Some standard content:
ICS 07.100
National Standard of the People's Republic of China
GB15193.6—2003
Replaces GB15193.6—1994
Mammalian bone marrow cell chromosome aberration test
Issued on September 24, 2003
Ministry of Health of the People's Republic of China
Standardization Administration of the People's Republic of China
Implemented on May 1, 2004
GB15193.6—2003
This standard is mandatory in its entirety.
This standard replaces GB15193.6-1994 "Bone marrow cell chromosome aberration test". Compared with GB15193.6-1994, the main changes of this standard are as follows: the standard name "Bone marrow cell chromosome aberration test" is changed to "Mammalian bone marrow cell chromosome aberration test"; the specific content of the test objects is 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 is added; the relevant contents under the title "5.1 Animal Handling" in the original operating procedures are divided into three chapters: "Test Animals", "Dosage and Grouping" and "Operation Procedures";
- Add a chapter title " - Test animals”, change “take healthy adult rats and mice” to “normally use healthy young adult rats or mice”; - Add the chapter title “Dosage and grouping”, and add a sentence to the design method of high-dose group: “When the maximum dose (maximum concentration and maximum oral gavage volume) of the test substance in the acute toxicity test cannot be used to obtain the I.Dss, the high-dose group shall be designed in the following order: a) 10g/kg body weight; b) 100 times the possible human intake; or c) a single maximum oral gavage dose, and add the reference positive control “mitomycin C (1.5mg/kg body weight to 2.0mg/kg body weight), or cyclophosphamide (40mg/kg body weight); - Put the original “5.1.2\Contents other than dose design” under the added title “Operation steps”, and add the relevant content of “Preparation of test substance” under this title;
Add the content of “Result judgment”.
From the date of implementation of this standard, GB15193.6-1994 shall 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, Harbin Medical University, and Hangzhou Municipal Health and Epidemic Prevention Station.
The main drafters of this standard are: Han Chi, Tang Lingguang, and Yuan Zhenhua. This standard was first issued in 1994, and this is the first revision. 50
1 Scope
Chromosome aberration test of mammalian bone marrow cells This standard specifies the basic technical requirements for chromosome aberration test of mammalian bone marrow GB15193.6——2003
This standard is applicable to the evaluation of the genetic toxicity of chemical, biological and physical factors that may cause health hazards to rat and mouse bone marrow cells during the production, processing, storage, transportation and sales of food. 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, and microorganisms used in the food industry. This standard does not apply to situations where there is evidence that the test substance or reaction metabolites cannot reach the target tissue (bone marrow). 2 Principle
Chromosomes are small bodies with special structures and genetic functions in the cell nucleus. When chemical substances act on the G1 and S phases of the cell cycle, they induce chromosome type aberrations, while when they act on the G2 phase, they induce chromosome monomer type aberrations. Colchicine is injected into the abdominal cavity of experimental rats and mice to inhibit the formation of spindles during cell division, so as to increase the proportion of cells in the metaphase division phase and shorten, disperse the chromosome fibers, and make the outline clear. The number and morphology of chromosomes are observed under a microscope. 3 Instruments and reagents
All reagents are analytically pure unless otherwise specified, and the test water is distilled water. 3.10.1% colchicine: Place in a brown bottle and store in a refrigerator. 3.22.2% sodium citrate.
3.3pH7.4 phosphate buffer.
3.3.1 1/15mol/L disodium hydrogen phosphate solution: 9.47g disodium hydrogen phosphate (NaHPO.) is dissolved in 1000mL distilled water. 3.3.2 1/15mol/L potassium dihydrogen phosphate solution: 49.07g potassium dihydrogen phosphate (KHzPO) is dissolved in 1000mL distilled water. 3.3.3 Mix 80ml disodium hydrogen phosphate solution with 20mL potassium dihydrogen phosphate solution, measure and adjust the pH to 7.4 with a pH meter. 3.4 0.075 ol/L potassium chloride solution.
3.5 Methanol (analytical grade): glacial acetic acid (analytical grade) in a ratio of 3:1, prepare immediately before use. 3.6 Giemsa solution.
3.6.1 Giemsa stock solution: Take 3.8g Giemsa dye, put it in an agate mortar, add a small amount of methanol to grind, and gradually add methanol to 375ml. After dissolving, add 125mL pure glycerol and keep warm in a 37℃ overflow box for 48h. During this period, shake several times, place for 1~2 weeks and filter for use. 3.6.2 Giemsa application solution: Take 1mL of stock solution and add 10mL pH7.4 phosphate buffer. 3.7 Instruments and equipment: Common laboratory equipment, constant temperature water bath (37℃±5℃), centrifuge, biological microscope 4 Experimental animals
Usually use healthy young adult rats or mice. Each group uses at least 5 animals of both sexes. After purchasing, the animals should adapt to the environment for at least 3 days.
