Some standard content:
ICS07.100
National Standard of the People's Republic of China
GB15193.14—2003
Replaces GB15193.14-1994
Issued on September 24, 2003
Teratogenicity study
Implemented on May 1, 2004
Ministry of Health of the People's Republic of China
Standardization Administration of the People's Republic of China
GB15193.14-2003
The full text of this standard is mandatory.
This standard replaces GB15193.14—1994 Teratogenicity study. Compared with GB15193.14-1994, this standard has been modified as follows: In the "Scope", the specific content of the test objects has been added: chemical, biological and physical factors involved in the production, processing, storage, transportation and sales of food that may cause harm to health; the objects of inspection include food additives (including nutritional fortifiers), new food resources and their ingredients, new resource foods, irradiated foods, food containers and packaging materials, food tools, equipment, detergents, disinfectants, pesticide residues, veterinary drug residues, microorganisms used in the food industry, etc.; In the reagents, the following items have been added: Alizarin red solution for skinning; In "Experimental Animals": removed the content about mice and rabbits; In "Dose and Grouping": added the principle requirements for the design of each dose group and the dose design method when the maximum dose (maximum concentration and maximum gavage volume) of the test substance is given to the acute toxicity test without death and the LDso cannot be calculated; In "Operation Procedures": removed the content about mice and rabbits in the killing and examination of pregnant mice; Added the preparation method after skinning for the preparation and examination of fetal mouse bone specimens; Added 4 tables of "Contents to be checked and recorded in teratogenic tests". From the date of implementation of this standard, GB15193.14-1994 will be abolished at the same time. This standard is proposed and managed by the Ministry of Health of the People's Republic of China. Drafting units of this standard: Jiang Medical University, Institute of Nutrition and Food Safety, Chinese Center for Disease Control and Prevention. The main drafters of this standard: Huang Xingxian, Li Youhui, Geng Guiying This standard was first issued in 1994, and this is the first revision 90
1 Scope
Teratogenicity test
This standard specifies the test methods for predicting embryotoxicity and teratogenicity of environmental harmful substances to humans. GB15193.14-2003
This standard is applicable to the evaluation of the teratogenic effects of chemical, biological and physical factors that may cause harm to health during the production, processing, storage, transportation and sales of food. The objects of inspection include food additives (including nutritional fortifiers), new food resources and their ingredients, new resource foods, irradiated foods, food containers and packaging materials, food tools, equipment, detergents, disinfectants, pesticide residues, veterinary drug residues, microorganisms used in the food industry, etc.
2 Principle
During pregnancy, the mother is exposed to certain harmful substances that can pass through the placental barrier, which affects the differentiation and development of the embryo's organs, leading to structural and functional defects and fetal malformations. Therefore, when the test substance is administered to the pregnant animal after the embryo has implanted and has begun to enter the cell and organ differentiation stage, the teratogenic effect of the substance on the fetus can be detected. 3 Instruments and reagents
3.1 Instruments and equipment
Common laboratory equipment, biological microscope and stereo microscope, vernier caliper (percentile caliper). 3.2 Reagents
The experimental water is distilled water.
3.2.1 Formaldehyde, glacial acetic acid, 2,4,6-trinitrophenol, potassium hydroxide, glycerol, chloral hydrate, alizarin red. 3.2.2 Alizarin red stock solution: Alizarin red saturated solution, 5.0mL 50% acetic acid saturated solution, 10.0mL glycerol, 60.0mL 1% chloral hydrate are mixed and placed in a brown bottle.
3.2.3 Alizarin red application solution: Take 3mL~5mL of stock solution, dilute to 1000mL with 1g/100mL~2g/100mL potassium hydroxide solution, and store in a brown bottle.
3.2.4 Alizarin red solution: 0.1g Alizarin red, 10g potassium hydroxide, 1000mL distilled water. (Skinning dye solution) 3.2.5 Transparent solution A: Mix 200mL glycerol, 10g potassium hydroxide, and 790mL distilled water. 3.2.6 Transparent solution B: Mix glycerol and distilled water in equal amounts. 3.2.7 Fixative (Bouins solution): 75 parts of 2,4,6-trinitrophenol (saturated solution of picric acid), 20 parts of formaldehyde, and 5 parts of glacial acetic acid. Experimental animals
Common experimental animals are rats. Select healthy, sexually mature (90-100 days) rats, 80-90 female unmated rats, and half the number of males.
