Multiple-dose Intramuscular Toxicity Study of Shinbaro Pharmacopuncture in Sprague-Dawley Rats over a 4-week period

Jin Ho Lee; In Hyuk Ha; Me Riong Kim; Min Jeong Kim; Jae Woong Lee; In Hee Lee; Hwa Jin Chung; Eun Jee Kim

doi:10.13045/acupunct.2015063

J Acupunct Res > Volume 32(4); 2015 > Article

Lee, Ha, Kim, Kim, Lee, Lee, Chung, and Kim: Multiple-dose Intramuscular Toxicity Study of Shinbaro Pharmacopuncture in Sprague-Dawley Rats over a 4-week period※

Original Article

Acupuncture 2015; 32(4): 77-89.

DOI: https://doi.org/10.13045/acupunct.2015063

Multiple-dose Intramuscular Toxicity Study of Shinbaro Pharmacopuncture in Sprague-Dawley Rats over a 4-week period^※

Jin Ho Lee, In Hyuk Ha, Me Riong Kim, Min Jeong Kim, Jae Woong Lee, In Hee Lee, Hwa Jin Chung, Eun Jee Kim^*

Jaseng Spine and Joint Research Institute, Jaseng Medical Foundation

^*Corresponding author: Jaseng Spine and Joint Research Institute Jaseng Medical Foundation, 5, Eonju-ro 170-gil, Gangnam-gu, Seoul, Republic of Korea,
Tel: +82-10-5342-7720, E-mail: letmelove57@hanmail.net

^※ The authors are grateful to korea promotion institute for traditional medicine industry (KOT-MIN) for their support (No. 20150602383).

Received November 13, 2015 Revised December 09, 2015 Accepted December 11, 2015

See the Original "".

Abstract

Objectives:

To evaluate the safety of multiple-dose intramuscular Shinbaro Pharmacopuncture in male and female Sprague-Dawley(SD) rats over a period of 4 weeks(12 sessions)

Methods:

In order to test the safety of multiple-dose intramuscular Shinbaro Pharmacopuncture we used 40 healthy male and female 6-week old SD rats(male weight 171.79~196.37 g, female weight 127.93~146.43 g). Shinbaro Pharmacopuncture was administered intramuscularly to male and female SD rats at doses of 4.6 (low dose group, n=10), 9.2 (moderate dose group, n=10), and 18.5mg/kg(high dose group, n=10), respectively. General symptoms, body weight changes, blood tests, biochemical testing, necropsy, organ weight and histopathogical findings were examined over a 4-week period.

Results:

No mortalities or adverse effects were caused by the investigational substance were observed during the study period.
There was no significant difference in body weight caused by the the investigational substance across all groups.
No significant between-group difference was found to be caused by the investigational substance in blood tests and biochemical testing.
No abnormalities were detected by a necropsy examination with the unaided eye at the macro level after treatment with the investigational substance.
Difference in organ weight between groups caused by the investigational substance was not found.
All groups did not exhibit pathological findings caused by the investigational substance in histopathogical examination.

Conclusions:

According to these results, Shinbaro Pharmacopuncture has no systemic or organ toxicity with multiple-dose intramuscular administrations in male and female SD rats over a 4-week period (12 sessions). These results imply that no adverse effects are observed at a level (NOAEL) of Shinbaro Pharmacopuncture of 18.5 mg/kg.

Key Words: Shinbaro; Pharmacopuncture; Multiple-dose toxicity test; Spraque-Dawley rat.

Fig. 1

Body weight changes of male and female SD rat with shinbaro pharmacopuncture for four weeks

^*: Significant difference between control and 18.5 mg/kg group value by Dunnett’s T test, p<0.0

Table 1

Group composition for assessment of four weeks repeated-dose toxic test in SD rats

Group	Sex	Animal No.¹	Dose (mg/kg/day)
G1²	Male	G1-1~G1-10	0
G1²	Female	G1-51~G1-60	0
G2	Male	G2-16~G2-25	4.6
G2	Female	G2-66~G2-75	4.6
G3	Male	G3-26~G3-35	9.2
G3	Female	G3-76~G3-85	9.2
G4	Male	G4-36~G4-45	18.5
G4	Female	G4-86~G4-95	18.5

