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New Drug (Chemical Drug) Application Materials (14th)
Zhongzhou Ointment
Materials of Acute Toxicity Tests and References
Application Unit: Developer Zhao Zhongzhou
Yunnan Zhongzhou Pharmaceutical Co., Ltd.
New Drug (Chemical Drug) Application Materials (14th)
Zhongzhou Ointment
Materials of Animal Acute Toxicity Tests and References
1.
Animal test of acute oral toxicity of Zhongzhou Ointment
Reported conclusion: actual non-toxic grade
2, 3. Animal test of acute oral toxicity of each single drug in Zhongzhou Ointment
Reported conclusion: actual non-toxic grade
4.
Animal test of acute percutaneous toxicity of Zhongzhou Ointment
Reported conclusion: actual non-toxic grade
5, 6. Animal test of acute percutaneous toxicity of each single drug in Zhongzhou Ointment
Reported conclusion: actual non-toxic grade
Materials of animal acute toxicity tests and references are as follows:
Acute Oral Toxicity Test
I. Objective: mainly to investigate the acute toxic reaction of Zhongzhou Ointment after oral administration.
II. Drugs: prepare a 0.2 g/ml suspension by adding 0.5% carboxymethylcellulose sodium into the original
powder of Zhongzhou Ointment, which is to be used for gastric perfusion (oral administration).
III.Animals: 50 rats of Kunming species, with the weight of 20g ±2g, 25 males and 25 females, were
randomized into 5 groups, 10 in each group. Fasting for 12h before administration. Rats were provided
by the Animal Center of Henan Medical University. Room temperature: 22ºC.
IV.Dosage and Results (see table 1)
Table 1:
Oral LD50 of Zhongzhou Ointment
Group
1
2
3
4
5
i=log
Dosage
(g/kg)
5.0
6.0
7.2
8.6
10.4
r=0.0792
log10.4
Number of
Animals
10
10
10
10
10
1.017
Deaths
Death Rate
P2
0
3
5
8
10
(P)
0
0.3
0.5
0.8
1.0
∑P=2.6
0
0.09
0.25
0.64
1
2
∑P =1.98
The median lethal dose (LD50) is calculated according
to Sun’s Comprehensive Calculation Method.
LD50=7.1 (g/kg) (95% CI: 7.422/6.778 (g/kg)) Conclusion: the oral LD50 (LD50=7.1 g/kg) in rats
was evaluated according to the acute toxicity classification standard for chemicals: the acute oral
toxicity of Zhongzhou Ointment in rats is classified as “actual non-toxic grade”.
Animal test of acute oral toxicity oriented at both single drugs (zinc oxide and sublimed sulfur) and
the compound preparation (Zhongzhou Ointment) indicates that, the toxicity of the compound
preparation (Zhongzhou Ointment) (LD50=7.1 g/kg) is stronger than that of both single drugs (zinc
oxide: no death at 15g/kg; sublimed sulfur: no death at 20 g/kg).
Appendix: Acute Toxicity (LD50) Classification Standard for Chemicals
Grade
Extremely toxic
High toxic
Moderate toxic
Low toxic
Actual non-toxic
Oral LD50 in mice (mg/kg)
<1
>1-50
>50-500
>50-5000
>5000
References
1. Procedures and methods of safety evaluation for cosmetics. National Standard of the
People’s Republic of China.
UDC 668.58.001.4 C51 GB 7919-87
Procedures and methods of safety evaluation for cosmetics.
2. OECD toxicity test guidelines for chemicals.
Drug Management Bureau, Ministry of Health; Institute for the Control of Pharmaceutical and
Biological Products [NICPBP], Ministry of Health; Pharmacopoeia Commission of the
Ministry of Health, Apr. 1984
3. Measures for new drug approval.
Ministry of Health, People’s Republic of China. Jul. 1985
4. Experiment Unit: Pharmacology & Toxicology Experiment Center of Henan College of
Traditional Chinese Medicine
Experiment Period: from Jul. 1990 to Jul. 1991
Principal Researcher: Li Zongduo
Original Data Stored at: Pharmacology & Toxicology Experiment Center of Henan College of
Traditional Chinese Medicine.
Acute Oral Toxicity Test
I. Objective: mainly to investigate the acute toxic reaction of “zinc oxide” after oral administration.
II. Drugs: prepare a 0.3 g/ml suspension by adding 0.5% carboxymethylcellulose sodium into
“zinc oxide”, which is to be used for gastric perfusion (oral administration).
III. Animals: 40 rats of Kunming species, with the weight of 20g ±2g, 20 males and 20 females, were
randomized into 4 groups, 10 in each group. Fasting for 12h before administration
(free drinking was allowed). Rats were provided by the Animal Center of Henan Medical University.
