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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