¹ No: Numbers

² G1: Control group(0 mg/kg), G2~G4: Experimental group (4.6, 9.2, 18.5 mg/kg)

Table 2

Hematological values of male and female SD rat with shinbaro pharmacopuncture for four weeks

sex: Male
Hematological	G124(n = 10)	G2 (n = 10)	G3 (n = 10)	G4 (n = 10)
Hematological	0 mg/kg	4.6 mg/kg	9.2 mg/kg	18.5 mg/kg
wbc1(K/μL)	8.99±2.42	11.04±2.82	10.16±2.72	9.24±3.26
NE2(K/μL)	0.89±0.24	1.37±0.47^*	1.38±0.40^*	1.09±0.23
ly3(K/μL)	7.68±2.15	9.27±2.42	8.41 ±2.35	7.84±2.95
mo4(K/μL)	0.18±0.05	0.22±0.08	0.18±0.08	0.16±0.08
eo5(K/μL)	0.05±0.01	0.05±0.02	0.05±0.02	0.05±0.03
BA6(K/μL)	0.00±0.00	0.01 ±0.00	0.01 ±0.01	0.01±0.01
LUC7(K/μL)	0.19±0.15	0.12±0.06	0.13±0.10	0.10±0.07
NEP8(%)	10.25±2.42	12.35±2.75	13.77±2.51^*	12.36±2.28
LYP9(%)	85.14±2.94	84.04±2.78	82.49±2.91	84.32±2.41
MOP10(%)	1.95±0.37	1.92±0.48	1.84±0.71	1.68±0.44
EOP11(%)	0.63±0.21	0.49±0.31	0.53±0.23	0.51±0.23
BAP12(%)	0.05±0.05	0.08±0.04	0.07±0.08	0.11±0.12
LUP13(%)	1.97±1.23	1.12±0.70	1.26±1.22	0.99±0.41
rbc14(M/μL)	7.90±0.20	8.18±0.32	8.12±0.36	7.94±0.24
Hb15(g/dL)	17.22±0.47	17.86±0.67	17.58±0.78	17.35±0.62
HCT16(%)	47.30±1.49	48.55±2.02	48.17±1.57	47.72±1.48
MCV17(fL)	59.90±1.64	59.43±2.12	59.37±1.93	60.13±1.65
MCH18(pg)	21.80±0.55	21.82±0.60	21.69±0.85	21.88±0.66
MCHC19(g/dL)	36.38±0.46	36.76±0.74	36.51±0.67	36.35±0.97
RDW20(%)	11.39±0.30	11.13±0.30	11.37±0.36	11.17±0.40
PLT21(K/μL)	1077.90±89.33	1154.80±129.89	1115.10±218.20	1117.50±92.20
MPV22(fL)	6.76±0.36	6.48±0.24	6.91±0.39	6.69±0.54
Reti23(%)	2.61±0.48	2.46±0.37	2.47±0.48	2.65±0.23

sex: Female
Hematological	G124(n = 10)	G2 (n = 10)	G3 (n = 10)	G4 (n = 10)
Hematological	0 mg/kg	4.6 mg/kg	9.2 mg/kg	18.5 mg/kg
wbc1(K/μL)	7.02±0.88	7.72±1.43	6.41 ±1.06	7.11±2.40
ne2(K/μL)	0.76±0.31	1.59±2.36	0.69±0.21	0.51 ±0.21
ly3(K/μL)	5.91±0.68	5.66±1.88	5.48±0.94	6.35±2.36
mo4(K/μL)	0.15±0.05	0.27±0.36	0.12±0.03	0.11±0.03
eo5(K/μL)	0.07±0.04	0.05±0.03	0.05±0.01	0.05±0.02
BA6(K/μL)	0.01±0.01	0.01±0.01	0.01±0.00	0.01±0.00
LUc7(K/μL)	0.12±0.13	0.14±0.14	0.08±0.03	0.09±0.06
NEP8(%)	10.70±3.80	18.09±20.42	10.74±2.51	8.05±4.43
LYP9(%)	84.46±4.90	75.96±22.76	85.40±2.54	88.31±4.83
MOP10(%)	2.17±0.50	3.01±3.12	1.85±0.44	1.67±0.44
EOP11(%)	0.99±0.56	0.71±0.36	0.76±0.24	0.72±0.26
BAP12(%)	0.09±0.07	0.12±0.09	0.10±0.04	0.09±0.05
LUP13(%)	1.59±1.35	2.09±2.29	1.15±0.36	1.15±0.48
rbc14(M/μL)	7.84±0.34	7.76±0.36	7.81±0.39	7.83±0.37
Hb15(g/dL)	17.26±0.80	16.63±0.79	16.88±0.83	16.93±0.94
HCT16(%)	45.44±2.43	44.56±2.23	45.68±2.41	45.30±2.89
MCV17(fL)	57.92±1.10	57.48±2.01	58.52±1.70	57.86±1.56
MCH18(pg)	22.00±0.38	21.45±0.70	21.64±0.36	21.65±0.53
MCHC19(g/dL)	38.02±0.84	37.34±0.45^*	37.00±0.72^*	37.40±0.57
RDW20(%)	10.62±0.98	10.51±0.38	10.43±0.28	10.33±0.24
PLT21(K/μL)	1240.30±147.30	1209.30±129.69	1165.00±169.52	1193.50±88.04
MPV22(fL)	6.63±0.29	6.69±0.42	6.76±0.28	6.66±0.25
Reti23(%)	2.50±1.16	2.28±0.45	2.22±0.35	2.11±0.45