Room temperature: 22ºC.
IV. Dosage (see table)
Table: Survivals in Acute Toxicity Test of Zinc Oxide on Rats
Dosage (g/kg)
Number of Animals
Survivals after 14d
3000
10
All survived
6000
10
All survived
9000
10
All survived
15000
10
All survived
V. Results: After gastric perfusion with the dosage of 15000 mg/kg in rats, there was no difference in the
psychosis, respiration, activities of the four limbs and all the other behaviors, compared with the preadministration conditions.
VI. Conclusion: In the single oral administration of zinc oxide suspension in rats, when the perfusion
reached the maximum dosage (15000 mg/kg) and maximum volume (1 ml/20 g body weight), there is
no case of death and no acute toxic symptoms after 14d observation period. According to the
evaluation standard, the acute oral toxicity of zinc oxide in rats is classified as “actual non-toxic grade”
Appendix: Acute Toxicity (LD50) Classification Standard for Chemicals
Grade
Oral LD50 in mice (mg/kg)
Extremely toxic
<1
High toxic
>1-50
Moderate toxic
>50-500
Low toxic
>50-5000
Actual non-toxic
>5000
1. Procedures and methods of safety evaluation for cosmetics. National Standard of the People’s
Republic of China.
UDC 668.58.001.4 C51 GB 7919-87
Procedures and methods of safety evaluation for cosmetics.
2. OECD toxicity test guidelines for chemicals. Drug Management Bureau, Ministry of Health; Institute
for the Control of Pharmaceutical and Biological Products [NICPBP], Ministry of Health;
Pharmacopoeia Commission of the Ministry of Health. Apr. 1984
3. Measures for new drug approval.
Ministry of Health, People’s Republic of China. Jul. 1985
4. Experiment Unit: Pharmacology & Toxicology Experiment Center of Henan
College of Traditional Chinese Medicine
Experiment Period: from Jul. 1990 to Jul. 1991
Principal Researcher: Li Zongduo
Original Data Stored at: Pharmacology & Toxicology Experiment Center of Henan College of
Traditional Chinese Medicine.
Acute Oral Toxicity Test
I. Objective: Mainly to investigate the acute toxic reaction of “sublimed sulfur” after oral administration.
II. Drugs: Prepare a 0.1 g/ml suspension by adding 0.5% carboxymethylcellulose sodium into
“sublimed sulfur”, which is to be used for gastric perfusion (oral administration)
III. Animals: 40 rats of Kunming species, with the weight of 20g ±2g, 20 males and 20 females, were
randomized into 4 groups, 10 in each group. Fasting for 12h before administration
(free drinking was allowed). Rats were provided by the Animal Center of Henan Medical University.
Room temperature: 22ºC.
IV. Dosage (see table)
Table: Survivals in Acute Toxicity Test of sublimed sulfur on Rats
Dosage (g/kg)
Number of Animals
Survivals after 14d
4000
10
All survived
8000
10
All survived
12000
10
All survived
20000
10
All survived
V. Results: After gastric perfusion with the dosage of 20000 mg/kg in rats, there was no difference in the
psychosis, respiration, activities of the four limbs and all the other behaviors, compared with the pre-ad
ministration conditions.
VI. Conclusion: In the single oral administration of sublimed sulfur suspension in rats, when the perfusion
reached the maximum dosage (20000 mg/kg) and maximum volume (1 ml/20 g body weight), there is no
case of death and no acute toxic symptoms after 14d observation period. According to the evaluation
standard, the acute oral toxicity of sublimed sulfur in rats is classified as “actual non-toxic grade”.
Appendix: Acute Toxicity (LD50) Classification Standard for Chemicals
Grade
Oral LD50 in mice (mg/kg)
Extremely toxic
<1
High toxic
>1-50
Moderate toxic
>50-500
Low toxic
>50-5000
Actual non-toxic
>5000
References
1. Procedures and methods of safety evaluation for cosmetics. National Standard of the People’s
Republic of China.
UDC 668.58.001.4 C51 GB 7919-87
Procedures and methods of safety evaluation for cosmetics.
2. OECD toxicity test guidelines for chemicals.
Drug Management Bureau, Ministry of Health; Institute for the Control of Pharmaceutical and Biological
Products[NICPBP], Ministry of Health; Pharmacopoeia Commission of the Ministry of Health. Apr. 1984
3. Measures for new drug approval.
Ministry of Health, People’s Republic of China. Jul. 1985.
4. Experiment Unit: Pharmacology & Toxicology Experiment Center of Henan College of Traditional Chi
nese Medicine.
