This standard specifies the basic technical requirements for the mouse testicular chromosome aberration test. This standard is applicable to the evaluation of the damage to the chromosomes of the testicular germ cells of mammals as a whole by chemical, biological and physical factors that may cause health hazards 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 15193.8-2003 Mouse testicular chromosome aberration test GB15193.8-2003 standard download decompression password: www.bzxz.net

ICS 07.100
National Standard of the People's Republic of China
GB15193.8—2003
Replaces GB15193.8—1994
Mice testicle cells chromosome aberration test
Mice testicle cells chromosome aberration test2003-09-24Promulgated
Ministry of Health of the People's Republic of China
Standardization Administration of the People's Republic of China
Implemented on 2004-05-01
GB15193.8-2003
This standard is mandatory in its entirety.
This standard replaces GB15193.8—1994 "Mice testicle cells chromosome aberration test". Compared with GB15193.81994, 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.; and the scope of inapplicability has been increased; in the "Dose Grouping": the design method of the high-dose group has been added with an "acute toxicity test" When the animals do not die at the maximum dose (maximum concentration and maximum oral volume) of the test substance and the LDso cannot be determined, 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 dose", and the design method of positive and negative controls shall be added;
In "Operational Procedures": change the administration time of colchicine from "6 days before sacrifice" to "6 hours before sacrifice"; the "administer the test substance at 0.1-0.2mL/10g body weight" in brackets shall be changed to \ injection volume: 0.1mL/10g body weight ~ 0.2mL/10g body weight". From the date of implementation of this standard, GB15193.8-1994 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 Zhejiang Medical University and Beijing Municipal Health and Epidemic Prevention Station. The main drafters of this standard are Xu Weian, Yao Xiaoman and Zhu Huijuan. This standard was first issued in 1994 and this is the first revision. 60
1 Scope
Mouse testicular chromosome aberration test
This standard specifies the basic technical requirements for mouse testicular chromosome aberration test. GB 15193.8—2003
This standard is applicable to the evaluation of the damage of chemical, biological and physical factors involved in the production, processing, storage, transportation and sales of food that may cause health hazards to the chromosomes of testicular germ cells of whole mammals. 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. 2 Terms and definitions
The following terms and definitions apply to this standard. 2.1
Testicular chromosome aberration Chromosome aberration in lesticle cells Abnormalities in the number and structure of testicular chromosomes, including gaps, fragments, translocations, microbodies, autosomal monovalent bodies, and sex chromosome monovalent bodies. 3 Principle
Male germ cells in different cycles have different sensitivities to chemical substances. In most cases, chemical mutagens must undergo DNA replication to induce chromosome aberrations, so they are treated in the proleptotene stage. Samples were collected on the 12th to 14th day to observe the effects of the proleptotene on the chromosome aberration of spermatocytes.
4 Instruments and reagents
All reagents are analytical grade unless otherwise specified, and the test water is distilled water. 4.1 Common laboratory equipment.
4.21% trisodium citrate (analytical grade): Take 1g trisodium citrate and add distilled water to 100mL. 4.360% glacial acetic acid (analytical grade): Take 60ml. glacial acetic acid and add distilled water to 100ml. All should be freshly prepared. 5 Experimental animals
Select healthy adult male mice, weighing 25g to 30g, with at least 5 mice per group. After purchasing, the animals were adapted to the environment for 3 to 5 days before the test.
6 Dosage and grouping
The test substance should be divided into three groups. In principle, the highest dose group is the dose at which the animals show severe poisoning and/or individual animals die. Generally, 1/2LDo can be taken. The low dose group should not show toxicity, and 1/4LDso and 1/8LDso are taken as the medium and low doses respectively. When the acute toxicity test gives the test substance the maximum dose (maximum use concentration and maximum gavage capacity) and the animals do not die, but the LDs 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 gavage dose at a time, and then the medium and low dose groups are set up. At the same time, a solvent control group and a positive control group are set up. At least 5 animals survive in each group. The selected positive substance should be able to cause chromosome structural aberration in sperm cells in vivo. A different route of administration from the test substance can be used - once for the positive substance. Mitomycin C (1.5mg/kg～2mg/kg·intraperitoneal injection, once) or cyclophosphamide (40mg/kg, intraperitoneal injection, once a day, for 5 consecutive days) can be used.