¹ White blood cell

² Neutrophils

³ Lymphocyte

⁴ Monocyte

⁵ Eosinophil

⁶ Basophil

⁷ Large unstained cell

⁸ Percent of neutrophils

⁹ Percent of lymphocyte

¹⁰ Percent of monocyte

¹¹ Percent of eosinophil

¹² Percent of basophil

¹³ Percent of large unstained cel

¹⁴ Red blood cell

¹⁵ Hemoglobin

¹⁶ Hematocrit

¹⁷ Mean corpuscular volume

¹⁸ Mean corpuscular hemoglobin

¹⁹ Mean corpuscular hemoglobin concentration

²⁰ Red cell distribution width

²¹ Platelet

²² Mean platelet volume

²³ Reticulocyte

²⁴ G1: Control group(0 mg/kg), G2~G4: Experimental group(4.6, 9.2, 18.5 mg/kg),

^* Significant difference compared with control value by Dunnett’s T test, p<0.05

¹ White blood cell

² Neutrophils

³ Lymphocyte

⁴ Monocyte

⁵ Eosinophil

⁶ Basophil

⁷ Large unstained cell

⁸ Percent of neutrophils

⁹ Percent of lymphocyte

¹⁰ Percent of monocyte

¹¹ Percent of eosinophil

¹² Percent of basophil

¹³ Percent of large unstained cel

¹⁴ Red blood cell

¹⁵ Hemoglobin

¹⁶ Hematocrit

¹⁷ Mean corpuscular volume

¹⁸ Mean corpuscular hemoglobin

¹⁹ Mean corpuscular hemoglobin concentration

²⁰ Red cell distribution width

²¹ Platelet

²² Mean, platelet volume

²³ Reticulocyte

²⁴ G1: Control group(0 mg/kg), G2~G4: Experimental group(4.6, 9.2, 18.5 mg/kg), Group(n) / Dose(mg / kg)

^* Significant difference compared with control value by Dunnett’s T test, p<0 05

Table 3

Serum biochemical values of male and female SD rat with shinbaro pharmacopuncture for four weeks

