Table. 1. Grade criteria for agar diffusion test ...Table. 3. Pyrogen test. Bodyweight and temperature elevation before administration • Body weights 1.5 and above are qualified

Table. 1. Grade criteria for agar diffusion test

Grade Reactivity Description of reactivity zone

0 None No detectable zone around or under specimen

1 Slight Some malformed or degenerated cells under the specimen

2 Mild Zone limited to area under specimen

3 Moderate Zone extending specimen size up to 1.0 cm

4 Severe Zone extending farther than 1.0 cm beyond specimen

Table. 2. Reactivity grades for agar diffusion test

Blank n1 n2 n3 Mean

Grades 0 0 0 0

Reactivity None None None None

Negative n1 n2 n3 Mean

Grades 0 0 0 0

Reactivity None None None None

Positive n1 n2 n3 Mean

Grades 4 4 4 4

Reactivity Severe Severe Severe Severe

Collagen Matrix n1 n2 n3 Mean

Grades 0 0 0 0

Reactivity None None None None

Table. 3. Pyrogen test. Bodyweight and temperature elevation before

administration

• Body weights 1.5 and above are qualified for the test.

Table. 4. Pyrogen test. Body temperature elevation after administration

Animal No.

Body temperature (°C)

Temperature elevation

1 39.3 39.3 39.5 39.4 39.1 0.3

2 39.2 39.1 39.2 39.1 39.3 0.1

3 39.1 39.1 38.9 38.7 38.7 <0

• Body temperatures were measured 1h and 3h after administration with 30 min interval

Animal No. Dose (mL/kg)

Sex Weight (kg)

Body temperature (°C)

Control group

1 10 Female 1.836 39.2

2 10 Female 2.051 39.2

3 10 Female 1.995 39.3

Table. 5. In vitro mammalian cell mutation test. The mutation frequency of negative control,

positive control and test article polar extract with S9 mixture were listed below:

Treatment RS (%) RTG (%)

Colonies in TFT MF ( x 106) p-value

Large Small Large

Small

Total

Negative 1 22 15 156.5 106.7 263.1

Negative 2 20 9 138.9 62.5 201.4

Negative 3 100.0 100.0 17 18 114.5 121.3 235.8 -

Negative 4 24 12 163.2 81.6 244.8

Avg of Negative 20.8 13.5 142.9 93.0 235.8

Positive 1

122.1 78.1

24 29 221.3 267.4 488.7

0.000

Positive 2 24 30 230.8

288.5

519.3

1X - 1

96.9 122.1

18 14 104.1 81.0 185.0

0.522

1X - 2 20 19 127.6

121.2

248.9

1/2X - 1

106.6 113.3

17 19 105.3 117.7 223.0

0.188

1/2X - 2 15 12 93.6

74.9

168.4

1/4X - 1

126.7 147.5

17 22 88.1 114.0 202.0

0.093

1/4X - 2 19 14 103.8

76.5

180.2

1/8X - 1

131.6 151.9

28 15 141.4 75.8 217.2

0.729

1/8X - 2 27 15 154.3

85.7

240.1

Negative control: RPMI 1640 medium with 5% horse serum. Positive control: μg/mL3 cylophosphamide (CP)

RS: relative survival; RTG: Relative total growth; MF: mutation frequency; The p values were

compared with the average of the Negative control using the t-test. There was no significant

difference with Negative control when p > 0.05.

Table. 6. In vitro mammalian cell mutation test. The mutation frequency of negative control,

positive control and test article polar extract without S9 mixture were listed below:

Treatment RS (%) RTG (%)

Colonies in TFT

MF ( x

106) p-value

Large Small Large

Small

Total

Negative 1 13 8 92.2 56.8 149.0

Negative 2 11 6 76.1 41.5 117.6

Negative 3 100.0 100.0 17 7 123.0 50.6 173.6 -

Negative 4 17 9 124.6 66.0 190.6

Avg of Negative 14.5 7.5 103.5 53.5 157.1

Positive 1

92.8 70.4

25 17 214.4 145.8 360.2

0.001

Positive 2 23 20 192.7

167.5

360.2

1X - 1

115.1 97.2

22 4 149.6 27.2 176.8

0.781

1X - 2 15 7 104.6

48.8

153.3

1/2X - 1

147.8 91.0

16 10 129.9 81.2 211.1

0.129

1/2X - 2 14 11 97.4

76.5

173.9

1/4X - 1

123.0 147.3

24 4 104.5 17.4 122.0

0.208

1/4X - 2 23 5 100.2

21.8

122.0

1/8X - 1

123.0 167.2

17 6 61.1 21.6 82.6

0.056

1/8X - 2 21 3 89.0

12.7

101.8

Negative control: RPMI medium with 5% horse serum. Positive control: 10μg/mL Methylmethanesulfonate (MMS) RS: relative survival; RTG:

