This standard specifies the basic technical requirements for 30-day or 90-day feeding tests. This standard is applicable to the evaluation of the harmful effects of chemical, biological and physical factors that may cause health hazards to animals during the production, processing, storage, transportation and sales of food. 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. GB 15193.13-2003 30-day and 90-day feeding tests GB15193.13-2003 Standard download decompression password: www.bzxz.net

ICS 07.100
National Standard of the People's Republic of China
GB15193.13—2003
Replaces GB15193.13—1994
30-day and 90-day feeding study
Thirty and ninety days feeding 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 15193.13—2003
This standard is mandatory in its entirety. www.bzxz.net
This standard replaces GB15193.13-1994 "30-day and 90-day feeding study". Compared with GB15193.13-1994, this standard has the following major revisions: In the "scope", the specific content of the test substances is 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.: - "Animal selection" is uniformly changed to "experimental animals", and "preferred rats" is changed to "rats"; - In the dose and grouping: add a 30-day feeding test that should cover dose groups that are 100 times the possible human intake as much as possible. For test substances with a large human intake, the high-dose group can be designed according to the maximum tolerated dose. The dosage of the 90-day feeding test shall be determined based on the results of the 30-day feeding test";
-- "Test methods" shall be uniformly changed to "Operation procedures", and the following content shall be added to "Operation procedures": "When gavage is performed, the volume shall generally not exceed 1 mL/(100 g body weight·day), and the gavage volume of each dose group shall be consistent. The time points of gavage every day shall be similar" and the animals shall be kept in single cages";
--- In the observation indicators: "spleen and testicles" shall be added to the organs that should be weighed, "spleen, testicles and ovaries" shall be added to the organs that should be subjected to pathological histological examination, and "if necessary, other sensitive indicators may be added according to the nature of the test substance and the toxic reactions observed" shall be added; in the histopathological examination: "when no obvious lesions are found in the gross examination of the animals in each dose group" shall be changed to "when no obvious lesions are found in the gross examination of the animals in each dose group and no abnormal changes are found in the biochemical indicators". GB15193.13-1994 shall be abolished from the date of implementation of this standard. This standard is proposed and managed by the Ministry of Health of the People's Republic of China. This standard was drafted by: Institute of Nutrition and Food Safety, Chinese Center for Disease Control and Prevention, Beijing Municipal Health and Epidemic Prevention Station. The main drafters of this standard: Dai Yin, Xu Jinkang, Yao Xiaoman. This standard was first issued in 1994 and this is the first revision. 86
1 Scope
30-day and 90-day feeding test
This standard specifies the basic technical requirements for 30-day or 90-day feeding test. GB15193.13—2003
This standard is applicable to the evaluation of the harmful effects of chemical, biological and physical factors that may cause harm to health on animals during the production, processing, storage, transportation and sales of food. 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.
2 Terms and definitions
The following terms and definitions apply to this standard. 2.1
No-observed-adverse-effect-level, NOAEL The maximum dose at which no toxic effects related to the test substance are observed by existing technical means and detection indicators through animal experiments. 2.2
Target organ
Any organ in which the test substance causes obvious toxic effects in experimental animals. 3 Principle
When evaluating the toxic characteristics of a test substance, after understanding the purity, solubility characteristics, stability and other physical and chemical properties of the test substance and the preliminary data on toxicity, a 30-day or 90-day feeding test can be conducted to propose the dose, toxicity and target organ of the test substance that causes adverse effects on animals when fed with different doses for a longer period of time, and to estimate the hazard of subchronic intake. The maximum no-observed-adverse-effect dose determined by the 90-day feeding test can provide a basis for the dose selection and observation indicators of chronic tests. When the maximum no-observed-adverse-effect dose reaches a certain multiple of the possible intake of humans, it can be extrapolated to humans based on this basis to provide a basis for determining the safe dose for human consumption. 4 Experimental animals
Select animal species and strains that have been proven to be sensitive to the test substance in acute toxicity tests. Generally, rodent rats are selected. In order to observe the effects of the test substance on growth and development, weaned rats of both sexes (4 weeks after birth) are used. The difference in animal weight at the beginning of the test should not exceed ±20% of the average weight.
