Some standard content:
ICS 07.100
National Standard of the People's Republic of China
GB15193.15—2003
Replaces GB15193.15-1994
Issued on 2003-09-24
Reproductive study
Ministry of Health of the People's Republic of China
Standardization Administration of the People's Republic of China
Implemented on 2004-05-01
GB15193.15—2003
The full text of this standard is mandatory.
This standard replaces GB15193.15—1994 "Reproductive test". Compared with GB15193.15-1994, 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 test objects 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.; "experimental rats" have been changed to "experimental animals", and specific requirements for the age and number of experimental animals have been added; some principle requirements for the setting of each group have been added in the "Dosage and Grouping"; in the "Operation Procedures": the formulation of the test objects has been added. The contents of the control, handling of experimental animals, mating, standardization of each litter and the number of generations observed;
In the "Observation indicators", add clinical observation, water consumption, organ weighing and pathological examination indicators; change the original "3.3.2 Calculation" content to "Data processing"; and add "live birth rate, sex ratio", and modify the pregnancy rate count to:
Pregnancy rate (%) - number of animals giving birth to live pups/number of pregnant animals × 100-add report content".
From the date of implementation of this standard, GB15193.15-1994 will be abolished at the same time. This standard was proposed and managed by the Ministry of Health of the People's Republic of China. The drafting unit of this standard: Nutrition and Food Safety Institute of China Center for Disease Control and Prevention. The main drafters of this standard: Han Chi, Li Youhui. This standard was first issued in 1994 and this is the first revision. 96
1 Scope
Reproduction test
This standard specifies the basic technical requirements for reproduction test. GB15193.15-2003
This standard is applicable to the evaluation of the effects of chemical, biological and physical factors that may cause health hazards during the production, processing, storage, transportation and sales of food on the reproductive function of test animals. The test objects include food additives (including nutritional enhancers), 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||t t||Any test substance that can cause reproductive dysfunction, interfere with the formation of gametes or damage reproductive cells, in addition to affecting the implantation of fertilized eggs or pregnant eggs and causing infertility, can also affect the occurrence of embryos and fetal development, such as embryonic death leading to spontaneous abortion, fetal development retardation and fetal malformation. If it causes adverse effects on the mother, there will be abnormalities in pregnancy, delivery and milk secretion, and abnormal development of the fetus after birth.
3 Experimental Animals
Select rats aged 5 to 9 weeks. The difference in animal weight at the beginning of the experiment should not exceed ±20% of the average weight. At least 3 days should be allowed after purchase. Each group should have enough female and male mice to pair up to produce about 20 pregnant female mice. For this reason, generally 30 animals (F.) of each sex are required in each group at the beginning of the experiment; in the continued experiment, 25 animals of each sex are required for mating in each group (at least 1 male and female per litter, and at most 2 male and female per litter). The parental female mice selected should be non-parous and non-pregnant mice. 4 Dosage and grouping
At least 3 doses of test substance groups and one control group should be set up. Healthy animals are randomly divided into treatment groups and control groups. The difference in animal body weight at the beginning of the test should not exceed ±20% of the average body weight. The design of high-dose groups for certain test substances should take into account their effects on nutrient balance. The dose of non-nutritional test substances should not exceed 5% of the feed. The maximum tolerated dose or embryotoxic dose can be selected as the high dose for its dose design. The low-dose group should not produce systemic toxicity or reproductive toxicity to parental animals (it can be 1/30 of the maximum no-observed-adverse-effect dose or 100 times the possible intake). At the same time, a control group should be set up. The feeding and treatment methods of the control group are the same as those of the test substance group. Depending on the situation, the control group can be an untreated control or a sham-treated control. If a certain medium is used when the test substance is administered, a medium control should be set up. If the test substance is administered by adding it to the feed and causes a decrease in food intake and utilization, it is necessary to consider using a paired control group. 5 Operation steps
5.1 Preparation of test substance
Generally, distilled water is used as solvent. If the test substance is insoluble in water, edible oil, medical starch, carboxymethyl cellulose, etc. can be used to prepare emulsion or suspension. The test substance should be freshly prepared before oral administration, unless there is information indicating that it is stable when stored as a solution (or suspension, emulsion, etc.). At the same time, the possible effects of the medium used on the absorption, distribution, metabolism or retention of the test substance should be considered; the effects on the chemical properties and the toxic characteristics caused by them; the effects on the feed or drinking water consumption or the nutritional status of the animals. 5.2 Treatment of experimental animals
5.2.1 Administration of test substance: Oral administration, which can be added to feed, drinking water or oral administration. If the test substance is administered by oral administration, the body weight should be weighed twice a week97
GB15193.15—2003
, and the volume of the test substance administered should be calculated based on the body weight. 5.2.2 Parents and offspring receive the same dose of the test substance (administered by animal body weight, mg/kg body weight or g/kg body weight), feed and drinking water. Female and male F1 generation rats are administered daily after weaning. Rats of both sexes (parents and Fi generation) should be administered the test substance daily for at least 10 consecutive weeks before mating, and continue to be administered the test substance until the end of the study. 5.2.3 Administration of the test substance regimen: During the study, all animals should be administered the test substance in the same manner; the test substance should be administered continuously, 7 days a week.