Experiment Period: from Jul. 1990 to Jul. 1991
Principal Researcher: Li Zongduo
Original Data Stored at: Pharmacology & Toxicology Experiment Center of Henan College of Traditional
Chinese Medicine.
Acute Percutaneous Toxicity Test
Objective: To investigate the short-term percutaneous adverse reaction after single administration of
Zhongzhou Ointment applied on skin, also to provide an experimental basis for long-term toxicity test.
Drugs: Zhongzhou Ointment, pale yellow powder, with vaseline as the excipient, containing 30g drug in
every 100g ointment, is prepared to 60% (g/g) to meet the test demands, which is twice of the original
concentration.
I. Acute Toxicity Test on Complete Skin
1. Animals: 30 albino healthy rabbits, weight range 2.0-3.0 kg, 15 males and 15 females (unlabored and
unpregnant), were provided by the Animal Center of Henan Medical University. Before the test, the rab
bits were raised in separate rooms for 5 days in order to make them accustomed to the test environment.
24h before administration, cut furs on the back trunk of each rabbit, which should be no less than 10% of
the body surface area.
2. Dosage and Grouping: rabbits of both sexes were randomized into 3 groups, 10 in each group.
(1) Excipient group: 30 mg/ kg Vaseline were applied on the skin area without furs
(equivalent to the clinical dosage)
(2) Low-dosage group: 600 mg/kg Zhongzou Ointment were applied on the skin area without furs
(equivalent to 20 folds of the clinical dosage)
(3) High-dosage group: 6000 mg/kg Zhongzou Ointment were applied on the skin area without furs
(equivalent to 200 folds of the clinical dosage)
3. Methods: Evenly apply high-dosage, low-dosage Zhongzhou Ointment and vaseline on the back skin area
without furs respectively, and then cover this area with oilpaper and two layers of gauze. Single
administration was taken in all of the 3 groups. After 24h, remove the coverage and clean this area with
normal saline to observe the local reaction. Keep observing animals’ behaviors after administration, i.e.
whether or not the ointment has been percutaneously absorbed by the animals, any toxic reaction or death
has been caused by the ointment. Observation period lasted for 14 days. Weigh each rabbit before
administration, 7d after administration and 14d after administration respectively. Rabbits were killed at
the end of the observation period in order to detect any pathological changes in the major organs with
naked eyes.
4. Results: During 14 days of observation, no systemic toxic reaction was observed in the excipient group
and both of the Zhongzhou Ointment groups after administration. Compared with the control group, no
differences were observed on the local skin area. At the end of the test, animals from all of the 3 groups
all gained some weight, with no significant differences among the 3 groups (P>0.05) (see Table 1). No
pathological changes were detectable in the killed animals with naked eyes.
5. Conclusion: When the single dosage of Zhongzhou Ointment reached 6000mg/kg, no toxic reaction was
observed in animals. According to relevant state regulations, Zhongzhou Ointment, as a dermatological
agent, could be classified as “actual non-toxic grade”.
Appendix І: Calculating method of body surface area of test animals: S=K• W2/3
S1=body surface area, cm2, K1=constant, 10 rabbits, W1=animal’s body weight (g)
Appendix Π: Acute Toxicity (LD50) Classification Standard for Chemicals
Grade
Percutaneous LD50 in rabbits (mg/kg)
Extremely toxic
High toxic
Moderate toxic
Low toxic
Actual non-toxic
<5
>5-44
>44-350
>350-2180
>2180
Appendix І and Appendix Π: from “Procedures and methods of safety evaluation for cosmetics”, formulated
by the Ministry of Health.
References
(1) Procedures and methods of safety evaluation for cosmetics. National Standard of the
People’s Republic of China.
UDC 668.58.001.4 C51 GB 7919-87
Procedures and methods of safety evaluation for cosmetics.
(2) OECD toxicity test guidelines for chemicals.
Drug Management Bureau, Ministry of Health; Institute for the Control of Pharmaceutical and
Biological Products［NICPBP］, Ministry of Health; Pharmacopoeia Commission of the Ministry of
Health. Apr. 1984
(3) Measures for new drug approval.
Ministry of Health, People’s Republic of China. Jul. 1987.