sex: Male
Serum	G123(n = 10)	G2 (n = 10)	G3 (n = 10)	G4 (n = 10)
Serum	0 mg/kg	4.6 mg/kg	9.2 mg/kg	18.5 mg/kg
AST1 (IU/L)	133.20±29.70	130.10±35.62	121.60±28.92	121.00±32.86
ALT2 (IU/L)	31.10±4.01	37.70±7.39	34.60±6.90	34.20±6.14
ALP3 (IU/L)	707.80±57.14	785.10±137.19	681.50±83.95	719.70±174.69
GGT4 (IU/L)	0.90±0.30	0.80±0.40	0.90±0.30	1.00±0.00
LDH5 (IU/L)	1288.80±709.57	1061.70±915.57	847.20±770.71	921.40±766.96
BUN6 (mg/dL)	17.69±2.34	19.09±2.06	17.94±1.33	17.50±2.34
CRE7 (mg/dL)	0.52±0.05	0.55±0.05	0.55±0.05	0.51±0.02
GLU8 (mg/dL)	213.80±22.10	218.40±27.10	203.60±25.80	206.30±41.68
CHO9 (mg/dL)	68.40±12.00	80.90±10.25	72.00±11.94	73.50±11.67
TP10 (g/dL)	6.68±0.27	6.88±0.26	6.79±0.31	6.77±0.21
CPK11 (U/L)	843.30±404.87	726.90±519.65	584.60±392.44	648.80±447.59
ALB12 (g/dL)	2.75±0.11	2.81±0.13	2.78±0.14	2.76±0.10
T-BIL13 (mg/dL)	0.04±0.02	0.03±0.02	0.03±0.20	0.04±0.02
A/G ratio14	0.70±0.04	0.69±0.04	0.69±0.03	0.69±0.03
TG15 (mg/dL)	50.20±22.17	51.30±25.41	62.90±16.92	64.00±26.59
UA6 (mg/dL)	2.63±0.61	3.24±0.74	3.06±0.59	2.88±0.49
Ca17 (mg/dL)	13.13±0.61	13.18±0.41	13.05±0.47	13.35±0.67
IP18 (mg/dL)	12.54±0.97	12.72±0.75	12.65±0.99	12.87±0.75
Cl19 (mmol/L)	105.70±1.42	106.10±1.14	106.20±1.33	106.00±1.41
Mg20 (mg/dL)	3.10±0.26	3.36±0.34	3.20±0.32	3.21±0.28
Na21 (mmol/L)	144.80±1.17	144.90±1.45	145.40±0.92	146.10±1.64
K22 (mmol/L)	5.41 ±0.62	5.72±0.28	6.06±0.91	5.88±0.67

sex: Female
Serum	G123(n = 10)	G2 (n = 10)	G3 (n = 10)	G4 (n = 10)
Serum	0 mg/kg	4.6 mg/kg	9.2 mg/kg	18.5 mg/kg
AST1 (IU/L)	163.40±57.47	129.60±36.10	116.70±18.32	139.70±31.07
ALT2 (IU/L)	37.10±13.20	34.80±5.74	30.10±4.66	34.80±7.53
ALP3 (IU/L)	427.60±103.61	408.70±116.02	460.80±86.69	438.10±85.90
GGT4 (IU/L)	0.30±0.46	0.30±0.46	0.50±0.50	0.60±0.49
LDH5 (IU/L)	1316.50±704.54	1164.40±941.13	835.20±518.99	1346.80±716.47
BUN6 (mg/dL)	20.55±2.39	20.00±3.35	21.51 4.38	19.49±1.75
CRE7 (mg/dL)	0.59±0.06	0.56±0.05	0.61±0.07	0.57±0.05
GLU8 (mg/dL)	165.40±40.76	167.10±26.18	160.90±28.77	170.30±31.23
CHO9 (mg/dL)	85.10±13.58	96.40±9.33	99.00±23.32	102.00±17.98
TP10 (g/dL)	7.35±0.37	7.11 ±0.41	7.18±0.51	7.17±0.38
CPK11 (U/L)	807.50±321.17	682.90±472.68	488.60±217.21	731.90±297.78
ALB12 (g/dL)	3.17±0.24	3.09±0.23	3.12±0.27	3.16±0.21
T-BIL13 (mg/dL)	0.06±0.04	0.06±0.03	0.05±0.02	0.05±0.02
A/G ratio14	0.76±0.10	0.77±0.05	0.77±0.05	0.79±0.06
TG15 (mg/dL)	26.80±11.14	29.00±6.32	33.40±14.57	26.10±11.16
UA6 (mg/dL)	2.67±0.52	2.55±0.47	2.64±0.45	2.96±0.62
Ca17 (mg/dL)	13.10±1.06	13.67±2.23	13.71±1.62	12.90±0.52
IP18 (mg/dL)	11.46±1.27	12.09±0.70	12.09±0.87	11.59±0.83
Cl19 (mmol/L)	106.20±1.99	105.30±1.55	106.20±1.83	106.90±4.18
Mg20 (mg/dL)	3.37±0.17	3.38±0.21	3.46±0.39	3.40±0.47
Na21 (mmol/L)	143.50±1.69	141.70±1.55	143.40±1.91	144.30±5.50
K22 (mmol/L)	5.30±1.00	5.90±1.04	5.35±0.86	5.63±0.91