The negative control group should be given a solvent or medium in the same way as the test substance group. If there is data showing that the solvent or medium used has no mutagenic effect, an untreated control may not be used. Otherwise, an untreated control should be set up separately. 7 Operation steps
7.1 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 oral administration, unless there is data indicating that it is stable when stored as a solution (or suspension, emulsion, etc.). 7.2 Treatment of experimental animalswww.bzxz.net
Administer the test substance by oral administration, once a day, for 5 consecutive days. The test animals in each group were killed on the 12th to 14th day after the first administration of the test substance for preparation of slices. 6 hours before the animals were killed, colchicine was injected intraperitoneally with 4mg/kg to 6mg/kg body weight (injection volume: 0.1mL/10g body weight 0.2mL/10g body weight). Colchicine should be freshly prepared on the same day. Mice were killed by cervical dislocation. 7.3 Preparation of specimens
7.3.1 Collection
Take out the testicles on both sides, remove the fat, wash off the hair and blood stains in hypotonic solution, and place them in a small flat III containing 1% trisodium citrate or 0.4% potassium chloride solution.
7.3.2 Slide preparation
7.3.2.1 Hypotonicity: Use ophthalmic forceps to tear open the membrane and gently separate the seminiferous tubules. Hypotonicity is maintained at room temperature. The hypotonicity time depends on the specific conditions. 7.3.2.2 Fixation: Carefully aspirate the hypotonic solution, add 10mL of fixative solution (methanol: glacial acetic acid = 3:1) and fix for a week. The first time should not exceed 15min. After pouring out the fixative solution, add new fixative solution and fix for more than 20min. 7.3.2.3 Centrifuge to aspirate the fixative solution. Add 1mL~2mL of 60% glacial acetic acid. After most of the seminiferous tubules are softened, immediately add double the amount of fixative solution, tick, transfer to a centrifuge tube, and centrifuge at 1000r/min for 10min. 7.3.2.4 Drop slide: Discard most of the supernatant, leaving about 0.5mL1.0mL, fully mix to make a cell suspension, and drop the cell suspension evenly on the ice water slide. Prepare 2 to 3 slides for each sample. Air dry or heat dry for 1.7.3.2.5 Staining: Use 1:10 Giemsa solution (pH6.8) for 20min40min. 7.4 Slide reading
7.4.1 Slide reading requirements
Search for metaphases with clear background, good dispersion and moderate chromosome contraction in sequence under low power microscope, and then analyze under oil microscope.
7.4.2 Observation items: In the structural aberration of chromosomes, in addition to cracks, fragments and micro-exosomes (according to GB15193.6). In addition, the analysis of translocations, XY and autosomal univalents is also required. Reciprocal translocations: Reciprocal translocations involve the exchange of terminal fragments between non-homologous chromosomes. It requires secondary breakage and repair. There are translocations between autosomes and between sex chromosomes and autosomes. Autosomal translocations can produce circular multivalents or chain multivalents. For example, a single translocation can form a circular quadrivalent, a chain quadrivalent, a trivalent and a monovalent (cⅢ+"); if it is translocated twice, three times or four times, a hexavalent, an octavalent or a decavalent can be observed. The translocation of sex chromosomes can be X chromosome or Y chromosome and autosome translocation. The recurrence rate of translocation in control adult animals is extremely low, less than 0.01%. It may increase slightly in elderly animals. XY and autosome monovalent: also known as premature separation. XY monovalents are more common in control animals, about 0~10%. Because X and Y chromosomes are at the far end of the long arm, non-homologous fragments are connected. The separation of X and Y can often cause infertility. Autosomes The monovalent bodies are caused by asynapsis (lack of paired synapses between homologous segments) or loss of synapsis (separation due to failed crossing over). They are less common in control animals because crossing over is formed in the diplotene stage and the normal pairing is combined until the end of metaphase I. They often occur in the smallest pair of autosomes. 62
Data processing
GB15193.8-2003
The fragments, translocations, aberrant cell rates, autosomal monovalent bodies, sex chromosome monovalent bodies, etc. of the experimental group and the negative control group were statistically processed according to the method described by Kastenbaum and Bowman, as shown in P
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