Relative total growth; MF: mutation frequency; The p values were compared with the average

of the Negative control using the t-test. There was no significant difference with Negative

control when p > 0.05.

Table. 7. In vitro mammalian cell mutation test. The mutation frequency of negative control,

positive control and test article non-polar extract with S9 mixture were listed below:

Treatment RS (%) RTG (%)

Colonies in TFT

MF ( x

106) p-value

Large Small Large

Small

Total

Negative 1 15 9 99.1 59.5 158.6

Negative 2 18 2 115.9 12.9 128.8

Negative 3 100.0 100.0 15 5 96.6 32.2 128.8 -

Negative 4 21 5 140.7 33.5 174.2

Avg of Negative 17.3 5.3 112.9 34.4 147.3

Positive 1

62.2 48.2

25 22 224.3 197.4 421.7

0.000

Positive 2 26 27 233.9

242.9

476.7

1X - 1

89.9 125.4

11 9 59.4 48.6 108.0

0.067

1X - 2 11 8 59.0

42.9

101.9

1/2X - 1

78.9 111.1

15 8 88.2 47.0 135.3

0.938

1/2X - 2 15 14 84.4

78.7

163.1

1/4X - 1

108.0 97.7

15 6 80.3 32.1 112.5

0.088

1/4X - 2 15 7 72.2

33.7

105.9

1/8X - 1

136.0 113.7

10 8 50.0 40.0 90.1

0.168

1/8X - 2 15 9 83.1

49.9

132.9

Negative control: RPMI medium with 0.25% DMSO and 5% horse serum. Positive control: μg/mL3 cyclophosphamide (CP) RS: relative survival;RTG: Relative total growth; MF: mutation frequency;The p values were compared with the average of the Negative control using t test. There was no significant difference with Negative control when p > 0.05.

Table. 8. In vitro mammalian cell mutation test. The mutation frequency of negative control,

positive control and test article non-polar extract without S9 mixture were listed below:

Treatment RS (%) RTG (%)

Colonies in TFT

MF ( x

106)

p

value

Large Small Large

Small

Total

Negative 1 14 7 70.8 35.4 106.1

Negative 2 11 10 58.7 53.4 112.1

Negative 3 100.0 100.0 14 4 77.9 22.3 100.2 -

Negative 4 12 9 64.1 48.1 112.1

Avg of Negative 12.8 7.5 67.8 39.9 107.6

Positive 1

105.1 68.1

21 15 178.9 127.8 306.7

0.000

Positive 2 25 15 221.6

132.9

354.5

1X - 1

95.3 128.5

16 10 76.3 47.7 124.0

0.604

1X - 2 17 5 77.8

22.9

100.7

1/2X - 1

108.8 126.5

14 7 68.4 34.2 102.6

0.082

1/2X - 2 15 6 69.3

27.7

97.1

1/4X - 1

70.6 97.1

23 3 119.7 15.6 135.3

0.189

1/4X - 2 15 4 86.0

22.9

108.9

1/8X - 1

89.5 122.9

9 9 42.0 42.0 84.0

0.083

1/8X - 2 13 7 65.4

35.2

100.7

Negative control: RPMI medium with 0.25% DMSO and 5% horse serum.

Positive control: 10μg/mL Methylmethanesulfonate (MMS), RS: relative survival; RTG:

Relative total growth; MF: mutation frequency; The p values were compared with the average

of the Negative control using the t-test. There was no significant difference with Negative

control when p > 0.05.