5 Dosage and grouping
At least 3 test groups should be set up. In principle, the high dose should cause toxic effects in some pregnant mice (and/or fetuses), such as weight loss, etc. The low dose group should not cause obvious toxic effects. Each dose group can use 1/4.1/16, 1/64LDs. When there is no death in the acute toxicity test when the animal is given the maximum dose (maximum concentration and maximum gavage capacity) of the test substance, the maximum no-observed-adverse-effect dose of the 30-day feeding test is the high dose group, and the following 2 dose groups are set up, and a negative control group is set up. For a new animal, a positive control group can be set up for the first test. Each group should have at least 12 pregnant mice. Commonly used positive controls include diclofenac (0.5mg/kg body weight ~ 1.0mg/kg body weight), sodium pentachlorophenol (30mg/kg body weight), aspirin (250mg/kg body weight ~ 300mg/kg body weight) and vitamin A (7500μg/kg body weight 13000μg/kg body weight) etc.
6 Operation steps
6.1 Inspection of "pregnant animals" and administration of test substances After sexually mature female and male rats are caged in a ratio of 1:1 (or 2:1), observe the vaginal plug (or vaginal smear) every morning. If the vaginal plug (or sperm) is found, it is considered that the rat has mated, and the day is regarded as "pregnancy day zero". If there is no mating within 5 days, the male rat should be replaced. The detected "pregnant mice" were randomly divided into groups, weighed and numbered. From the 7th to the 16th day of pregnancy, the test substance was orally administered every day (calculated as 0.5mL/100g body weight to 1.0mL/100g body weight). The body weight was weighed on the 0th, 7th, 12th, 16th and 20th days of pregnancy, and the amount of the test substance was calculated. The room temperature during the mating period of the animals should be 20℃ to 25℃, and the environment should be quiet. If necessary, nutrients such as malt and cake should be added. 6.2 Sacrifice and examination of pregnant mice
The rats were directly decapitated and killed on the 20th day of pregnancy. The uterus was removed by laparotomy and weighed, and the number of absorbed fetuses, early fetal deaths, late fetal deaths and live fetuses was recorded and examined.
6.3 Examination of live fetuses
The sex, weight and body length of the fetuses were recorded one by one, and the fetal appearance was checked for abnormalities. The fetal body surface examination items are shown in Table 1. Table 1 Items of body surface examination of fetal mice in teratogenic test
Anencephaly
Encephalocele
Cranial fissure
Hydrocephalus
Microcephaly
Facial cleft
Microphthalmia
Protruding eyeball
Anotia
Microtia
Low-set ears
Microfrontal disease
Inferior frontal cleft
Cleft lip and mouth
6.4. Preparation and examination of fetal mouse bone specimens
Drive
Sternal cleft
Chestal cleft
Spinabida
Scoliosis
Kymphalocele
Hypospadias
No anus
Short tail, dreaded tail
Put 1/2 of the live fetuses (odd or even number) in 95% (volume fraction) ethanol and fix them for 2 to 3 weeks. Take out the fetuses and rinse them with running water for a few minutes. Then put them in 1g/100mL~2g/100mL potassium hydroxide solution (at least 5 times the volume of the fetus) for 8h~72h. After it becomes transparent, put them in Alizarin Red-S application solution for staining for 6h~48h, and shake them gently 1~2 times/day until the skull is stained red. Then put it in transparent liquid A for 1 to 2 days, and in transparent liquid B for 2 to 3 days. When the bones are stained red and the soft tissues are basically faded, put the specimen in a small dish, use a transmitted light source, and observe it as a whole under a stereo microscope, and then check the bones step by step. Measure the defects of the parietal bone and the occipital bone, and then check the number of sternums, missing or fused (there are 6 sternums, and when ossification is incomplete, the 5th sternum is missing first, followed by the 2nd sternum). Ribs usually have 12 to 13 pairs, and common deformities include fused ribs, bifurcated ribs, wavy ribs, short ribs, multiple ribs, missing ribs, and interrupted ribs. Check the development of the spine and the number of vertebrae (7 cervical vertebrae, 12 to 13 thoracic vertebrae, 5 to 6 lumbar vertebrae, 4 basal vertebrae, and 3 to 5 coccygeal vertebrae) for fusion and longitudinal fissures. Finally, check the bones of the limbs.