5 Dosage 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, 1/2 LDso can be taken. The low dose group should not show any poisoning. Take 1/4 and 1/8 LDs as the medium and low doses respectively. When the maximum dose (maximum concentration and maximum oral gavage volume) of the test substance is given to the acute toxicity test and the animals do not die, but the LDso cannot be calculated, the high-dose group is designed according to 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 substance can be given orally or intraperitoneally (preferably orally) with mitomycin C (1.5mg/kg body weight to 2.0mg/kg body weight) or cyclophosphamide (40 mg/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, or carboxymethyl cellulose can be used to prepare an emulsion or suspension. The test substance should be freshly prepared before oral administration, unless there is data indicating that it is stable when stored as a solution (or suspension, emulsion, etc.). 6.2 Treatment of experimental animals
Give the test substance orally 2 to 4 times, with an interval of 24 hours between each dose, and collect samples 18 to 24 hours after the last dose of the test substance. If necessary, three animals of one dose can be used first, and the animals can be killed and collected at 6, 24, and 48 hours after the test substance is given, so as to select the optimal time to kill the animals. When the test substance is given once, 15 animals can also be used in each dose group, and 5 animals can be killed and collected at 6, 24, and 48 hours after each dose. 2 to 4 hours before the animals are killed, colchicine is injected intraperitoneally at 4 mg/kg body weight. Rats are killed by decapitation, and mice are decapitated by cervical vertebrae. 6.3 Specimen preparation
6.3.1 Sampling
Take the femur, remove the attached muscles, cut off the bones at both ends, use a syringe with a needle to draw 2mL~4mL 2.2% sodium citrate solution, wash the bone marrow into a 10 mL centrifuge tube, rinse repeatedly several times until the cross section of the femur changes from red to pink, then centrifuge at 1000 r/min~1500 r/min for 10 minutes, and discard the supernatant. 6.3.2 Slice preparationbzxZ.net
Add 4mL 0.0.75mol/L potassium chloride solution, mix well and place in a 37℃ water bath or thermostat for 10min～20min, then centrifuge at 1000r/min1500r/min, and discard the supernatant. Add 4mL of newly prepared methanol-glacial acetic acid fixative to the test substance along the tube wall. After 10min～15min, use a pipette to break up the cell clumps and continue to fix for 10min～15min, centrifuge at 1000r/min for 10min, discard the supernatant, add 4mL of fixative, let stand for 20min, then centrifuge, discard the supernatant, and mix well with a pipette to make 0.5ml～1.0mL cell suspension. First, store the washed slide in ice water for later use. Take out the slide from the ice water, tilt it at 30℃, immediately absorb 3 drops of cell suspension on 1/3 of the slide, blow gently to spread the cell suspension on the slide. Prepare 2～3 slides for each specimen and dry them naturally in the air.
When using, take 1mL of Giemsa stock solution and 10mL of phosphate buffer, place in a staining jar, immerse the smear in the staining solution for about 15 minutes, take out the slide, rinse with water, and dry naturally in the air. 6.4 Slide reading
6.4.1 Slide reading requirements
Check the slide quality under a low power microscope. The slide should be relatively concentrated, with all chromosomes dispersed, not overlapping, moderately shrunk, two monomers separated, clearly showing the centromere position, and the chromosomes are reddish purple. Use an oil immersion lens to analyze cell metaphase chromosomes. Analyze 100 metaphase cells for each animal, and no less than 1000 metaphase cells for each dose group. 6.4.2 Observation items
6.4.2.1 Changes in chromosome number
6.4.2.1.1 Aneuploidy: hypodiploidy or hyperdiploidy. 6.4.2.1.2 Polyploidy: chromosomes increase exponentially. 6.4.2.1.3 Endoreplication: a special form of polyploidization within the envelope. 6.4.2.2 Changes in chromosome structure
6.4.2.2.1 Break: the length of the damage is greater than the width of the chromosome. 6.4.2.2.2 Microbodies: smaller than the fragments and round in shape. 6.4.2.2.3 Centromere ring: with centromere parts, forming a ring structure at both ends and accompanied by a pair of acentric fragments. 52
6.4.2.2.4 Acentric ring: a ring structure. 5 Monomer exchange: forming images of three spokes, four spokes or various shapes. 6. 4.2. 2. 5
5 Double microbodies: paired chromatin microbodies. 6. 4. 2. 2. 6
Gap: the length of the damage is less than the width of the chromatid. 6. 4. 2. 2. 7
6. 4. 2. 2. 8
Non-specific phenotypic changes: such as fragmentation, centromere elongation, adhesion, etc. Data processing
Statistical processing uses the X2 test.
Result determination
GB15193.6—2003
When the chromosome aberration rate in the test group is compared with the control group, and there is an obvious dose-response relationship and statistical significance, it can be confirmed as a positive result. If the difference is statistically significant but there is no dose-response relationship, the test should be repeated. The results that can be repeated can be determined as positive.
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