¹ Aspartate aminotransferase

² Alanine aminotransferase

³ alkaline phosphatase

⁴ Gamma(γ)-glutamyl transferase

⁵ Lactate dehydrogenase

⁶ Blood urea nitrogen

⁷ Creatinine

⁸ Glucose

⁹ Total cholesterol

¹⁰ Total protein

¹¹ Creatine phosphokinase

¹² Albumin

¹³ Total bilirubin

¹⁴ Albumin/Globulin ratio

¹⁵ Triglyceride

¹⁶ Uric acid

¹⁷ Calcium

¹⁸ Inorganic phosphorus

¹⁹ Chloride

²⁰ Magnesium

²¹ Sodium

²² Potassium

²³ G1: Control group(0 mg/kg), G2~G4: Experimental group(4.6, 9.2, 18.5 mg/kg), Group(n) / Dose(mg / kg)

^* : Significant difference compared with control value by Dunnett’s T test, p<0.05

¹ Aspartate aminotransferase

² Alanine aminotransferase

³ alkaline phosphatase

⁴ Gamma(γ)-glutamyl transferase

⁵ Lactate dehydrogenase

⁶ Blood urea nitrogen

⁷ Creatinine

⁸ Glucose

⁹ Total cholesterol

¹⁰ Total protein

¹¹ Creatine phosphokinase

¹² Albumin

¹³ Total bilirubin

¹⁴ Albumin/Globulin ratio

¹⁵ Triglyceride

¹⁶ Uric acid

¹⁷ Calcium

¹⁸ Inorganic phosphorus

¹⁹ Chloride

²⁰ Magnesium

²¹ Sodium

²² Potassium

²³ G1: Control group(0 mg/kg), G2~G4: Experimental group(4.6, 9.2 18.5 mg/kg), Group (n) / Dose(mg / kg)

^* : Significant difference compared with control value by Dunnett’s T test, p<0.05

Table 4

Gross findings of male and female SD rat with shinbaro pharmacopuncture for four weeks

Sex	Organ	Sign	Group (mg/kg)
Sex	Organ	Sign	G11 (n = 10, 0)	G2 (n = 10, 4.6)	G3 (n = 10, 9.2)	G4 (n = 10, 18.5)
Male	All organs	Normal	10 / 10	10 / 10	10 / 10	10 / 10
Female	All organs	Normal	10 / 10	10 / 10	10 / 10	10 / 10

^* Number of animals with the sign / Number of animals examined

¹ G1: Control group(0 mg/kg), G2~G4: Experimental group(4.6, 9.2, 18.5 mg/kg)

Table 5

Organ weights of male and female SD rat with shinbaro pharmacopuncture for four weeks

sex: Male (g)
Organ	G11 (n = 10)	G2 (n = 10)	G3 (n = 10)	G4 (n = 10)
Organ	0 mg/kg	4.6 mg/kg	9.2 mg/kg	18.5 mg/kg
Body weigh	362.46±30.55	349.16±32.27	347.94±33.89	356.71±37.30
Testis(Lt.)	1.61±0.08	1.69±0.15	1.64±0.13	1.71±0.11
Testis(Rt.)	1.64±0.09	1.68±0.13	1.64±0.12	1.69±0.12
Prostate	0.58±0.12	0.62±0.13	0.62±0.10	0.66±0.10
Spleen	0.77±0.09	0.76±0.06	0.69±0.07	0.75±0.12
Liver	10.53±1.05	10.15±1.15	10.42±1.35	10.75±1.54
Adrenal gland(Lt.)	0.03±0.00	0.14±.34	0.03±0.00	0.03±0.00
Adrenal gland(Rt.)	0.03±0.01	0.03±0.01	0.03±0.00	0.03±0.00
Kidney(Lt.)	1.26±0.12	1.19±0.39	1.28±0.10	1.33±0.12
Kidney(Rt.)	1.30±0.11	1.31±0.09	1.33±0.11	1.36±0.12
Heart	1.33±0.11	1.23±0.10	1.28±0.14	1.34±0.14
Lung	1.47±0.14	1.52±0.14	1.45±0.19	1.53±0.20
Brain	2.02±0.09	2.06±0.10	203±0.08	204±0.06
Pituitary	0.01±0.00	0.01±0.00	0.01±0.00	0.01±0.00
Thymus	0.51±0.15	0.49±0.14	0.48±0.09	0.46±0.10