After removing the skin, internal organs and fat of the fetus, put it into the alizarin red solution for dyeing. Shake the glass bottle 2 to 3 times on the same day until the bones are dyed red. Change the fetus to transparent liquid A for 1 to 2 days, and change to transparent liquid B for 2 to 3 days. When the fetal bones have been dyed red and the purple-red color of the soft tissue has basically faded, it can be transferred to glycerol. (Skinning method) 92
See Table 2 for fetal bone examination items.
Spinal column
Bone of limbs
6.5 Examination of viscera of fetal mice
Table 2 Examination items of skeletal structure of fetal mice in teratogenicity test Ossification centre, dysplasia
Number, fusion, longitudinal fissure, partial fissure, number of ossification centres, dysplasia, constriction, detachment, shape Number of arches, number of ossification centres, abnormal shape, fusion, crack, constriction, detachment Shape, number
Number of ossification centres
Number, shape, fusion, bifurcation, defect, dysplasia Shape, complete defect, sternal segment fusion, crack, abnormal shape, dysplasia GB 15193.14—2003
Half of the live fetuses in each litter were fixed in Bouins solution for 2 weeks and then the viscera were examined. First, flush the fixative with tap water, place the mouse on its back on the paraffin board, cut off the limbs and tail, and use a blade to make 5 horizontal or vertical cuts on the head, and then open the chest and abdomen. Observe the size, shape and relative position of the organs according to the sections of different parts. The normal section is shown in Figure 1. Figure 1 Frontal section of the head of a rat
Make a horizontal cut from the tongue and the two corners of the mouth to the occipital part through the mouth (section 1), and you can observe the brain, diencephalon, orthencephalon, tongue and frontal fissure. b)
Make a vertical vertical cut in front of the eye (section 2), and you can see the nose. Make a vertical cut from the head vertically through the center of the eyeball (section 3). c)
Make a section through the brain along the maximum horizontal position of the head (section 4). The purpose of the above sections is to observe tongue fissure, bifurcated tongue, cleft palate, eyeball, nasal deformity, brain and ventricle abnormalities. e) Make a horizontal cross section 5 through the middle of the neck along the lower level, and you can observe the trachea, esophagus and medulla oblongata or spinal cord. After that, the chest and abdomen were cut open from the midline of the abdomen, and the size and position of the heart, lungs, transverse membrane, liver, stomach, intestines and other organs were checked in turn. After the inspection, they were removed, and the position and development of the kidneys, ureters, bladder, uterus or testicles were checked. Then the kidneys were cut open to observe whether there was hydronephrosis and enlargement of the renal pelvis. The internal organs of the experimental fetal mice were examined in Table 3.
Table 3 Examination items of fetal rat viscera in teratogenicity test Head (spinal cord)
Galvanic hypoplasia
Dilatation of lateral ventricles
Dilatation of the third ventricle
Anencephaly
Anophthalmia
Microphthalmia
Corneal defect
Monophthalmia
Dextrocardia
Atrial septal defect
Ventricular septal defect
Aortic arch
Esophageal atresia
Tracheal stenosis
Aplasia
Polypulmonary
Fusion of lung lobes||t t||Tracheoesophageal basket
Viscera heterotopia
Abnormal liver lobes
Adrenal gland loss
Polycystic kidney
Horseshoe kidney
Urinary bladder loss
Testicular loss
Ovarian loss
Ovarian ectopia
Uterine loss
Uterine hypoplasia
Hydronephrosis
Kidney loss
Hydrosalpinx
GB15193.14—2003
6.6 Test records
See Table 4 for the contents of the teratogenic test record.