5.3 Mating
At each mating, each female rat should be caged with a single male rat randomly selected from the same dose group (1:1 mating) until a vaginal plug is detected, or after 3 estrus periods or two weeks. After the vaginal plug is detected, the female and male rats should be separated as soon as possible. If mating has not occurred after 3 estrus periods or two weeks, the female and male rats should also be separated and no longer caged together. Paired male and female rats in the same cage should be marked. All female mice should be checked for sperm or vaginal plugs every day during the mating period until mating is confirmed. The day when the vaginal plug is found is considered day 0 of conception. Pregnant female mice should be placed in breeding cages separately, and bedding should be provided for nesting when pregnant mice are about to give birth. 5.4 Standardization of the number of pups per litter
Adjust the number of pups per litter to the same number on the 4th day after birth (generally 8 to 10 per litter, and no less than 8), and try to make the number of females and males in each litter equal. The number of females and males in a litter can also be different, but the number of mice of both sexes should be the same between litters. Excess mice in the original litter should be randomly selected, and should not be selected according to weight. 5.5 Observation generations
The observation generations vary with the purpose of the test, and can be one, two, three or more generations. If the test substance is observed to have obvious reproductive, morphological or toxic effects on the offspring in the two-generation reproduction test, a third-generation reproduction test is required to determine the accumulation effect of the test substance. 5.6 First, second and third generation breeding test methods 5.6.1 First generation breeding test method
The schematic diagram of the first generation breeding test method is shown in Figure 1.
Male (20 or 10) - Fo - Eagle (20) Give the test substance feed after weaning for three months 2 weeks | Mating
Fo Female (8-9) Delivery
Fo Female (8-9) 20 days Antenatal examination for malformation 3 weeks weaning, each litter of pups is divided into two (the mother mouse is killed) Fia Give the test substance
Fia Do not give the test substance
60 days to 90 days' Observe the various aspects of the surviving mice Figure 1 Schematic diagram of the first-generation reproduction test method
5.6.2 Two-generation reproduction test method
5.6.2.1 Schematic diagram of the two-generation reproduction test method See Figure 2. 98
Stand (20 or 10)
Fo (5)
Prenatal examination F1b
Check for teratogenesis
Female (20) are fed with the test substance feed for three months after weaning, and mated GB15193.15—2003
F1aAfter weaning, the rats were fed the basic diet for three months for observation. Some animals can be selected for the second mating. F1a10 days after weaning, Fo performed the second mating Fo (5 rats)
Natural delivery, postpartum
Observe the pups
Fo (10 rats)
F1bAfter weaning, the rats were fed the diet containing the test substance for three months, and mated with male rats (20 or 10 rats)
|Fib (10 rats)
F2b postpartum examination of teratogenicity
Female rats (20 rats)
F2a treated as F1a
F2a 10 days after weaning, F1b mated for the second time F1b (10 rats) gave birth naturallybzxZ.net
F2b was fed with a feed containing the test substance for three months after weaning. Figure 2 Schematic diagram of the two-generation breeding test method
5.6.2.2 Parent F. After weaning, feed the feed containing the test substance for three months, and then the male and female can mate, and the offspring are F1a. After weaning, F1a was fed with a basic feed without the test substance and observed for three months. 5.6.2.3 Ten days after weaning, F1a was fed with a basic feed without the test substance and observed for three months. After mating again, the offspring were F16. Among the 20 pregnant mice (F1), 5 were subjected to caesarean section 2 to 3 days before delivery to check whether the fetuses were deformed; the other 5 gave birth naturally to observe the condition of the offspring after delivery; 10 pregnant mice gave birth naturally, and the offspring F1 continued to breed.