Changes of Animals’ Weight in Acute Toxicity Test of Zhongzhou Ointment on Complete Skin
Groups
Drugs
Sex
Weight be- Weight
fore admin- after 7d
istration
(kg)
(kg)
Excipient
group
30mg/kg
♀
Vaseline,
Single Administration
♂
2.11±0.12
2.16±0.25 _ _
2.53±0.71
__
2.21±0.13
2.30±0.27 _ _
2.62±0.38
__
Low
dosage
group
600mg/kg
♀
Zhongzhou Ointment
Single Administration
♂
2.10±0.21
2.23±0.28 _ _
2.50±0.47
__
2.13±0.30
2.31±0.23 _ _
2.60±0.68
__
High
dosage
group
6000mg/kg
♀
Zhongzhou Ointment
Single Administration
♂
1.97±0.27
2.20±0.23 _ _
2.49±0.41
__
1.99±0.18
2.17±0.29 _ _
2.45±0.27
__
Toxic
Weight
Toxic
reaction after 14d (kg) reaction
II. Acute Toxicity Test on Damaged Skin
1. Drug preparation, fur-cutting procedure on animals, grouping and dosage are the same as those in “Acute
Toxicity Test on Complete Skin”.
2. Skin preparation of animals: with a 7# needle, horizontally draw 7 traces (15cm long) on the skin area
without furs of each rabbit, to the degree of no bleeding.
3. Methods: Evenly apply high-dosage, low-dosage Zhongzhou Ointment and excipient vaseline on the
damaged skin area of each rabbit respectively, cover this area with oilpaper and two layers of gauze, and
then use non-irritant adhesive tapes for fixation to ensure a close contact between the test preparations and
the damaged skin. Single administration was taken in all of the 3 groups. After 24h, remove the coverage
and observe the local reaction. After local application, drugs could be absorbed percutaneously to
produce systemic effects, so carefully observe animals’ behaviors after administration, i.e. any toxic
reaction or death has been caused after the drugs were percutaneously absorbed by the animals.
Observation period lasted for 14 days. Weigh each rabbit before administration, 7d after administration
and 14d after administration respectively. Rabbits were killed at the end of the observation period in order
to detect any pathological changes in the major organs with naked eyes.
4 Results: During consecutive 14 days of observation, no local or systemic toxic reaction was observed in
the low-dosage Zhongzhou Ointment group and excipient vaseline group after administration. Compared
with the control group, no differences were observed on the local skin area. At the end of the test, animals
from all of the 3 groups all gained some weight, with no significant differences among the 3 groups
(P>0.05) (see Table 1).
5 Conclusion: When the single dosage of Zhongzhou Ointment applied on the damaged skin reached
6000mg/kg, no local or systemic toxic reaction was observed in animals. According to relevant state
regulations, Zhongzhou Ointment for Demodex Rosacea, as a dermatological agent, could be classified as
“actual non-toxic grade”.
Appendix І: Calculating method of body surface area of test animals: S=K• W2/3
S1=body surface area, cm2, K1=constant, 10 rabbits, W1=animal’s body weight (g)
Appendix Π: Acute Toxicity (LD50) Classification Standard for Chemicals
Grade
Percutaneous LD50 in rabbits (mg/kg)
Extremely toxic
High toxic
Moderate toxic
Low toxic
Actual non-toxic
<5
>5-44
>44-350
>350-2180
>2180
Appendix І and Appendix Π: from “Procedures and methods of safety evaluation for cosmetics”, formulated
by the Ministry of Health.
References
(1)
Procedures and methods of safety evaluation for cosmetics. National Standard of the People’s Republic of China.
UDC 668.58.001.4 C51 GB 7919-87
Procedures and methods of safety evaluation for cosmetics.
(2)
OECD toxicity test guidelines for chemicals. Drug Management Bureau, Ministry of Health; Institute
for the Control of Pharmaceutical and Biological Products [NICPBP], Ministry of Health; Pharmacopoeia
Commission of the Ministry of Health. Apr. 1984
(3)
Measures for new drug approval.
Ministry of Health, People’s Republic of China. Jul. 1985
Changes of Animals’ Weight in Acute Toxicity Test of Zhongzhou Ointment on Damaged Skin
Groups
Drugs
Sex
Weight be- Weight
fore admin- after 7d
istration
(kg)
(kg)
Excipient
group
30mg/kg
♀
Vaseline,
Single Administration
♂
2.21±0.41
2.39±0.28 _ _
2.58±0.21
__
2.25±0.13
2.41±0.31 _ _
2.66±0.30
__
Low
dosage
group
600mg/kg
♀
Zhongzhou Ointment
Single Administration
♂
2.33±0.19
2.59±0.19 _ _
2.71±0.27
__
2.38±0.37
2.58±0.22 _ _
2.81±0.31
__
High
dosage
group
6000mg/kg
♀
Zhongzhou Ointment
Single Administration
♂
2.22±0.38
2.47±0.32 _ _
2.69±0.31
__
2.41±0.28
2.65±0.41 _ _
2.77±0.37
__
10
Toxic
Weight
Toxic
reaction after 14d (kg) reaction
Acute Percutaneous Toxicity Test
Objective: To investigate the short-term percutaneous adverse reaction after single administration of
“zinc oxide” applied on skin, also to provide an experimental basis for long-term toxicity test.
