This standard specifies the basic technical requirements for the micronucleus test. This standard is applicable to the evaluation of the mutagenic effects of chemical, biological and physical factors that may cause harm to health 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. This standard does not apply to situations where there is evidence that the test substance or its metabolites cannot reach the target tissue. GB 15193.5-2003 Bone marrow cell micronucleus test GB15193.5-2003 standard download decompression password: www.bzxz.net

ICS07.100
National Standard of the People's Republic of China
GB15193.5—2003
Replaces GB15193.5-1994
Bone marrow cell micronucleus test
Bone marrow cell micronucleus test2003-09-24 Issued
Ministry of Health of the People's Republic of China
Standardization Administration of the People's Republic of China
Implementation on 2004-05-01
GB15193.5—2003
This standard is mandatory in its entirety.
This standard replaces GB15193.5—1994 "Bone marrow micronucleus test". Compared with GB15193.5-1994, this standard has been modified as follows: First, the specific content of the test substance 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.; the scope of inapplicability has been added; in the "dose grouping content": the design method of the high-dose group has been added as follows: "When the maximum dose (maximum use concentration and maximum gavage capacity) of the test substance given in the acute toxicity test cannot determine the LD50, the high-dose group shall be designed in the following order: a) 10 g/kg body weight; b) 100 times the possible human intake; or c) a maximum gavage dose at a time"; First, the "result evaluation" has been uniformly changed to "result determination", and "the micronucleus rate of the general negative control group is <5%o, for reference" has been added. However, the spontaneous micronucleus rate of experimental animals used in this laboratory should be used as a reference". From the date of implementation of this standard, GB15193.5-1994 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. The drafting units of this standard are: Institute of Nutrition and Food Safety, Chinese Center for Disease Control and Prevention, Zhejiang Provincial Health and Epidemic Prevention Station. The main drafters of this standard are: Xu Fengdan, Han Chi, Zhao Shuo. This standard was first issued in 1994 and this is the first revision. 46
1 Scope
Bone marrow cell micronucleus test
This standard specifies the basic technical requirements for the micronucleus test. GB 15193.52003
This standard is applicable to the evaluation of the mutagenic effects of chemical, biological and physical factors that may cause harm to health 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. wwW.bzxz.Net
This standard does not apply to situations where there is evidence that the test substance or its metabolites cannot reach the target tissue. 2 Principle
Micronuclei are chromatids or chromosomes that remain in the cytoplasm when chromosomes regularly enter daughter cells to form cell nuclei in the late stage of cell mitosis. After the late stage, it forms one or several regular secondary nuclei alone and is contained in the cytoplasm of the cell. Because it is much smaller than the nucleus, it is called micronucleus. This situation often occurs as a result of the action of chromosome clastogens. In addition, it is also possible that when the spindle poison is applied, the main nucleus cannot be formed and is replaced by a group of small nuclei. In this case, the small nuclei are often slightly larger than typical micronuclei.
3 Instruments and reagents
3.1 Instruments
Dissecting instruments, biological microscopes, glass slides, etc. 3.2 Reagents
All reagents are analytical grade unless otherwise specified, and the test water is distilled water. 3.2.1 Calf serum: After filtering the calf serum, place it in a 56°C constant temperature water bath for 1 hour to inactivate it. Usually Store in a 4℃ refrigerator. Rat and mouse serum can also be used instead.
3.2.2 Giemsa stain: Weigh 3.8g Giemsa stain, add 375mL methanol (analytical grade) and grind. After it is completely dissolved, add 125mL glycerol. Keep it in a 37℃ constant temperature box for 48h and shake it several times. Filter it and use it after two weeks. 3.2.3 Giemsa application solution: Mix 1 part of Giemsa stain with 6 parts of phosphate buffer. Prepare it before use. 3.2.41/15 mol/L phosphate buffer (pH 6.8) potassium dihydrogen phosphate (KH2PO4)
sodium dihydrogen phosphate (NazHPO4·12H2O) add distilled water to
3.2.5 methanol (analytical grade).