Period of animal mating with test substance
Animal number
Animal species and strain
Age at mating
Confirmation of pregnancy
Clinical symptoms
Food and water intake
Test substance and solvent
Sampling route
Sampling period
7 Data processing
Table 4 Record contents of teratogenicity test
During solvent examination
Maternal autopsy findings
Ovaries Weight
Number of corpora lutea
Number of implantations
Placental weight
Position of fetus in uterus
Number of dead fetuses
Number of live fetuses
Fetal weight
Fetal examination
Body surface examination
Bone examination
Visceral examination
The statistics of various rates are tested by X-test, the weight gain of pregnant mice is tested by variance analysis or non-parametric statistics, and the length, weight, average number of live fetuses in a litter, and the weight of uterus and fetuses are tested by t-test. The data of fetuses are statistically analyzed in litters. The results should be able to show whether the test substance has maternal toxicity, embryotoxicity, and teratogenicity, and it is best to get the minimum teratogenic dose. In order to compare the teratogenicity intensity of different harmful substances, the teratogenicity index can be calculated. Teratogenicity index 1
LDao of female rat
Minimum teratogenic dose
Teratogenicity hazard index 2
8Result judgment
Maximum non-teratogenic dose
Maximum possible human intake
...(2)
Teratogenicity index below 10 is non-teratogenic, 10-100 is teratogenic, and above 100 is strong teratogenic. To indicate the probability of human harm when harmful substances exist in food, the teratogenicity hazard index can be calculated. If the index is greater than 300, it means that the substance is less harmful to humans, 100-300 is medium, and less than 100 is severe.14—2003
Half of the live fetuses in each litter were fixed in Bouins solution for 2 weeks and examined for internal organs. First, flush the fixative with tap water, place the rat on its back on a paraffin plate, cut off the limbs and tail, and use a blade to make 5 cross-sectional or longitudinal cuts on the head, and then open the chest and abdomen. Observe the size, shape and relative position of the organs according to the sections of different parts. The normal section is shown in Figure 1. Figure 1 Frontal section of the head of a rat
A cross-sectional cut from the tongue and the two corners of the mouth to the occipital part through the mouth (section 1) can observe the cerebrum, diencephalon, orthencephalon, tongue and frontal fissure. b)
Make a vertical longitudinal cut in front of the eye (section 2) to see the nose. Make a vertical longitudinal cut from the head through the center of the eyeball (section 3). c)
Make a section through the brain along the maximum horizontal position of the head (section 4). The purpose of the above sections is to observe cleft tongue, bifurcated tongue, cleft palate, eyeball, nasal deformity, brain and ventricle abnormalities. e) Make a cross section 5 along the lower level through the middle of the neck to observe the trachea, esophagus and medulla oblongata or spinal cord. After that, cut the chest and abdominal cavity from the midline of the abdomen, and check the size and position of the heart, lungs, transverse membrane, liver, stomach, intestines and other organs in turn. After the inspection, remove them, and then check the position and development of the kidneys, ureters, bladder, uterus or testicles. Then cut the kidneys to observe whether there is hydronephrosis and enlargement of the renal pelvis. The internal organs of the experimental fetal mice are shown in Table 3.
Table 3 Examination items of fetal rat viscera in teratogenicity test Head (spinal cord)
Galvanic hypoplasia
Dilatation of lateral ventricles
Dilatation of the third ventricle
Anencephaly
Anophthalmia
Microphthalmia
Corneal defect
Monophthalmia
Dextrocardia
Atrial septal defect
Ventricular septal defect
Aortic arch
Esophageal atresia
Tracheal stenosis
Aplasia
Polypulmonary
Fusion of lung lobes||t t||Tracheoesophageal basket
Viscera heterotopia
Abnormal liver lobes
Adrenal gland loss
Polycystic kidney
Horseshoe kidney
Urinary bladder loss
Testicular loss
Ovarian loss
Ovarian ectopia
Uterine loss
Uterine hypoplasia
Hydronephrosis
Kidney loss
Hydrosalpinx
GB15193.14—2003www.bzxz.net
6.6 Test records
See Table 4 for the contents of the teratogenic test record.