5 Dosage and grouping
At least three dose groups and one control group should be set up. Each dose group should have at least 20 animals, 10 females and 10 males. In principle, animals in the high-dose group should show obvious poisoning symptoms during feeding the test substance but not cause death or serious damage, and the low-dose group should not cause toxic effects. Estimate or determine the maximum dose without observed adverse effects. Set one or more dose groups between these two doses in order to obtain a clearer dose-response relationship. The design of dosage can refer to the following principles: 5.1 For test substances that can obtain LDso: 10% to 25% of LDso is used as the highest dose group for 30-day or 90-day feeding test. The selection of this LDsc percentage mainly refers to the slope of the LDso dose-response curve. Then set up several dose groups at this dose, and the lowest dose group is at least 3 times the possible human intake.
5.2 For test substances that cannot obtain LDso: 30-day feeding test should cover dose groups that are 100 times the possible human intake as much as possible. For test substances with a large human intake, the high-dose group can be designed according to the maximum tolerated dose. The 90-day feeding test determines the dose based on the results of the 30-day feeding test, or takes 100 to 300 times the possible human intake as the maximum no-observed-adverse-effect dose, and then sets up several dose groups above this dose, and if necessary, sets up dose groups below this dose. 6 Operation steps
6.1 Method of administering the test substance
Preferably, the test substance should be mixed with feed for feeding (pay attention to the stability of the test substance in feed). If there are difficulties, it can also be added to drinking water or gavage. Animals are kept in single cages. When the test substance is mixed with feed, the test substance dose should be converted into feed amount (mg/kg) based on the intake per 100g body weight. For a 30-day feeding test, it should be converted to 10% of body weight, and for a 90-day feeding test, it should be converted to 8% of body weight. 6.2 Gavage volume
When gavage is performed, the volume should generally not exceed 1mL/(100g body weight·day). The gavage volume of each dose group should be consistent. The time point of gavage should be similar every day.
6.3 Observation indicators
Due to differences in the test substance and research purpose, the following items can generally be included: 6.3.1 General observation: Observe and record the general performance, behavior, poisoning symptoms and death of the animals every day. Weigh body weight once a week and food intake twice a week, calculate weekly and total food utilization. These are all items that must be observed and measured. 6.3.2 Hematological indices: Determine hemoglobin, red blood cell count, white blood cell count and classification, and determine platelet count and reticulocyte count, etc., if necessary, according to the test substance. 30-day feeding is generally measured once at the end of the experiment, and 90-day feeding is generally measured once in the middle and at the end of the experiment.
6.3.3 Blood biochemical indices: ALT or SGPT, AST or SGOT, urea nitrogen (BUN), creatinine (Cr), blood sugar (Glu), serum albumin (Alb), total protein (TP), total cholesterol (TCH) and triglycerides (TG) are all mandatory indicators.
6.3.4 Pathological examination:
6.3.4.1 Gross anatomy: At the end of the experiment, all animals must be subjected to a gross examination, and important organs and tissues must be fixed and preserved. 6.3.4.2 Organ weighing: The absolute weight and relative weight (organ/body ratio) of the liver, kidney, spleen, and testicles are mandatory indicators. Other organs may be weighed if necessary.
6.3.4.3 Histopathological examination: When no obvious lesions are found in the gross examination of the animals in each dose group and no abnormal changes are found in the biochemical indicators, only the histopathological examination of the main organs of the animals in the highest dose group and the control group can be carried out. After the lesions are found, the corresponding organs and tissues of the lower dose groups can be examined. Histopathological examination of the liver, kidney, spleen, gastrointestinal tract, testis and ovary is a mandatory test item. The examination of other tissues and organs needs to be determined according to different situations. 6.3.5 Other indicators: When necessary, other sensitive indicators can be added according to the properties of the test substance and the observed toxic reactions. 7 Data processing
All observed results, whether count data or measurement data, should be evaluated by appropriate statistical methods. The statistical method to be used should be selected when the experiment is designed. Analysis of variance or t-test is used for measurement data, and Chi-square test, Poisson distribution, etc. are used for count data. 88
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