Some standard content:
ICS 07. 100
National Standard of the People's Republic of China
GB15193.17—2003
Replaces GB15193.17--1994
Chronic toxicity and carcinogencity study
Chronic toxicity and carcinogencity study2003-09-24Promulgated
Ministry of Health of the People's Republic of China
Standardization Administration of the People's Republic of China
Implementation on 2004-05-01
GB 15193.17—2003
This standard is mandatory in its entirety.
This standard replaces GB15193.17-1994 "Chronic toxicity and carcinogencity study". Compared with GB15193.17-1994, this standard has been modified as follows: First, the specific content of the test objects has been added in the "Scope": 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.; First, in "Experimental Animals": "Selection of Animal Species", "Starting Age and Weight of Animals", "Tumor Spontaneous Rate", "Sex and Number of Animals" and other corresponding items have been added. - The content of "Selection of Animal Species" was revised to "Rats are used for chronic toxicity tests on rodents, and mice and rats are used for carcinogenicity tests"; - The "number of groups" and "number of animals" in the original standard "Grouping" were merged and changed to "Dosage and Grouping", and "The design of the high-dose group is determined based on a 90-day feeding test, and the low-dose group does not cause any toxic effects" and "If the test substance uses a medium of unknown toxicity, an untreated control and a medium (vehicle) control should be set up at the same time"; in "Operational Procedures": feed, feeding management, test substances, experimental period, observation and testing of test animals, pathological examination were added, and the content of "administration method of the test substance and preparation of the test substance" was added. —In “Observation indicators”:
1) Blood biochemical test: changed to alanine aminotransferase (ALT or SGPT), aspartate aminotransferase (AST or SGOT), urea nitrogen (BUN), creatinine (Cr), blood glucose (Glu), serum albumin (Alb), total protein (TP), total cholesterol (TCH) and triglycerides (TG) are all mandatory indicators. In addition, alkaline phosphatase (AI.P), lactate dease (I.DH), bile acid and other indicators can also be considered.
2) Gross examination: add “measurement of the absolute weight and organ-to-body ratio of important organs, including at least the liver, kidney, adrenal gland, spleen, testis, epididymis, ovary, uterus, brain, heart and other organs. Other organs should also be selected if necessary. "3) Biological microscope examination: add digestive system, nervous system, glands, respiratory system, cardiovascular system and facial system, urinary and reproductive system and others (all tissues, masses, skin, etc. that are damaged by gross observation). From the date of implementation of this standard, GB15193.17-1994 will be abolished at the same time. This standard is proposed by the Ministry of Health of the People's Republic of China and is under the jurisdiction of the Institute of Nutrition and Food Safety of China Center for Disease Control and Prevention and Shanghai Medical University. The main drafters of this standard are Dai Yin, Xu Jinkang and Yan Wen. This standard was first issued in 1994 and this is the first revision. 110
1 Scope
Chronic toxicity and carcinogenicity test
This standard specifies the basic technical requirements for chronic toxicity and carcinogenicity tests. GB15193.17-2003
This standard is applicable to the evaluation of food production, The chronic toxicity and/or carcinogenicity of chemical, biological and physical factors involved in the processing, preservation, transportation and sales that may cause harm to health. The inspection 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. If necessary, the two can be combined. 2 Terms and Definitions
The following terms and definitions apply to this standard
Maximum no-observed-adverse-effect-level, NOAEL is the maximum dose at which no toxic effects related to the test substance are observed through animal experiments using existing technical means and detection indicators. 2.2
Target organtarget organ
Any organ in which the test substance causes significant toxic effects in experimental animals. 2.3
Carcinogenicity
Carcinogenicity
The development of tumors in experimental animals caused by repeated daily exposure to the test substance. 2.4
Chronic toxicity toxicity
Adverse effects of long-term and daily repeated exposure of experimental animals to the test substance 3 Principle
During most of the animal's life, the chronic toxic effects and their dose-response relationship are observed after repeated administration of the test substance, especially the progressive irreversible toxic effects and tumor diseases. The no observed adverse effect level (NOAEL) of the test substance is determined as the basis for the final assessment of whether the test substance can be used in food. 4 Experimental animals
4.1 Selection of animal species
