inhibition of allergic inflammatory responses and has an anti - inflammation effect [ 9 , 10 ] . thus , the component herbs of eun - bi san pharmacopuncture have been reported to have many effects on several disorders . although it is used in clinics , safety studies on eun - bi san pharmacopuncture are insufficient , so more safety studies are needed . the toxicity study is an essential data base and is important for evaluating the safety of test substances in medications . this study was carried out to provide objective safety data for eun - bi san pharmacopuncture . doses of 0.25 , 0.5 and 1.0 ml of eun - bi san pharmacopuncture were administered to the experimental groups , and a 1.0-ml dose of normal saline solution was administered to the control group . no deaths occurred , and no abnormalities were found in any of the four groups . no significant changes in the clinical signs , weights , hematologic examination results and blood chemical test results were observed ; all results were within normal ranges . when the rats were dissected to check for abnormalities in organs and tissue , no significant histopathological findings were suggested . to analyze the toxicity of eun - bi san pharmacopuncture , we need more study on its acute and chronic harmful effects and its relations with capacity reaction . the korea food & drug administration has testing protocol guidelines for the study of toxicity , and all experiments should be carried out following glp regulations . the results showed that treatment with 1.0-ml / animal of eun - bi san pharmacopuncture produced no changes in weight or in the results of hematological , blood chemical , and autopsy examinations . because eun - bi san pharmacopuncture had no risks associated with its use in this study , eun - bi san pharmacopuncture administration can be considered to be a safe treatment ; however , additional study is needed to confirm this result . the results suggest that an intramuscular injection of 1.0-ml / animal of eun - bi san pharmacopuncture did not cause any changes in weight or in the results of hematological , blood chemical , and necropsy examinations . these results indicates that eun - bi san pharmacopuncture intramuscular injection can be used as a safe treatment .
objectives : this study was carried out to analyze the single - dose toxicity of eun - bi san pharmacopuncture injected into the muscle of sprague - dawley ( sd ) rats.methods:all experiments were performed at biotoxtech , an institution certified to conduct non - clinical studies under the good laboratory practice ( glp ) regulations . 
 six week old sd rats reared by orientbio were chosen for this pilot study . 
 the reason sd rats were chosen is that they have been widely used in safety tests in the field of medicine , so the results can be easily compared with many other databases . 
 the eun - bi san pharmacopuncture was made in a clean room at the korean pharmacopuncture institute ( kpi , k - gmp ) . 
 the constituents of the eun - bi san pharmacopuncture are angelicae gigantis radix , strychni semen and glycyrrhizae radix . 
 these were extracted at low temperature and low pressure in an aseptic room at the kpi . 
 doses of eun- bi san pharmacopuncture , 0.25 , 0.5 and 1.0 ml , were administered to the experimental groups , and a dose of normal saline solution , 1.0 ml , was administered to the control group . 
 this study was performed under the approval of the institutional animal ethics committee of biotoxtech co. , ltd.results:no deaths or abnormalities occurred in any of the four groups . 
 no significant changes in weight , hematological parameters or clinical chemistry between the control group and the experimental groups were observed . to determine if abnormalities existed in any organs and tissues , we used microscopy to examine representative histological sections of each specified organ ; the results showed no significant differences in any of the organs or tissues.conclusion:the results of this study suggest that treatment with eun - bi san pharmacopuncture is relatively safe and that its clinical use may be beneficial . 
 further evaluations and studies on this subject will be needed to provide more concrete evidence in support of these conclusions .