Drugs: “zinc oxide”, pale white powder, with vaseline as the excipient, containing 30g drug in every 100g
ointment, is prepared to 60% (g/g) to meet the test demands, which is twice of the original concentration.
I. Acute Toxicity Test on Complete Skin
1. Animals: 30 albino healthy rabbits, weight range 2.0-3.0 kg, 15 males and 15 females (unlabored and
unpregnant), were provided by the Animal Center of Henan Medical University. Before the test, the
rabbits were raised in separate rooms for 5 days in order to make them accustomed to the test
environment. 24h before administration, cut furs on the back trunk of each rabbit, which should be no less
than 10% of the body surface area.
2. Dosage and Grouping: rabbits of both sexes were randomized into 3 groups, 10 in each group.
(1) Excipient group: 15 mg/kg Vaseline were applied on the skin area without furs
(equivalent to the clinical dosage).
(2) Low-dosage group: 300 mg /kg zinc oxide were applied on the skin area without furs
(equivalent to 20 folds of the clinical dosage).
(3) High-dosage group: 3000 mg/kg zinc oxide were applied on the skin area without furs
(equivalent to 200 folds of the clinical dosage).
3. Methods: Evenly apply high-dosage, low-dosage “zinc oxide” and vaseline on the back skin area without
furs respectively, and then cover this area with oilpaper and two layers of gauze, and then use non-irritant
adhesive tapes for fixation to ensure a close contact between the test preparations and the skin. Single administration was taken in all of the 3 groups. After 24h, remove the coverage and clean this area with normal
saline to observe the local reaction. Keep observing animals’ behaviors after administration, i.e. whether
or not the ointment has been percutaneously absorbed by the animals, any toxic reaction or death has been
caused by the ointment. Observation period lasted for 14 days. Weigh each rabbit before administration, 7d
after administration and 14d after administration respectively. Rabbits were killed at the end of the observation period in order to detect any pathological changes in the major organs with naked eyes.
4. Results: During 14 days of observation, no systemic toxic reaction was observed in the excipient group
and both of the “zinc oxide” ointment groups after administration. Compared with the control group, no
differences were observed on the local skin area. At the end of the test, animals from all of the 3 groups all
gained some weight, with no significant differences among the 3 groups (P>0.05) (see Table 1). No pathological changes were detectable in the killed animals with naked eyes.
5. Conclusion: When the single dosage of “zinc oxide” reached 3000mg/kg, no toxic reaction was observed
in animals. According to relevant state regulations, “zinc oxide” ointment, as a dermatological agent, could
be classified as “actual non-toxic grade”.
11
Appendix І: Calculating method of body surface area of test animals: S=K• W2/3
S1=body surface area, cm2, K1=constant, 10 rabbits, W1=animal’s body weight (g)
Appendix Π: Acute Toxicity (LD50) Classification Standard for Chemicals
Grade
Percutaneous LD50 in rabbits (mg/kg)
Extremely toxic
High toxic
Moderate toxic
Low toxic
Actual non-toxic
<5
>5-44
>44-350
>350-2180
>2180
Appendix І and Appendix Π: from “Procedures and methods of safety evaluation for cosmetics”, formulated
by the Ministry of Health.
References
(1) Procedures and methods of safety evaluation for cosmetics. National Standard of the
People’s Republic of China.
UDC 668.58.001.4 C51 GB 7919-87
Procedures and methods of safety evaluation for cosmetics.
(2) OECD toxicity test guidelines for chemicals.
Drug Management Bureau, Ministry of Health; Institute for the Control of Pharmaceutical and
Biological Products [NICPBP], Ministry of Health; Pharmacopoeia Commission of the Ministry of
Health. Apr. 1984.
(3) Measures for new drug approval.
Ministry of Health, People’s Republic of China. Jul. 1987
Experiment Unit: Pharmacology & Toxicology Experiment Center of Henan
College of Traditional Chinese Medicine
Experiment Period: from Jul. 1990 to Jul. 1991
Principal Researcher: Li Zongduo
Original Data Stored at: Pharmacology & Toxicology Experiment Center of Henan
College of Traditional Chinese Medicine
12
Changes of Animals’ Weight in Acute Toxicity Test of “zinc oxide” on Complete Skin
Groups
Drugs
Sex
Weight be- Weight
fore admin- after 7d
istration
(kg)
(kg)
Excipient
group
15mg/kg
♀
Vaseline,
Single Administration
♂
2.21±0.11
2.37±0.45 _ _
2.58±0.31
__
2.20±0.23
2.41±0.38 _ _
2.64±0.37
__
Low
dosage
group
300mg/kg
♀
zinc oxide
Single Administration
♂
2.13±0.17
2.26±0.31 _ _
2.49±0.41
__
2.17±0.22
2.10±0.41 _ _
2.61±0.59
__
High
dosage
group
3000mg/kg
♀
zinc oxide
Single Administration
♂
2.09±017
2.22±0.81 _ _
2.57±0.44
__
2.15±0.27
2.20±0.24 _ _
2.62±0.23
__
13
Toxic
Weight
Toxic
reaction after 14d (kg) reaction
II. Acute Toxicity Test on Damaged Skin
1 Drug preparation, fur-cutting procedure on animals, grouping and dosage are the same as those in “Acute
Toxicity Test on Complete Skin”.