In principle, experimental animals with metabolic characteristics close to those of humans should be selected, because the characteristics of various strains of rats and mice and their sensitivity to inducing tumors have been mastered, and they can be used preferentially in chronic toxicity and carcinogenicity tests; rats are used as rodents for chronic toxicity tests, and rats and mice are used for carcinogenicity tests. For test substances with unknown activity, rodents and non-rodents of both sexes should be used. 4.2 Starting age and weight of animals
Generally, weaned rats or mice of both sexes are used. The range of variation in the weight of individual animals should not exceed 20% of the average weight of each sex. bzxZ.net
4.3 Spontaneous tumor rate
The principle of natural tumor incidence of experimental animals is to control it as low as possible. However, at the end of the test, the evaluation is mainly based on the observation of the tumor incidence of the control group and each dose group under the same conditions and the dose-response relationship to determine its carcinogenicity. 111
GB15193.17—2003
4.4 Gender and number of animals
Each group shall have at least 50 animals, half of which are male and half are female. Female mice shall be non-parous and non-pregnant. There shall be at least 4 animals of each sex in each group of non-rodents. If it is planned to perform regular slaughter during the test, the number of animals shall be increased accordingly. When chronic toxicity and carcinogenicity tests are conducted together, it is advisable that each group of animals shall have more than 50 male and female animals. If it is planned to perform regular slaughter during the test, the number of animals shall be increased accordingly. 5 Dosage and grouping
Except for the control group, the general test group can be divided into 3 to 5 groups. The high dose should cause some toxic manifestations or damage, but will not affect its normal growth, development and life span. The design of the high dose group is determined based on a 90-day feeding test, and the low dose group does not cause any toxic effects. The control group should be the same as the test group in all aspects except that the test substance is not given. If the test substance uses a medium of unknown toxicity, an untreated control and a medium (vehicle) control should be set up at the same time. The dose of the test group can be divided according to geometric series or other rules. 6 Operation steps
6.1 Administration of the test substance
Oral administration can be added to feed, drinking water or gavage. If the test substance is given by gavage, the body weight should be weighed twice a week, and the volume of the test substance administered should be calculated based on the body weight.
6.2 Preparation and storage of test substances
6.2.1 Distilled water is generally used as a solvent. If the test substance is insoluble in water, edible vegetable oil, medical starch, carboxymethyl cellulose, etc. can be used to prepare an emulsion or suspension. The test substance should be freshly prepared before gavage, 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 physical and chemical properties and the toxic characteristics caused by them; the effects on the food intake or water intake or the nutritional status of the animal. 6.2.2 The amount of non-nutritional test substances added to the feed should not exceed 5% of the feed amount; the nutritional test substances should be used at a high dose as much as possible, and the nutritional balance of the experimental animals should be ensured or the feeding method should be adopted. 6.2.3 When preparing or storing the test substance, it is required not to affect the nutritional content and properties of the feed. 6.2.4 When the amount of the test substance added to the feed is very small, it is advisable to first add the test substance to a small amount of feed and mix thoroughly, then add a certain amount of feed and mix again, and repeat this process 3 to 4 times. 6.3 Feed
6.3.1 The nutritional components in the feed should be able to meet the nutritional needs of the experimental animals. 6.3.2 The content of pollutants in feed, such as residual pesticides, polycyclic aromatic hydrocarbons, estrogens, heavy metals, nitrosamine compounds, etc., should be controlled; the content of unsaturated fatty acids and selenium should be limited, and they should not affect the test results of the test substance. 6.4 Feeding management
In addition to referring to the requirements for experimental animals and feeds in food toxicology tests, it is also necessary to do the following: a) Two types of experimental animals shall not be placed in the same animal room, and toxicity tests of two test substances cannot be carried out at the same time; b) Disinfectants and pesticides and other drugs shall not be used; c) The feed in the animal feed tank shall be replaced at least twice a week. 6.5 Test period
6.5.1 In general, the test period of carcinogenicity test is set at 18 months for mice and 24 months for rats; it can be appropriately extended for individual animals with longer life spans and lower spontaneous tumor rates.