5.6.2.4 After weaning, F1b was fed with feed containing the test substance for three months, and mated. The offspring F2a were fed with feed not containing the test substance after weaning and observed for three months.
5.6.2.5 Offspring F2. Ten days after weaning, the F1 was mated again. Before giving birth to F2b, the F1 pregnant mice were divided into two groups, each with 10, the same as 5.6.2.3. 5.6.3 Three-generation breeding test method
The schematic diagram of the three-generation breeding test method is shown in Figure 3.
GB15193.15—2003
Male rats (20 or 10)——Fo——Female rats (20) were fed with the test substance-containing feed for three months after weaning and mated. Fia was fed with the basic feed for three months after weaning and observed. F1a was mated for the second time 10 days after weaning. F1b was fed with the test substance-containing feed for three months after weaning and mated. Male rats
(20 or 10)
(20 or 10)
(20)
F2a was fed with the basic feed for three months after weaning and observed. 10 days after weaning, F2a and Fib were mated for the second time. F2b was fed with the test substance feed for three months after weaning, and then mated with female mice
(20 mice)
F3a was fed with the basic feed for three months and observed. 10 days after weaning, F2b was mated for the second time. F3b was fed with the test substance feed for 1 month after weaning, and observed. Figure 3 Schematic diagram of the three-generation breeding test method
5.7 Parental, first, second and third generation breeding tests 5.7.1 Parental, first, second and third generation breeding tests can be carried out with reference to 5.6.2.2 and 5.6.2.3. 5.7.2 More than two litters can be bred according to the situation. 6 Observation Index
6.1 General Observation: Perform a comprehensive clinical examination, record the general health status, all symptoms of toxicity and efficacy of the test substance, related behavioral changes, signs of difficult or delayed delivery, all toxicity indicators and mortality, and estimate the length and normal state of the sexual cycle by daily examination (F., F1 generation female mice) vaginal plug. 6.2 Weigh the body weight. The parent animals (P, F1 generation, and F2 generation, determined by the number of generations) are weighed on the first day of administration of the test substance, and then weighed weekly. The female mice should be weighed on the 0th, 7th, 14th and 21st days of pregnancy, and the litter weight of the pups should be weighed at the same time during the lactation period. 6.3 Before mating and during the pregnancy period, the food consumption should be weighed at least once a week. If the test substance is mixed in drinking water, the water consumption should be measured at least once a week.
6.4 At the end of the test, all parental (F.) and F1 generation (F generation, F3 generation, determined according to the number of generations) male mice should be examined for sperm attached to the larvae, and the activity, shape and number of sperm should be evaluated. The activity of sperm can be observed under a microscope; the sperm shape can only be examined for parental and offspring male mice in the control group and the high-dose group, and at least 200 sperms should be examined for each animal. 6.5 Offspring observation indicators: The number, sex, number of stillbirths, number of live births and visible abnormalities of each litter should be examined as soon as possible after delivery (0 days of lactation). For those who died on the day of birth, their defects and causes of death should be examined as much as possible. The number and sex of live births should be recorded, and each live birth should be weighed at birth (or as soon as possible). Thereafter, the weight should be weighed at least on the 4th, 7th, 14th and 21st days of lactation, when the vagina is open or the foreskin of the glans penis is separated, and at the end of the test. The age of vaginal opening or foreskin separation of F1 generation weaned mice used for mating should be recorded, and the sex ratio and sexual maturity should be observed.