Table. 9. Systemic toxicity study. Organ weight variation of male rats

Groups Control group Treatment group

Dosage(ml/kg) 40 40

Animals 10 10

Brain Weight(g) 2.1260±0.1100 2.1811±0.0775

Liver Weight(g) 9.8768±0.9884 10.9968±0.7417 Ratio 4.6439±0.3780 5.0506±0.4162

Heart Weight(g) 1.4108±0.1193 1.4416±0.1313 Ratio 0.6637±0.0462 0.6615±0.0622

Kidney Weight(g) 2.9770±0.3950 3.0224±0.1385 Ratio 1.4057±0.2247 1.3869±0.0727

Spleen Weight(g) 0.6153±0.1074 0.7581±0.0773 Ratio 0.2895±0.0477 0.3478±0.0356

Testes Weight(g) 3.0769±0.2257 3.1292±0.2887 Ratio 1.4473±0.0782 1.4356±0.1334

Epididymis Weight(g) 0.9390±0.0578 0.9378±0.0576 Ratio 0.4422±0.0273 0.4304±0.0297

Adrenal

gland

Weight(g) 0.0978±0.1298 0.0561±0.0121

Ratio 0.0464±0.0626 0.0258±0.0059

Thymus Weight(g) 0.4497±0.0680 0.4562±0.0809 Ratio 0.2116±0.0302 0.2094±0.0378

Mean ± S.D., n=10

Ratio : Organ weight/Brain weight; Ratio (%) : (Organ weight/Brain weight ) × 100%

Table. 10. Systemic toxicity study. Organ weight variation in female rats

Groups Control group Treatment group

Dosage(ml/kg) 40 40

Animals 10 10

Brain Weight(g) 2.0134±0.1088 2.0353±0.0936

Liver Weight(g) 6.9409±0.5021 7.2933±0.8223

Ratio 3.4506±0.2361 3.5796±0.3152

Heart Weight(g) 0.9668±0.0586 0.9473±0.0866

Ratio 0.4808±0.0295 0.4656±0.0414

Kidney Weight(g) 1.9054±0.2202 1.8636±0.1228

Ratio 0.9450±0.0805 0.9153±0.0336

Spleen Weight(g) 0.4472±0.0585 0.5492±0.1195

Ratio 0.2222±0.0267 0.2241±0.0531

Testes Weight(g) 0.5122±0.2751 0.4744±0.2062

Ratio 0.2552±0.1396 0.2340±0.1044

Epididymis Weight(g) 0.1282±0.0120 0.1264±0.0139

Ratio 0.0638±0.0063 0.0621±0.0061

Adrenal

gland

Weight(g) 0.0651±0.0096 0.0662±0.0100

Ratio 0.0324±0.0049 0.0325±0.0047

Thymus Weight(g) 0.3904±0.0910 0.4474±0.0827

Ratio 0.1939±0.0428 0.2196±0.0365

Mean ± S.D., n=10

Ratio : Organ weight/Brain weight; Ratio (%) : (Organ weight/Brain weight ) × 100%

Table. 11. Systemic toxicity study. Haematology assay

Groups Control Treatment Control Treatment

Gender Male Male Female Female

Dosage(ml/kg) 40 40 40 40

Animals 10 10 10 10 RED BLOOD CELLS

RBC (106 /µl) 8.87±0.61 8.57±0.45 8.26±0.56 8.19±0.42

HGB (g/dl) 17.40±0.90 16.56±0.95 15.81±0.70 16.05±0.59

HCT (%) 55.30±2.54 52.64±3.01 48.87±1.77 49.25±1.85

MCV (fl) 62.50±2.38 61.44±1.56 59.29±2.25 60.19±2.57

MCH (pg) 19.65±0.68 19.32±0.43 19.16±0.63 19.60±0.76

MCHC (g/dl 31.47±0.38 31.45±0.34 32.34±0.48 32.58±0.34

RDW-SD (fL) 31.25±1.06 31.11±0.84 27.74±1.12 28.12±1.24

RDW-CV (%) 15.86±1.57 15.80±0.69 13.91±0.88 13.85±0.58 WHITE BLOOD CELLS

WBC (103 /

µl)