6.5.2 During the test period, when the number of surviving animals in the lowest dose group or control group is only 25% of the number at the beginning, the test can be terminated in time; however, if the animals in the high dose group die prematurely due to obvious toxic effects of the test substance, the test should be continued; if the animal death rate caused by poor management is greater than 10% and the survival rate of each group is less than 50% when the test period is 18 months for mice or 24 months for rats, the test should also be terminated. 6.6 Observation indicators
6.6.1 General observation of experimental animals
6.6.1.1 The general health status of experimental animals should be carefully observed and recorded at least once a day. Dead animals should be dissected in time; sick or dead animals should be placed separately or killed, and various indicators should be tested. GB15193.17--2003
6.6.1.2 If animals show abnormalities, detailed records of what is seen with the naked eye, the nature of the lesions, time, location, size, appearance and development, etc., and a detailed description of animals that are dying.
6.6.1.3 During the first 13 weeks of the test period, i.e. the first three months, all animals should be weighed every week, and then every four weeks. The amount of feed consumed by each animal should be checked and recorded every week. If there is no abnormal change in health status or weight, it can be checked every three months. 6.6.2 Hematological examination
Hemoglobin, hematocrit, red blood cell count, white blood cell count and classification, platelet and blood coagulation tests, etc. should be checked routinely in the third and sixth months of the test and every six months thereafter. Ten animals of each sex should be checked in each group of mice and rats, and the same animal should be arranged as much as possible for each examination. All non-rodents should be checked.
6.6.3 Blood biochemical test
It shall be carried out at the time specified in 6.6.2. The indicators include: alanine aminotransferase (ALT or SGPT), aspartate aminotransferase (AST or SGOT), urea nitrogen (BUN), creatinine (Cr), blood glucose (Glu), serum albumin (AIb), total protein (TP), total cholesterol (TCH) and triglycerides (TG), which are all mandatory indicators. In addition, alkaline phosphatase (ALP), lactate dehydrogenase (IDH), bile acid, etc. may also be considered. 6.6.4 Pathological examination
6.6.4.1 Gross examination
6.6.4.1.1 All experimental animals, including those that died or were dying during the experiment and those that were killed at the end of the experiment, shall be dissected and comprehensively and systematically observed with the naked eye. Samples of suspicious lesions and tumor sites shall be kept for further histological examination. 6.6.4.1.2 Determine the absolute weight and organ-to-body ratio of important organs, including at least the liver, kidney, adrenal gland, spleen, testis, epididymis, ovary, uterus, brain, heart, etc. Other organs should also be selected when necessary. 6.6.4.2 Biological microscope examination
Biological microscope examination is the main mandatory inspection item of chronic toxicity test, and it should be carried out on all animals that are moribund during the test. The organs and tissues that should be preserved for further testing when necessary include: digestive system: esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, pancreas, liver; nervous system: brain, pituitary gland, peripheral nerves, spinal cord, eyes; glands: adrenal glands, thyroid gland (including parathyroid glands), thymus gland; respiratory system: trachea, lungs, pharynx, larynx, nose; cardiovascular system and hematopoietic system: aorta, heart, bone marrow, lymph nodes, spleen; urinary and reproductive system: kidney, bladder, prostate, testis, epididymis, seminal vesicle, uterus, ovary, mammary gland of female rats; others: all tissues, masses, and skin that show damage in gross observation. 6.6.4.3 Electron microscopic examination
It can be carried out as appropriate when conditions permit and when necessary.