GB15193.15-2003
6.6 Organ weighing: At the end of the experiment, all P and F1 generation parent animals should be weighed: uterus (including fallopian tubes and cervix), ovaries; testicles, epididymis (total weight of both sides); brain, liver, kidney, spleen and known target tissues. 6.7 Pathological examination: All parent animals that died at the end of the experiment and during the experiment should be grossly dissected and examined under a microscope to observe various morphological structural abnormalities and pathological changes, with special attention to reproductive organs. If the number of pups in each litter is sufficient, at least 3 mice of each sex in each litter of F1 generation, F2 generation (and F3 generation) should be taken for the same examination. The organs and tissues to be examined are: uterus, ovaries, testicles, epididymis, target organs (if the target organs are known), and abnormal tissues should be observed grossly. 7 Data processing
Conception rate (%) Number of pregnant animals/number of mated female animals × 100... Pregnancy rate (%) Number of animals giving birth to live offspring/number of pregnant animals × 100...
Live birth rate (%) Number of live offspring at birth/total number of offspring at birth × 100 (3) Birth survival rate (%) Number of offspring surviving 4 days after birth Number of live offspring at birth × 100·1....(49
Lactation survival rate (%) Number of offspring surviving at 21 days of weaning/number of offspring surviving 4 days after birth × 100... (5) Sex ratio = Number of male offspring at maturity/number of female offspring 8. Contents of the report
8.1 Test plan, test substance dose, absolute values of all values, complete data for each individual, data summary tables and analysis tables for each litter. 8.2 General health status, body weight, food intake, mortality, conception rate, pregnancy rate, live birth rate, sex ratio, birth survival rate, lactation survival rate (4-day and 21-day survival), total number of litters, uterine weight and average weight. 8.3 Discussion of all major parameters. When analyzing the results, comparison with historical controls can be made, but the experimental data of the historical controls should be provided, such as date, animal strain, test substance medium and route of administration of the test substance. 10115—2003
Male rats (20 or 10)——Fo——Female rats (20) were fed with the test substance-containing diet for three months after weaning and mated. Fia was fed with the basic diet for three months after weaning and observed. F1a was mated for the second time 10 days after weaning. F1b was fed with the test substance-containing diet for three months after weaning and mated. Male rats
(20 or 10)
(20 or 10)
(20)
F2a was fed with the basic diet for three months after weaning and observed. F2a was mated 1 0 days, Fib had a second mating. F2b was fed with the test substance feed for three months after weaning, and then mated with female mice
(20 mice)
F3a was fed with the basic feed for three months after weaning and observed. F3a was fed with the test substance feed for 1 month after weaning, and observed. Figure 3 Schematic diagram of the three-generation breeding test method
5.7 Parental, first, second and third generation breeding tests 5.7.1 Parental, first, second and third generation breeding tests can be carried out with reference to 5.6.2.2 and 5.6.2.3. 5.7.2 More than two litters can be bred according to the situation. 6 Observation Index
6.1 General Observation: Perform a comprehensive clinical examination, record the general health status, all symptoms of toxicity and efficacy of the test substance, related behavioral changes, signs of difficult or delayed delivery, all toxicity indicators and mortality, and estimate the length and normal state of the sexual cycle by daily examination (F., F1 generation female mice) vaginal plug. 6.2 Weigh the body weight. The parent animals (P, F1 generation, and F2 generation, determined by the number of generations) are weighed on the first day of administration of the test substance, and then weighed weekly. The female mice should be weighed on the 0th, 7th, 14th and 21st days of pregnancy, and the litter weight of the pups should be weighed at the same time during the lactation period. 6.3 Before mating and during the pregnancy period, the food consumption should be weighed at least once a week. If the test substance is mixed in drinking water, the water consumption should be measured at least once a week.