2 Skin preparation of animals: with a 7# needle, horizontally draw 7 traces (15cm long) on the skin area
without furs of each rabbit to the degree of no bleeding.
3 Methods: Evenly apply high-dosage, low-dosage “zinc oxide” and excipient vaseline on the damaged skin
area of each rabbit respectively, cover this area with oilpaper and two layers of gauze, and then use
nonirritant adhesive tapes for fixation to ensure a close contact between the test preparations and the
damaged skin. Single administration was taken in all of the 3 groups. After 24h, remove the coverage and
observe the local reaction. After local application, drugs could be absorbed percutaneously to produce
systemic effects, so carefully observe animals’ behaviors after administration, i.e. any toxic reaction or
death has been caused after the drugs were percutaneously absorbed by the animals. Observation period
lasted for 14 days.
Weigh each rabbit before administration, 7d after administration and 14d after administration respectively.
Rabbits were killed at the end of the observation period in order to detect any pathological changes in the
major organs with naked eyes.
4 Results: During consecutive 14 days of observation, no local or systemic toxic reaction was observed in
the low-dosage zinc oxide group and excipient vaseline group after administration. Compared with the
control group, no differences were observed on the local skin area. At the end of the test, animals from all
of the 3 groups all gained some weight, with no significant differences among the 3 groups (P>0.05)
(see Table ).
5 Conclusion: When the single dosage of “zinc oxide” ointment applied on the damaged skin reached 3000
mg/kg, no local or systemic toxic reaction was observed in animals. According to relevant state regulations,
“zinc oxide” ointment, as a dermatological agent, could be classified as “actual non-toxic grade”.
Appendix І: Calculating method of body surface area of test animals: S=K• W2/3
S1=body surface area, cm2, K1=constant, 10 rabbits, W1=animal’s body weight (g)
Appendix Π: Acute Toxicity (LD50) Classification Standard for Chemicals
Grade
Percutaneous LD50 in rabbits (mg/kg)
Extremely toxic
High toxic
Moderate toxic
Low toxic
Actual non-toxic
<5
>5-44
>44-350
>350-2180
>2180
Appendix І and Appendix Π: from “Procedures and methods of safety evaluation for cosmetics”, formulated
by the Ministry of Health.
14
Changes of Animals’ Weight in Acute Toxicity Test of “zinc oxide” on Damaged Skin
Groups
Drugs
Sex
Weight be- Weight
fore admin- after 7d
istration
(kg)
(kg)
Toxic
Weight
Toxic
reaction after 14d (kg) reaction
Excipient
group
15mg/kg
♀
Vaseline,
Single Administration
♂
2.23±0.43
2.40±0.31 _ _
2.60±0.32
__
2.27±0.27
2.43±0.41 _ _
2.65±0.17
__
Low
dosage
group
300mg/kg
♀
zinc oxide
Single Administration
♂
2.41±0.33
2.51±0.41 _ _
2.75±0.19
__
2.50±0.21
2.71±0.24 _ _
2.86±0.31
__
High
dosage
group
3000mg/kg
♀
zinc oxide
Single Administration
♂
2.33±0.19
2.61±0.23 _ _
2.76±0.29
__
2.39±0.25
2.63±0.43 _ _
2.83±0.43
__
References
(1) Procedures and methods of safety evaluation for cosmetics. National Standard of the
People’s Republic of China.
UDC 668.58.001.4 C51 GB 7919-87
Procedures and methods of safety evaluation for cosmetics.
(2) OECD toxicity test guidelines for chemicals.
Drug Management Bureau, Ministry of Health; Institute for the Control of Pharmaceutical and
Biological Products [NICPBP], Ministry of Health; Pharmacopoeia Commission of the Ministry of
Health. Apr. 1984.
(3) Measures for new drug approval.
Ministry of Health, People’s Republic of China. Jul. 1985.