6.7 Data collection
Daily observations on animals, feed, feeding, weather, personnel changes, and situations occurring during the experiment should be recorded in detail and properly preserved. It is necessary to accumulate tumor occurrence data of commonly used animals to provide a basis for the future formulation of corresponding natural tumor incidence rates. 7 Data processing
According to the test materials and data of each stage, statistical analysis should be conducted after the data are summarized; the differences in various indicators between the control group and the animals in each dose group should be fully and accurately described to demonstrate their toxic effects. Data processing should be carried out according to relevant statistical methods. 8 Test report
The report should explain the design, toxicity performance, and evaluation of the feasibility of the test substance for consumption. 1132 Biological microscope examination
Biological microscope examination is the main mandatory inspection item of chronic toxicity test, and it should be carried out on all animals that are moribund during the test. The organs and tissues that should be preserved for further inspection when necessary include: digestive system: esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, pancreas, liver; nervous system: brain, pituitary gland, peripheral nerves, spinal cord, eyes; glands: adrenal glands, thyroid glands (including parathyroid glands), thymus glands; respiratory system: trachea, lungs, pharynx, larynx, nose; cardiovascular system and hematopoietic system: aorta, heart, bone marrow, lymph nodes, spleen; urinary and reproductive system: kidney, bladder, prostate, testicle, epididymis, seminal vesicle, uterus, ovary, mammary gland of female mice; others: all tissues, masses, and skin that are damaged by gross observation. 6.6.4.3 Electron microscope examination
It can be carried out as appropriate when conditions permit and it is necessary.
6.7 Data collection
Daily observations on animals, feed, feeding, weather, personnel changes and situations occurring during the experiment should be recorded in detail and properly preserved. Tumor occurrence data of commonly used animals should be accumulated to provide a basis for the formulation of corresponding natural tumor incidence rates in the future. 7 Data processing
Statistical analysis should be conducted after the experimental data and data of each stage are summarized; the differences in various indicators between the control group and the animals in each dose group should be fully and accurately described to demonstrate their toxic effects. Data processing should be carried out according to relevant statistical methods. 8 Experimental report
The report should explain the design, toxicity performance, and evaluation of the feasibility of the test substance for consumption. 1132 Biological microscope examination
Biological microscope examination is the main mandatory inspection item of chronic toxicity test, and it should be carried out on all animals that are moribund during the test. The organs and tissues that should be preserved for further inspection when necessary include: digestive system: esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, pancreas, liver; nervous system: brain, pituitary gland, peripheral nerves, spinal cord, eyes; glands: adrenal glands, thyroid glands (including parathyroid glands), thymus glands; respiratory system: trachea, lungs, pharynx, larynx, nose; cardiovascular system and hematopoietic system: aorta, heart, bone marrow, lymph nodes, spleen; urinary and reproductive system: kidney, bladder, prostate, testicle, epididymis, seminal vesicle, uterus, ovary, mammary gland of female mice; others: all tissues, masses, and skin that are damaged by gross observation. 6.6.4.3 Electron microscope examination
It can be carried out as appropriate when conditions permit and it is necessary.
6.7 Data collection
Daily observations on animals, feed, feeding, weather, personnel changes and situations occurring during the experiment should be recorded in detail and properly preserved. Tumor occurrence data of commonly used animals should be accumulated to provide a basis for the formulation of corresponding natural tumor incidence rates in the future. 7 Data processing
Statistical analysis should be conducted after the experimental data and data of each stage are summarized; the differences in various indicators between the control group and the animals in each dose group should be fully and accurately described to demonstrate their toxic effects. Data processing should be carried out according to relevant statistical methods. 8 Experimental report
The report should explain the design, toxicity performance, and evaluation of the feasibility of the test substance for consumption. 113
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