sex: Female (g)
Organ	G11 (n = 10)	G2 (n = 10)	G3 (n = 10)	G4 (n = 10)
Organ	0 mg/kg	4.6 mg/kg	9.2 mg/kg	18.5 mg/kg
Body weigh	226.01 ±11.21	219.97±8.85	223.46±9.87	220.46±12.84
Testis(Lt.)	0.05±0.01	0.05±0.01	0.05±0.01	0.05±0.00
Testis(Rt.)	0.05±0.01	0.05±0.01	0.05±0.00	0.05±0.00
Prostate	0.38±0.07	0.53±0.24	0.49±0.10	0.51±0.15
Spleen	0.53±0.10	0.53±0.11	0.52±0.09	0.51±0.08
Liver	6.82±0.44	6.86±0.37	6.97±0.57	6.83±0.75
Adrenal gland(Lt.)	0.03±0.00	0.03±0.00	0.03±0.00	0.04±0.00
Adrenal gland(Rt.)	0.03±0.00	0.03±0.00	0.03±0.01	0.04±0.00
Kidney(Lt.)	0.78±0.09	0.79±0.06	0.83±0.07	0.83±0.09
Kidney(Rt.)	0.80±0.07	0.83±0.08	0.86±0.09	0.85±0.08
Heart	0.83±0.08	0.88±0.06	0.88±0.10	0.85±0.07
Lung	1.18±0.11	1.19±0.13	1.24±0.08	1.27±0.22
Brain	1.85±0.06	1.88±0.07	1.91±0.11	1.88±0.06
Pituitary	0.01±0.00	0.01 ±0.00	0.01±0.00	0.01±0.00
Thymus	0.45±0.06	0.45±0.06	0.46±0.06	0.42±0.07

¹ G1: Control group(0 mg/kg), G2~G4: Experimental group(4.6, 9.2, 18.5 mg/kg)

^* Significant difference compared with control value by Dunnett’s T test, p<0.05, Group(n) / Dose(mg/kg)

Table 6

Histopathological findings of male and female SD rat with shinbaro pharmacopuncture for four weeks

Organ / findings (Number of animals)		Male		Female
Organ / findings (Number of animals)		G11 (n=10, 0 mg/kg)	G4 (n=10, 18.5mg/kg)	G1 (n=10, 0mg/kg)	G4 (n=10, 18.5mg/kg)
Liver	–Bile duct hyperplasia	±2 (n=1)	NR3	NR	NR
	–Cell infiltration, mononuclear, perivascular	± (n=1)	NR	NR	NR
	–Cell infiltration, mononuclear, multifocal	± (n=2)	NR	NR	NR
	–Cell infiltration, mononuclear, focal	± (n=1)	± (n=4)	± (n=3)	± (n=3)
	–Necrosis, focal	NR	NR	± (n=2)	NR
Thyroid	–Ultimobranchial cyst	± (n=1)	± (n=1)	NR	NR
Thyroid	–Ectopic thymus	± (n=1)	NR	± (n=1)	NR
Lung	–Osseous metaplasia	± (n=1)	NR	NR	NR
Lung	–Accumulation, alveolarmacrophage, focal	NR	± (n=1)	NR	NR
Heart	–Edema, perivascular	NR	± (n=1)	NR	NR
Heart	–Cellinfiltration, mononuclear, focal	± (n=2)	NR	NR	NR
Kidney	–Basophilic tubule, with inflammatory cell infiltration, focal, cortex	NR	± (n=1)	NR	NR
Kidney	–Cyst, with inflammatory cell infiltration, cortex	NR	NR	NR	± (n=1)
Pituitary	–Cyst, parsdistalis	NR	± (n=1)	NR	NR
There were no remarkable changes in the other organ.

¹ G1: Control group(0 mg/kg), G2~G4: Experimental group (4.6, 9.2, 18.5 mg/kg), 2. Grade: minimal(±), mild(+), 3. NR: No remarkable lesions

V.

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