14.46±2.31 14.53±3.18 12.26±1.93 12.83±3.52

NEUT (%) 13.94±2.97 10.61±4.14 8.07±2.87 6.43±2.54

LYMPH (%) 81.10±3.14 85.96±4.48 88.44±3.52 90.30±2.91

MONO (%) 3.86±0.76 3.49±0.74 2.59±0.89 2.18±0.65

EOS (%) 0.96±0.21 0.82±0.27 0.75±0.27 0.95±0.19

BASO (%) 0.14±0.05 0.12±0.04 0.15±0.05 0.14±0.07 CLOTTING POTENTIAL

PLT (103 / µl) 1144.80±253.49 1433.00±240.72 1300.50±190.07 1363.00±266.92

MPV (fL) 7.96±0.30 8.01±0.58 7.52±0.25 7.55±0.21

CT (sec) 129.0±11.0 129.4±10.3 128.5±11.7 128.7±10.3

APTT (sec) 24.5±7.78 18.7±1.8 21.5±5.4 22.4±3.3

PT (sec) 12.2±2.41 10.4±0.4 10.1±0.5 11.0±2.2

n/n : Item number/Group number

Table 12. Systemic toxicity study. Clinical chemistry assay

Groups Control Treatment Control Treatment

Gender Male Male Female Female

Dosage(ml/kg) 40 40 40 40

Animals 10 10 10 10

AST (U/L) 255.80±30.48 216.80±21.09 148.60±20.81 134.60±11.72

ALT(U/L) 58.70±11.29 53.50±6.80 41.00±4.11 40.80±7.35

Alkaline P'ase

(U/L)

778.40±318.89 623.30±151.03 408.80±116.2

9

366.70±111.87

Bilirubin, Total

(mg/dl)

0.06±0.02a 0.04±0.01a 0.04±0b 0.04±0b

Protein, Total

(g/dl)

6.89±0.38 6.79±0.20 7.15±0.38 7.23±0.28

Albumin (g/dl) 4.35±0.19 4.21±0.12 4.59±0.18 4.58±0.15

Globulin(g/dl) 2.54±0.21 2.58±0.12 2.56±0.22 2.65±0.18

BUN (mg/dl) 18.78±1.86 17.14±1.55 18.71±3.23 17.98±2.15

Creatinine

(mg/dl)

0.60±0.05 0.55±0.04 0.51±0.03 0.50±0.03

ˠ-GT (U/L) <6c <6c <6c <6c

Glucose, AC

(mg/dl)

206.50±59.35 191.40±44.67 211.10±29.27 197.00±45.48

Sodium (mEq/L) 152.36±1.65 150.64±1.99 146.86±1.54 146.54±1.27

Potassium

(mEq/L)

7.80±0.92 7.91±0.77 7.32±0.97 7.43±0.93

Chloride (mEq/L) 100.12±2.43 101.50±1.30 102.67±1.53 102.14±1.42

Calcium (mg/dl) 12.09±0.62 12.21±0.52 12.07±0.43 12.49±0.44

Phosphorus

(mg/dl)

13.32±1.01 13.54±1.67 11.04±0.69 11.10±0.58

Triglyceride

(mg/dl)

45.90±12.03 42.00±6.02 47.90±7.67 48.80±10.61

Cholesterol

(mg/dl)

55.00±8.27 53.50±8.02 66.20±9.10 64.70±12.95

Uric Acid (mg/dl) 4.15±0.93 3.94±1.00 3.12±0.66 3.13±0.58

Amylase (U/L) 1539.80±218.69 1542.40±189.8

0

948.40±119.3

7

1015.10±163.9

5

Lipase (U/L) 103.70±7.53 112.60±6.48 97.90±6.28 95.70±4.85

Mean ± S.D., n=10

a:n=3

b:n=1

c:all raw data were <6 (under the minimum of detection machine)

Table. 13. Intracutaneous irritation test. Irritation grading of each region in clinical

observation of individual rabbit was recorded as follows

1. Polar group

Animal Test Control

Body

Items for

Animal No.