Period of animal mating with test substance
Animal number
Animal species and strain
Age at mating
Confirmation of pregnancy
Clinical symptoms
Food and water intake
Test substance and solvent
Sampling route
Sampling period
7 Data processing
Table 4 Record contents of teratogenicity test
During solvent examination
Maternal autopsy findings
Ovaries Weight
Number of corpora lutea
Number of implantations
Placental weight
Position of fetus in uterus
Number of dead fetuses
Number of live fetuses
Fetal weight
Fetal examination
Body surface examination
Bone examination
Visceral examination
The statistics of various rates are tested by X-test, the weight gain of pregnant mice is tested by variance analysis or non-parametric statistics, and the length, weight, average number of live fetuses in a litter, and the weight of uterus and fetuses are tested by t-test. The data of fetuses are statistically analyzed in litters. The results should be able to show whether the test substance has maternal toxicity, embryotoxicity, and teratogenicity, and it is best to get the minimum teratogenic dose. In order to compare the teratogenicity intensity of different harmful substances, the teratogenicity index can be calculated. Teratogenicity index 1
LDao of female rat
Minimum teratogenic dose
Teratogenicity hazard index 2
8Result judgment
Maximum non-teratogenic dose
Maximum possible human intake
...(2)
Teratogenicity index below 10 is non-teratogenic, 10-100 is teratogenic, and above 100 is strong teratogenic. To indicate the probability of human harm when harmful substances exist in food, the teratogenicity hazard index can be calculated. If the index is greater than 300, it means that the substance is less harmful to humans, 100-300 is medium, and less than 100 is severe.14—2003
Half of the live fetuses in each litter were fixed in Bouins solution for 2 weeks and examined for internal organs. First, flush the fixative with tap water, place the rat on its back on a paraffin plate, cut off the limbs and tail, and use a blade to make 5 cross-sectional or longitudinal cuts on the head, and then open the chest and abdomen. Observe the size, shape and relative position of the organs according to the sections of different parts. The normal section is shown in Figure 1. Figure 1 Frontal section of the head of a rat
A cross-sectional cut from the tongue and the two corners of the mouth to the occipital part through the mouth (section 1) can observe the cerebrum, diencephalon, orthencephalon, tongue and frontal fissure. b)
Make a vertical longitudinal cut in front of the eye (section 2) to see the nose. Make a vertical longitudinal cut from the head through the center of the eyeball (section 3). c)
Make a section through the brain along the maximum horizontal position of the head (section 4). The purpose of the above sections is to observe cleft tongue, bifurcated tongue, cleft palate, eyeball, nasal deformity, brain and ventricle abnormalities. e) Make a cross section 5 along the lower level through the middle of the neck to observe the trachea, esophagus and medulla oblongata or spinal cord. After that, cut the chest and abdominal cavity from the midline of the abdomen, and check the size and position of the heart, lungs, transverse membrane, liver, stomach, intestines and other organs in turn. After the inspection, remove them, and then check the position and development of the kidneys, ureters, bladder, uterus or testicles. Then cut the kidneys to observe whether there is hydronephrosis and enlargement of the renal pelvis. The internal organs of the experimental fetal mice are shown in Table 3.
Table 3 Examination items of fetal rat viscera in teratogenicity test Head (spinal cord)
Galvanic hypoplasia
Dilatation of lateral ventricles
Dilatation of the third ventricle
Anencephaly
Anophthalmia
Microphthalmia
Corneal defect
Monophthalmia
Dextrocardia
Atrial septal defect
Ventricular septal defect
Aortic arch
Esophageal atresia
Tracheal stenosis
Aplasia
Polypulmonary
Fusion of lung lobes||t t||Tracheoesophageal basket
Viscera heterotopia
Abnormal liver lobes
Adrenal gland loss
Polycystic kidney
Horseshoe kidney
Urinary bladder loss
Testicular loss
Ovarian loss
Ovarian ectopia
Uterine loss
Uterine hypoplasia
Hydronephrosis
Kidney loss
Hydrosalpinx
GB15193.14—2003
6.6 Test records
See Table 4 for the contents of the teratogenic test record.
Period of animal mating with test substance
Animal number
Animal species and strain
Age at mating
Confirmation of pregnancy
Clinical symptoms
Food and water intake
Test substance and solvent
Sampling route
Sampling period
7 Data processing
Table 4 Record contents of teratogenicity test
During solvent examination
Maternal autopsy findings
Ovaries Weight
Number of corpora lutea
Number of implantations
Placental weight
Position of fetus in uterus
Number of dead fetuses
Number of live fetuses
Fetal weight
Fetal examination
Body surface examination
Bone examination
Visceral examination
The statistics of various rates are tested by X-test, the weight gain of pregnant mice is tested by variance analysis or non-parametric statistics, and the length, weight, average number of live fetuses in a litter, and the weight of uterus and fetuses are tested by t-test. The data of fetuses are statistically analyzed in litters. The results should be able to show whether the test substance has maternal toxicity, embryotoxicity, and teratogenicity, and it is best to get the minimum teratogenic dose. In order to compare the teratogenicity intensity of different harmful substances, the teratogenicity index can be calculated. Teratogenicity index 1
LDao of female rat
Minimum teratogenic dose
Teratogenicity hazard index 2
8Result judgment
Maximum non-teratogenic dose
Maximum possible human intake
...(2)
Teratogenicity index below 10 is non-teratogenic, 10-100 is teratogenic, and above 100 is strong teratogenic. To indicate the probability of human harm when harmful substances exist in food, the teratogenicity hazard index can be calculated. If the index is greater than 300, it means that the substance is less harmful to humans, 100-300 is medium, and less than 100 is severe.
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