15
Acute Percutaneous Toxicity Test
Objective: to investigate the short-term percutaneous adverse reaction after single administration of
“sublimed sulfur” applied on skin, also to provide an experimental basis for long-term toxicity test.
Drugs: “sublimed sulfur”, pale yellow powder, with vaseline as the excipient, containing 30g drug in every
100g ointment, is prepared to 60% (g/g) to meet the test demands, which is twice of the original concentration.
I.
Acute Toxicity Test on Complete Skin
1 Animals: 30 albino healthy rabbits, weight range 2.0-3.0 kg, 15 males and 15 females (unlabored and
unpregnant), were provided by the Animal Center of Henan Medical University. Before the test, the rabbits
were raised in separate rooms for 5 days in order to make them accustomed to the test environment. 24h
before administration, cut furs on the back trunk of each rabbit, which should be no less than 10% of the
body surface area.
2 Dosage and Grouping: rabbits of both sexes were randomized into 3 groups, 10 in each group.
(1) Excipient group: 15 mg vaseline, applied on the skin area without furs (equivalent to the clinical dosage).
(2) Low-dosage group: 300 mg /kg sublimed sulfur were applied on the skin area without furs (equivalent to
20 folds of the clinical dosage)
(3) High-dosage group: 3000 mg /kg sublimed sulfur were applied on the skin area without furs (equivalent
to 200 folds of the clinical dosage)
3 Methods: Evenly apply high-dosage, low-dosage “sublimed sulfur” and vaseline on the back skin area
without furs respectively, and then cover this area with oilpaper and two layers of gauze, and then use
non-irritant adhesive tapes for fixation to ensure a close contact between the test preparations and the skin.
Single administration was taken in all of the 3 groups. After 24h, remove the coverage and clean this area
with normal saline to observe the local reaction. Keep observing animals’ behaviors after administration,
i.e. whether or not the ointment has been percutaneously absorbed by the animals, any toxic reaction or
death has been caused by the ointment. Observation period lasted for 14 days. Weigh each rabbit before
administration, 7d after administration and 14d after administration respectively. Rabbits were killed at
the end of the observation period in order to detect any pathological changes in the major organs with
naked eyes.
4 Results: During 14 days of observation, no systemic toxic reaction was observed in the excipient group
and both of the “sublimed sulfur” ointment groups after administration. Compared with the control group,
no differences were observed on the local skin area. At the end of the test, animals from all of the 3 groups
all gained some weight, with no significant differences among the 3 groups (P>0.05) (see Table 1). No
pathological changes were detectable in the killed animals with naked eyes.
5 Conclusion: When the single dosage of “sublimed sulfur” reached 3000mg/kg, no toxic reaction was
observed in animals. According to relevant state regulations, “sublimed sulfur” ointment, as a
dermatological agent, could be classified as “actual non-toxic grade”.
Appendix І: Calculating method of body surface area of test animals: S=K• W2/3
S1=body surface area, cm2, K1=constant, 10 rabbits, W1=animal’s body weight (g)
Appendix Π: Acute Toxicity (LD50) Classification Standard for Chemicals
16
Grade
Percutaneous LD50 in rabbits (mg/kg)
Extremely toxic
High toxic
Moderate toxic
Low toxic
Actual non-toxic
<5
>5-44
>44-350
>350-2180
>2180
Appendix І and Appendix Π: from “Procedures and methods of safety evaluation for cosmetics”, formulated
by the Ministry of Health.
References
(1) Procedures and methods of safety evaluation for cosmetics. National Standard of the
People’s Republic of China.
UDC 668.58.001.4 C51 GB 7919-87
Procedures and methods of safety evaluation for cosmetics.
(2) OECD toxicity test guidelines for chemicals.
Drug Management Bureau, Ministry of Health; Institute for the Control of Pharmaceutical and Biological
Products [NICPBP], Ministry of Health; Pharmacopoeia Commission of the Ministry of Health. Apr.
1984.
(3) Measures for new drug approval.
