GB 15193.12-2003 In vitro mammalian cell (V79/HGPRT) gene mutation test
Some standard content:
ICS07.100
National Standard of the People's Republic of China
GB15193.12—2003
Replaces GB15193.12—1994
In vitro mammalian cell (V79/HGPRT)
Gene mutation test
V79/HGPRT gene mutation test2003-09-24 Issued
Ministry of Health of the People's Republic of China
Standardization Administration of the People's Republic of China
Implemented on 2004-05-01
GB15193.12—2003
This standard is mandatory in its entirety.
This standard replaces GB15193.12—1994 "In vitro mammalian cell (V79/HGPRT) gene mutation test". Compared with GB15193.12-1994, this standard has been modified as follows: the specific content of the test objects has been added to the "scope": chemical, biological and physical factors involved in the production, processing, preservation, 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. From the date of implementation of this standard, GB15193.12-1994 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 unit of this standard: Nutrition and Food Safety Institute of China Center for Disease Control and Prevention. The main drafters of this standard: Feng Jinong, Gao Peng This standard was first issued in 1994, and this is the first revision. 82
1 Scope
GB 15193.12—2003
In vitro mammalian cell (V79/HGPRT) gene mutation test This standard specifies the basic technical requirements for in vitro mammalian cell (V79/HGPRT) gene mutation test. This standard is applicable to the evaluation of the genetic toxicity 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.
2 Principle
Under normal culture conditions, cells are sensitive to the toxic effects of 6-TG and cannot survive. Under the action of carcinogens and/or mutagens, the structural gene controlling hypoxanthine guanine phosphoribosyltransferase (HGPRT) on the X chromosome of some cells mutates and can no longer produce HGPRT, making the mutant cells resistant to 6-TG. These mutant cells can continue to divide and form colonies in the selective culture medium containing 6-TG. According to the number of mutant colonies formed, the mutation rate is calculated to determine the mutagenicity of the test substance. 3 Materials and Reagents
3.1 Cells: Chinese hamster lung (V79) cell line was used. In order to reduce the spontaneous mutation rate, the spontaneous HGPRT locus mutants present in the wild-type cell population were selectively killed before the formal experiment. The method is to inoculate the wild-type cells in MEM culture medium containing hypoxanthine, thymidine, methotrexate, and glycine for 1 week, and then re-inoculate them in MEM culture medium. 3.2 Culture medium: Use MEM (Eagle) basal culture medium or DMEM culture medium, supplemented with 10% calf serum and appropriate amount of antibiotics (penicillin, streptomycin).
3.3 Phosphate buffer (PBS without calcium and magnesium) Potassium dihydrogen phosphate (KHPO)
Sodium dihydrogen phosphate (Na2HPO4·12H2O)
Potassium chloride (KC1)
Sodium chloride (NaCI)
Double distilled water
Autoclave, 121℃, 0.103MPa.20min. 200mg
1000mL
3.4 Trypsin/EDTA solution: Prepared with PBS without calcium and magnesium, the concentration of trypsin is 0.05%, the concentration of EDTA is 0.02%, and the trypsin and EDTA solution are mixed in a ratio of 1:1. Store at -20℃. 3.5 Test substance: preferably soluble in culture medium. It can also be soluble in dimethyl sulfoxide (DMSO), and its concentration should be less than 0.5% (volume fraction). 3.6 Positive control substance: different positive control substances can be selected according to the properties and structure of the test substance, such as ethyl sulfonate (EMS), mitomycin C (MMC), methyl nitrosoguanidine (MNNG), benzo(a)pyrene (BP), etc. 3.76-TG: Prepare 1.0 mg/mL with 0.5% sodium bicarbonate solution and store at 4°C. 3.8 Rat liver homogenate S-9 mixture: Prepare according to the Ames test procedure. 4 Operation steps
4.1 Cell preparation: 5×105 cells are inoculated in a 100 mm diameter dish and placed in a 37°C, 5% carbon dioxide incubator for 24 hours.
4.2 Contact with test substance: aspirate the culture medium, wash twice with PBS, add serum-free culture medium and a certain concentration of test substance (add rat liver plasma S-9 mixture when metabolic activation is required), place in the incubator for 2 hours, aspirate the culture medium containing the test substance after the test, wash the cells twice with PBS, replace with culture medium containing 10% serum, and continue to culture for 19h~22h. 4.3 Expression: After the cells contacted with the test substance are cultured for 19h~22h, they are digested with trypsin-EDTA. After the cells fall off, 10% serum culture medium is added to terminate the digestion, the mixture is mixed, and the cells are placed in a centrifuge tube and centrifuged at a speed of 800r/min1000r/min for 5min~7min. The supernatant is discarded to prepare a cell suspension, count, and inoculate 5×105 cells in a 100mm diameter flat blood vessel. After 3 days, the cells are divided once and 5×105 cells are inoculated again for 3 days. bzxZ.net
4.4 Cytotoxicity determination: 200 cells after the first digestion and counting are inoculated in each dish, and each group has 5 dishes. They are cultured at 37℃ and 5% carbon dioxide for 7 days, fixed, stained with Giemsa, and the number of colonies in each group is counted. The cytotoxicity is expressed as the colony formation rate relative to the solvent control group. That is, the colony formation rate of the solvent control is 100% (1.00), and the relative value of each test group of the test product is calculated. A(%)
Wherein:
A-relative colony formation rate;
B——colony formation rate of the experimental group;
C—colony formation rate of the solvent control group.
.*(1 )
4.5 Selection of mutants and determination of colony formation rate: After the expression, the cells were digested and divided into 5 dishes per group, with 2×105 cells per dish. After the cells adhered to the wall, 6-TG was added to the final concentration of 5μg/mg. After 8 to 10 days of culture in an incubator, the cells were fixed and stained with Giemsa. The number of colonies per blood was counted, and the mutation rate was calculated. At the same time, the colony formation rate was determined. 200 cells were inoculated per dish without adding 6-TG. There were 5 dishes per group. After 7 days, the cells were fixed and stained, and the colony formation rate was calculated. D=number
where:
D——colony formation rate;
E—actual number of surviving cell colonies;
where:
number of inoculated cells.
G——mutation rate;
H—number of mutant colonies;
I—number of inoculated cells;
D—colony formation rate.
5 Result determination
5.1 If the colony formation rate in the negative control is less than 50%, the result should not be used. ...(2)
5.2 The mutation rate of the positive control selected by each laboratory has a certain range. If the result of the test substance is negative or weakly positive, the mutagenesis rate of the positive control should be above the lower limit of the normal value, otherwise the result cannot be established. 5.3 When the mutation rate is 3 times or more than the spontaneous mutation rate, or when the mutation rate has a dose-response relationship with increasing concentration within at least 3 concentration ranges, it can be judged as positive.
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