6.4 At the end of the test, all parental (F.) and F1 generation (F generation, F3 generation, determined according to the number of generations) male mice should be examined for sperm attached to the larvae, and the activity, shape and number of sperm should be evaluated. The activity of sperm can be observed under a microscope; the sperm shape can only be examined for parental and offspring male mice in the control group and the high-dose group, and at least 200 sperms should be examined for each animal. 6.5 Offspring observation indicators: The number, sex, number of stillbirths, number of live births and visible abnormalities of each litter should be examined as soon as possible after delivery (0 days of lactation). For those who died on the day of birth, their defects and causes of death should be examined as much as possible. The number and sex of live births should be recorded, and each live birth should be weighed at birth (or as soon as possible). Thereafter, the weight should be weighed at least on the 4th, 7th, 14th and 21st days of lactation, when the vagina is open or the foreskin of the glans penis is separated, and at the end of the test. The age of vaginal opening or foreskin separation of F1 generation weaned mice used for mating should be recorded, and the sex ratio and sexual maturity should be observed.
GB15193.15-2003
6.6 Organ weighing: At the end of the experiment, all P and F1 generation parent animals should be weighed: uterus (including fallopian tubes and cervix), ovaries; testicles, epididymis (total weight of both sides); brain, liver, kidney, spleen and known target tissues. 6.7 Pathological examination: All parent animals that died at the end of the experiment and during the experiment should be grossly dissected and examined under a microscope to observe various morphological structural abnormalities and pathological changes, with special attention to reproductive organs. If the number of pups in each litter is sufficient, at least 3 mice of each sex in each litter of F1 generation, F2 generation (and F3 generation) should be taken for the same examination. The organs and tissues to be examined are: uterus, ovaries, testicles, epididymis, target organs (if the target organs are known), and abnormal tissues should be observed grossly. 7 Data processing
Conception rate (%) Number of pregnant animals/number of mated female animals × 100... Pregnancy rate (%) Number of animals giving birth to live offspring/number of pregnant animals × 100...
Live birth rate (%) Number of live offspring at birth/total number of offspring at birth × 100 (3) Birth survival rate (%) Number of offspring surviving 4 days after birth Number of live offspring at birth × 100·1....(49
Lactation survival rate (%) Number of offspring surviving at 21 days of weaning/number of offspring surviving 4 days after birth × 100... (5) Sex ratio = Number of male offspring at maturity/number of female offspring 8. Contents of the report
8.1 Test plan, test substance dose, absolute values of all values, complete data for each individual, data summary tables and analysis tables for each litter. 8.2 General health status, body weight, food intake, mortality, conception rate, pregnancy rate, live birth rate, sex ratio, birth survival rate, lactation survival rate (4-day and 21-day survival), total number of litters, uterine weight and average weight. 8.3 Discussion of all major parameters. When analyzing the results, comparison with historical controls can be made, but the experimental data of the historical controls should be provided, such as date, animal strain, test substance medium and route of administration of the test substance. 10115—2003
Male rats (20 or 10)——Fo——Female rats (20) were fed with the test substance-containing diet for three months after weaning and mated. Fia was fed with the basic diet for three months after weaning and observed. F1a was mated for the second time 10 days after weaning. F1b was fed with the test substance-containing diet for three months after weaning and mated. Male rats
(20 or 10)
(20 or 10)
(20)
F2a was fed with the basic diet for three months after weaning and observed. F2a was mated 1 0 days, Fib had a second mating. F2b was fed with the test substance feed for three months after weaning, and then mated with female mice
(20 mice)
F3a was fed with the basic feed for three months after weaning and observed. F3a was fed with the test substance feed for 1 month after weaning, and observed. Figure 3 Schematic diagram of the three-generation breeding test method
5.7 Parental, first, second and third generation breeding tests 5.7.1 Parental, first, second and third generation breeding tests can be carried out with reference to 5.6.2.2 and 5.6.2.3. 5.7.2 More than two litters can be bred according to the situation. 6 Observation Index
6.1 General Observation: Perform a comprehensive clinical examination, record the general health status, all symptoms of toxicity and efficacy of the test substance, related behavioral changes, signs of difficult or delayed delivery, all toxicity indicators and mortality, and estimate the length and normal state of the sexual cycle by daily examination (F., F1 generation female mice) vaginal plug. 6.2 Weigh the body weight. The parent animals (P, F1 generation, and F2 generation, determined by the number of generations) are weighed on the first day of administration of the test substance, and then weighed weekly. The female mice should be weighed on the 0th, 7th, 14th and 21st days of pregnancy, and the litter weight of the pups should be weighed at the same time during the lactation period. 6.3 Before mating and during the pregnancy period, the food consumption should be weighed at least once a week. If the test substance is mixed in drinking water, the water consumption should be measured at least once a week.
