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
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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.
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