4 experimental animals
1 000 mL
mice are the conventional animals for micronucleus test, and rats can also be used. Usually, mice aged 7 to 12 weeks and weighing 25g to 30g or rats weighing 150g to 200g are used. Each group uses at least 5 animals of both sexes. Animals are allowed to adapt to the environment for at least 3 days after purchase. 5 dose and grouping
the test substance should be divided into three dose groups. In principle, the highest dose group is the dose at which animals show severe poisoning and/or individual animals die. Generally, 1/2LD5o can be taken. The low dose group should not show toxicity, and 1/4LD5o and 1/8LD5o are taken as the medium and low doses respectively. Acute toxicity test 47
GB15193.5-2003
When the animal does not die at the maximum dose (maximum concentration and maximum oral gavage volume) of the test substance and the LDso cannot be calculated, the high-dose group is designed in the following order: a) 10g/kg body weight; b) 100 times the possible human intake; or c) a maximum oral gavage dose, and then the medium and low dose groups are set up. A solvent control group and a positive control group are also set up. The positive control can be given orally or intraperitoneally (preferably orally) with cyclophosphamide 40mg/kg body weight.
6 Operation steps
6.1 Preparation of test substances
Generally, distilled water is used as the solvent. If the test substance is insoluble in water, edible 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 showing that it is stable when stored as a solution (or suspension, emulsion, etc.). 6.2 Treatment of experimental animals
Oral gavage. According to the cell cycle and the characteristics of the action of different substances, a preliminary test can be conducted to determine the time of sampling. The 30-hour test substance administration method is commonly used. That is, the test substance is administered twice with an interval of 24 hours. 6 hours after the second administration of the test substance, the animal is killed by cervical dislocation. 6.3 Specimen preparation
Take the sternum or femur, squeeze out the bone marrow fluid with hemostatic forceps and mix it with calf serum at one end of the glass slide, and make a conventional smear, or use calf serum to rinse the femoral bone marrow cavity to make a cell suspension smear. After the smear is naturally dried, it is placed in methanol for fixation for 5min~10min. It is fixed and stored on the same day. The fixed smear is placed in Giemsa application solution and stained for 10min~15min. Immediately rinse with pH6.8 phosphate buffer or distilled water and dry. Write a label and store in a cool and dry place. 6.4 Slide reading
Select the area with complete, evenly dispersed cells and appropriate coloring, and observe under the oil microscope. The morphology of nucleated cells is intact as the criterion for judging the quality of the film.
This method is to observe the micronuclei of polychromatic erythrocytes. Using the Giemsa staining method, polychromatic erythrocytes are gray-blue, and mature erythrocytes are royal pink. Typical micronuclei are mostly single, round, with smooth and neat edges, and the color is consistent with the nucleoplasm, purple-red or blue-purple, and the diameter is usually 1/20 to 1/5 of the erythrocyte.
The films are read in a double-blind method. Count 1000 polychromatic erythrocytes for each animal, observe the number of polychromatic erythrocytes containing micronuclei, and the micronucleus rate is expressed in thousandths.
Observe the ratio of polychromatic erythrocytes to mature erythrocytes (PCE/RBC), which can be used as one of the cytotoxicity indicators. Generally, 200 polychromatic erythrocytes are counted. The proportion of immature erythrocytes to the total number of erythrocytes in the test group should not be less than 20% of that in the control group. 7 Data processing
Generally, statistical methods such as chi-square test, Poisson distribution, or two-sided t-test are used for data processing, and statistics are separated by animal gender. 8 Result determination
When the micronucleus rate of the test group is compared with the control group, it can be confirmed as a positive result when there is an obvious dose-response relationship and statistical significance. If the difference is statistically significant but there is no dose-response relationship, the test must be repeated. Results that can be repeated can be determined as positive. Generally, the micronucleus rate of the negative control group is <5%, for reference. However, the spontaneous micronucleus rate of the experimental animals used in this laboratory should be used as a reference. 48
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