Ministry of Health, People’s Republic of China. Jul. 1985
Experiment Unit: Pharmacology & Toxicology Experiment Center of Henan College of Traditional
Chinese Medicine
Experiment Period: from Jul. 1990 to Jul. 1991
Principal Researcher: Li Zongduo
Original Data Stored at: Pharmacology & Toxicology Experiment Center of Henan College of Traditional
Chinese Medicine
17
Changes of Animals’ Weight in Acute Toxicity Test of “sublimed sulfur” on Complete Skin
Groups
Drugs
Sex
Weight be- Weight
fore admin- after 7d
istration
(kg)
(kg)
Excipient
group
15mg/kg
♀
Vaseline,
Single Administration
♂
2.10±0.21
2.23±0.27 _ _
2.57±0.32
__
2.23±0.41
2.41±0.29 _ _
2.60±0.42
__
Low
dosage
group
300mg/kg
♀
sublimed sulfur
Single Administration
♂
1.97±0.23
2.12±0.31 _ _
2.39±0.40
__
2.01±0.19
2.18±0.19 _ _
2.42±0.31
__
High
dosage
group
3000mg/kg
♀
sublimed sulfur
Single Administration
♂
2.15±0.18
2.21±0.31 _ _
2.47±0.27
__
2.17±0.21
2.28±0.40 _ _
2.49±0.38
__
18
Toxic
Weight
Toxic
reaction after 14d (kg) reaction
II. Acute Toxicity Test on Damaged Skin
1 Drug preparation, fur-cutting procedure on animals, grouping and dosage are the same as those in
“Acute Toxicity Test on Complete Skin”.
2 Skin preparation of animals: with a 7# needle, horizontally draw 7 traces (15cm long) on the skin area
without furs of each rabbit, to the degree of no bleeding.
3 Methods: Evenly apply high-dosage, low-dosage “sublimed sulfur” and excipient vaseline on the damaged
skin area of each rabbit respectively, cover this area with oilpaper and two layers of gauze, and then use
non-irritant adhesive tapes for fixation to ensure a close contact between the test preparations and the
damaged skin. Single administration was taken in all of the 3 groups. After 24h, remove the coverage
and observe the local reaction. After local application, drugs could be absorbed percutaneously to produce
systemic effects, so carefully observe animals’ behaviors after administration, i.e. any toxic reaction or
death has been caused after the drugs were percutaneously absorbed by the animals. Observation period
lasted for 14 days. Weigh each rabbit before administration, 7d after administration and 14d after
administration respectively. Rabbits were killed at the end of the observation period in order to detect any
pathological changes in the major organs with naked eyes.
4 Results: during consecutive 14 days of observation, no local or systemic toxic reaction was observed in the
low-dosage “sublimed sulfur” group and excipient vaseline group after administration. Compared with the
control group, no differences were observed on the local skin area. At the end of the test, animals from all
of the 3 groups all gained some weight, with no significant differences among the 3 groups (P>0.05)
(see Table).
5 Conclusion: when the single dosage of “sublimed sulfur” ointment applied on the damaged skin reached
3000mg/kg, no local or systemic toxic reaction was observed in animals. According to relevant state
regulations, “sublimed sulfur” ointment, as a dermatologic agent, could be classified as
“actual non-toxic grade”.
Appendix І: Calculating method of body surface area of test animals: S=K• W2/3
S1=body surface area, cm2, K1=constant, 10 rabbits, W1=animal’s body weight (g)
Appendix Π: Acute Toxicity (LD50) Classification Standard for Chemicals
Grade
Percutaneous LD50 in rabbits (mg/kg)
Extremely toxic
High toxic
Moderate toxic
Low toxic
Actual non-toxic
<5
>5-44
>44-350
>350-2180
>2180
Appendix І and Appendix Π: from “Procedures and methods of safety evaluation for cosmetics”, formulated
by the Ministry of Health
19
References
(1) Procedures and methods of safety evaluation for cosmetics. National Standard of the
People’s Republic of China.
UDC 668.58.001.4 C51 GB 7919-87
Procedures and methods of safety evaluation for cosmetics.
(2) OECD toxicity test guidelines for chemicals.
Drug Management Bureau, Ministry of Health; Institute for the Control of Pharmaceutical and Biological
Products [NICPBP], Ministry of Health; Pharmacopoeia Commission of the Ministry of Health. Apr.
1984
(3) Measures for new drug approval.
Ministry of Health, People’s Republic of China. Jul. 1985
Changes of Animals’ Weight in Acute Toxicity Test of “sublimed sulfur” on Damaged Skin
Groups
Drugs
Sex
Weight be- Weight
fore admin- after 7d
istration
(kg)
(kg)
Excipient
group
15mg/kg
♀
Vaseline,
Single Administration
♂
2.35±0.18
2.60±0.18 _ _
2.71±0.19
__
2.39±0.21
2.62±0.23 _ _
2.83±0.40
__
Low
dosage
group
300mg/kg
♀
sublimed sulfur
Single Administration
♂
2.23±0.19
2.38±0.34 _ _
2.58±0.31
__
2.26±0.28
2.47±0.41 _ _
2.70±0.29
__
High
dosage
group
3000mg/kg
♀
sublimed sulfur
Single Administration
♂
2.24±0.30
2.50±0.27 _ _
2.69±0.31
__
2.47±0.21
2.62±0.10 _ _
2.77±0.44
__
20
Toxic
Weight
Toxic
reaction after 14d (kg) reaction