Sex

Clinical Observation Clinical Observation

Weight Grading Time Point (h) Time Point (h)

(kg)

24 48 72 24 48 72

Erythema and 0

0 0 0 0 0

1

F

4.0790 eschar formation

Oedema

0

0 0 0 0 0

formation

Erythema and 0

0 0 0 0 0

2

2

F

3.5968 eschar formation

Oedema

0

0 0 0 0 0

formation

Erythema and 0

0 0 0 0 0

3

F

3.3424 eschar formation

Oedema

0

0 0 0 0 0

formation

2. Non-polar group

Animal Test Control

Body

Items for

Animal No. Sex Clinical Observation Clinical Observation

Weight Grading Time Point (h) Time Point (h)

(kg)

24 48 72 24 48 72

Erythema and 5

3 1 5 4 3

1

F

4.0790 eschar formation

Oedema

5

4 2 5 4 2

formation

Erythema and 5

5 4 5 5 5

2

F

3.5968 eschar formation

Oedema

5

4 2 3 3 1

formation

Erythema and 5

2 2 5 3 2

3

F

3.3424 eschar formation

Oedema

5

3 3 5 3 3

formation

Table. 14. Skin sensitization study. Individual animal grades skin reaction

(a) Polar group

Group

Sex

Number of animals 24 hrs. after 48 hrs. after

challenge phase challenge phase

1 0 0

Control 2 0 0

female

3 0 0

“0.9% saline”

4 0 0

5 0 0

Mean score 0 0

1 1 0

2 0 0

3 0 0

Test 4 1 1

5 0 0

Collagen Matrix

female

6 0 0

Extracts (saline)

7 1 1

8 0 0

9 0 0

10 0 1

Mean score 0.3 0.3

Table. 15. Skin sensitization study. Individual animal grades skin reaction

(b) Non-polar group

Group

Sex Number of animals 24 hrs. after 48 hrs. after

challenge phase challenge phase

1 0 0

Control 2 0 0

female 3 0 0

“cottonseed oil”

4 0 0

5 0 0

Mean score 0 0

1 0 0

2 0 0

male 3 0 0

Test 4 0 0

5 0 0

Collagen Matrix

Extracts (oil) 1 0 0

2 0 0

female 3 0 0

4 0 0

5 0 1

Mean score 0 0.1

The mean score of the test and control group in the polar and non-polar group at the

observation period, approximately in 24±2 and 48±2 hours after challenge phase, were

all less than 1.0. Neither the control nor the test group showed significant skin response

to the treated areas.

Table. 16. Muscle implantation study. Incidence of clinical observations in rabbits

Group Control (left side back muscle) Treatment (right side back muscle)

Number of animals 3 3

Test article HDPE ABCcolla®Collagen Matrix

Gross finding 0/3 0/3

Pathology 0/3 0/3

Remarks: the control and the test article were implanted on the same rabbit.

n/n: number of rabbits with clinical signs/ number of rabbits per group.

Table. 17. Muscle implantation study. Group total and the average score of microscopic

findings

Group

Route

Number of animals

Specimen for animal

Tissue findings

Muscle

Polymorphonuclear cell infiltration, implant

interface

TI

MSS

0/12

0.0

1/12

0.08

Mononuclear cell infiltration, implant

interface

TI

MSS

1/12

0.08

0/12

0.0

Fibroplasia/ tissue granulation, implant

interface

TI

MSS

7/12

0.58

2/12

0.17

Myofiber degradation/regeneration/atrophy,

incision site

TI

MSS

2/12

0.17

2/12

0.17

Group total TMSS 0.83 0.42

Average a

Test group total

Control group total

-0.41

Irritant ranking Non-irritant

IM: intramuscular; TI: total incidence=number of specimens with findings/number of specimens

examined per group; MSS: Mean Severity Score=sum of severity score (none =0, minimal = 1,

mild =2, moderate =3, severe =4)/ specimens examined per group; TMSS: Total mean severity

score=sum of mean severity scorea ; the score differences of findings at the tissue -implant

interface were used to determine the irritant ranking of the test material (a negative difference is

recorded as zero)_Non-irritant (0.0-2.9), slight irritant (3.0-8.9), moderate irritant (9.0-15.0),

severe irritant (>15.0)

Table. 18. Criteria for the histological scoring of wound healing

Score Epithelization Attenuated inflammation Collagenization

0 Absent 100% Neutrophils Absent

1 Comprising <25% > 75% Neutrophils Absent

2 Comprising 25 -50% 50% -75% Neutrophils Partial

3 Comprising 50-75% 25%-50% Neutrophils Complete, irregular

4 Covering 100% with

regular thickness <25% Neutrophils Complete, regular

Fig. 1. Histological scoring of wound healing at 4th week

Note: No significant difference was observed between the experimental groups, because of

natural healing occurs in the control group.