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

Article information

Acupunct. 2015;32(4):77-89
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 2015 November 13; Revised 2015 December 09; Accepted 2015 December 11.

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:

  1. No mortalities or adverse effects were caused by the investigational substance were observed during the study period.

  2. There was no significant difference in body weight caused by the the investigational substance across all groups.

  3. No significant between-group difference was found to be caused by the investigational substance in blood tests and biochemical testing.

  4. No abnormalities were detected by a necropsy examination with the unaided eye at the macro level after treatment with the investigational substance.

  5. Difference in organ weight between groups caused by the investigational substance was not found.

  6. 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.

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

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

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

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

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

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

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

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Article information Continued

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
Female G1-51~G1-60
G2 Male G2-16~G2-25 4.6
Female G2-66~G2-75
G3 Male G3-26~G3-35 9.2
Female G3-76~G3-85
G4 Male G4-36~G4-45 18.5
Female G4-86~G4-95
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)
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)
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)
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)
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)
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)
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)
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
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
–Ectopic thymus ± (n=1) NR ± (n=1) NR
Lung –Osseous metaplasia ± (n=1) NR NR NR
–Accumulation, alveolarmacrophage, focal NR ± (n=1) NR NR
Heart –Edema, perivascular NR ± (n=1) NR NR
–Cellinfiltration, mononuclear, focal ± (n=2) NR NR NR
Kidney –Basophilic tubule, with inflammatory cell infiltration, focal, cortex NR ± (n=1) NR NR
–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