6.4 At the end of the test, all parental (F.) and F1 generation (F generation, F3 generation, determined according to the number of generations) male mice should be examined for sperm attached to the larvae, and the activity, shape and number of sperm should be evaluated. The activity of sperm can be observed under a microscope; the sperm shape can only be examined for parental and offspring male mice in the control group and the high-dose group, and at least 200 sperms should be examined for each animal. 6.5 Offspring observation indicators: The number, sex, number of stillbirths, number of live births and visible abnormalities of each litter should be examined as soon as possible after delivery (0 days of lactation). For those who died on the day of birth, their defects and causes of death should be examined as much as possible. The number and sex of live births should be recorded, and each live birth should be weighed at birth (or as soon as possible). Thereafter, the weight should be weighed at least on the 4th, 7th, 14th and 21st days of lactation, when the vagina is open or the foreskin of the glans penis is separated, and at the end of the test. The age of vaginal opening or foreskin separation of F1 generation weaned mice used for mating should be recorded, and the sex ratio and sexual maturity should be observed.
GB15193.15-2003
6.6 Organ weighing: At the end of the experiment, all P and F1 generation parent animals should be weighed: uterus (including fallopian tubes and cervix), ovaries; testicles, epididymis (total weight of both sides); brain, liver, kidney, spleen and known target tissues. 6.7 Pathological examination: All parent animals that died at the end of the experiment and during the experiment should be grossly dissected and examined under a microscope to observe various morphological structural abnormalities and pathological changes, with special attention to reproductive organs. If the number of pups in each litter is sufficient, at least 3 mice of each sex in each litter of F1 generation, F2 generation (and F3 generation) should be taken for the same examination. The organs and tissues to be examined are: uterus, ovaries, testicles, epididymis, target organs (if the target organs are known), and abnormal tissues should be observed grossly. 7 Data processing
Conception rate (%) Number of pregnant animals/number of mated female animals × 100... Pregnancy rate (%) Number of animals giving birth to live offspring/number of pregnant animals × 100...
Live birth rate (%) Number of live offspring at birth/total number of offspring at birth × 100 (3) Birth survival rate (%) Number of offspring surviving 4 days after birth Number of live offspring at birth × 100·1....(49
Lactation survival rate (%) Number of offspring surviving at 21 days of weaning/number of offspring surviving 4 days after birth × 100... (5) Sex ratio = Number of male offspring at maturity/number of female offspring 8. Contents of the report
8.1 Test plan, test substance dose, absolute values of all values, complete data for each individual, data summary tables and analysis tables for each litter. 8.2 General health status, body weight, food intake, mortality, conception rate, pregnancy rate, live birth rate, sex ratio, birth survival rate, lactation survival rate (4-day and 21-day survival), total number of litters, uterine weight and average weight. 8.3 Discussion of all major parameters. When analyzing the results, comparison with historical controls can be made, but the experimental data of the historical controls should be provided, such as date, animal strain, test substance medium and route of administration of the test substance. 1012 General health status, body weight, food intake, mortality, conception rate, pregnancy rate, live birth rate, sex ratio, birth survival rate, lactation survival rate (4-day and 21-day survival), total number of litters, uterine weight and average weight. 8.3 Discuss all major parameters. When analyzing the results, you can compare with historical controls, but the experimental data of historical controls should be provided, such as date, animal strain, test medium and route of administration of the test substance. 1012 General health status, body weight, food intake, mortality, conception rate, pregnancy rate, live birth rate, sex ratio, birth survival rate, lactation survival rate (4-day and 21-day survival), total number of litters, uterine weight and average weight. 8.3 Discuss all major parameters. When analyzing the results, you can compare with historical controls, but the experimental data of historical controls should be provided, such as date, animal strain, test medium and route of administration of the test substance. 101
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