Some standard content:
ICS 07. 100
National Standard of the People's Republic of China
GB15193.10--2003
Replaces GB15193.10-1994bZxz.net
Unscheduled DNA synthesis test
Unscheduled DNA synthesis testIssued on 2003-09-24
Ministry of Health of the People's Republic of China
Standardization Administration of the People's Republic of China
Implemented on 2004-05-01
GB 15193.10-2003
The entire text of this standard is mandatory.
This standard replaces GB15193.10-1994 "Unscheduled DNA synthesis test". Compared with GB15193.10-1994, this standard has been modified as follows: - In "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.; In "Reagents", "Reference positive control substances" have been added: 7,12-dimethylbenzathracene, 2-acetylaminofluorene, 4-nitroquinolineoxide, N-methylnitrosamine;
In "Liquid scintillation counting display method of UDS": add "At least 6 culture bottles for each group (including the control group)". "In "UDS autoradiography display method", "a positive control group and a negative control group should be set up at the same time, including both the addition of S-9 and the absence of S-9" were added;
"Result determination" was added.
From the date of implementation of this standard, GB15193.10-1994 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: Zhejiang Medical University. The main drafters of this standard: Xu Yingnian and Ding Chen. This standard was first issued in 1994 and this is the first revision.70
1 Scope
Non-scheduled DNA synthesis test
This standard specifies the basic technical requirements for non-scheduled DNA synthesis test. GB 15193.10---2003
This standard is applicable to the evaluation of the mutagenicity and/or carcinogenicity 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 short-term screening method can detect some mutagens and/or carcinogens that cannot be detected by short-term in vitro tests.
2 Terms and Definitions
The following terms and definitions apply to this standard. 2.1
Unscheduled DNA synthesis, UDs When DNA is damaged, the DNA synthesis that repairs the damage mainly The DNA synthesis in other cell cycles other than the S phase is called unprogrammed DNA synthesis.
3 Principle
Under normal circumstances, in the cell mitosis cycle, only the S phase is the DNA synthesis period. When DNA is damaged, the DNA synthesis for damage repair is mainly in other cell cycles, which is called unprogrammed DNA synthesis, or UDS. Therefore, an increase in UDS indicates that DNA has been damaged.
In in vitro cultured cells, the key to using UDS measurement to display DNA repair synthesis is how to distinguish between very high levels of semiconservative DNA replication and low levels (at most 5% of semiconservative DNA replication) of UDS. This can be shown by blocking the cells in the G1 phase through synchronous culture and inhibiting the residual semiconservative DNA replication with drugs (commonly used hydroxyurea). Synchronous culture can use a culture medium lacking the essential amino acid arginine (ADM) to block the initiation of DNA synthesis and synchronize the cells in G1 period. In these cells where semiconservative DNA synthesis is significantly inhibited and blocked, UDS can be displayed by the increase in the incorporation of \H-thymidine nucleoside. It can be measured by autoradiography or liquid scintillation counting. 4 Reagents and Equipment
4.1 Reagents
All reagents are analytical grade unless otherwise specified, and the test water is double distilled water. 4.1.1 Reference Positive Control: 7,12-dimethylbenzathracene, 2-acetylaminofluorene, 4-nitroquinolineoxide, N-nitrosomethylamine (N-dimethyinitrasamine) can be used.
4.1.2 Culture medium for cell proliferation: 85 parts of Eagle's Minimal Essential Medium (EMEM), 15 parts of calf serum, 1 part of penicillin and streptomycin storage solution, so that the final concentrations of penicillin and streptomycin are 100 units and 100μg/mL respectively. EMEM culture medium can be prepared according to the information provided by the manufacturer and sterilized. Store in a refrigerator at 4℃.
4.1.3 Synchronous culture medium: 98 parts of Eagle's MEM culture medium (ADM) without arginine, 2 parts of calf serum, and the concentrations of penicillin and streptomycin are the same as those of cell proliferation culture medium.
GB15193.10—2003
4.1.4 Hanks balanced salt solution (HBSS) stock solution A: Dissolve 160g sodium chloride, 8g potassium chloride, 2g magnesium sulfate (MgSO.·7H.0) and 2g magnesium chloride (MgCl2·6H,O) in 800mL double distilled water (50℃～~60℃). Dissolve 2.8g anhydrous calcium chloride in 100mL double distilled water. Mix the above two solutions, add water to 1000mL, add 2mL chloroform, and store in a refrigerator at 4℃. Stock solution B: Dissolve 3.04 g of disodium hydrogen phosphate (NazHPO4·12HzO) (or 1.2 g of NazHPO.·2H2O), 1.2 g of potassium dihydrogen phosphate (KH,PO4·2H2O) (or 0.95 g of KH2PO4), and 20 g of glucose in 800 mL of double distilled water, add 100 mL of 0.4% phenol red solution (take 1 g of phenol red, dissolve it in 3 mL of 1 mol/I sodium hydroxide, wait for it to be completely dissolved, and add it to double distilled water to 250 mL), add water to 1000 mL, add 2 mL of chloroform, and store in a refrigerator at 4°C. Stock solution C: 1.4% sodium bicarbonate is prepared with double distilled water. Preparation of application solution: Take 1 part of solution A, 1 part of solution B, and 18 parts of water, mix and dispense into glass containers; sterilize under high pressure and store in a refrigerator at 4°C. Before use, add 1.4% sodium bicarbonate to adjust the pH to 7.2-7.4. 4.1.5 Phosphate buffer (Dulbecco's phosphate buffer without calcium and magnesium) (pH 7.4) Take 8.00g of sodium chloride, 0.20g of potassium dihydrogen phosphate, 0.20g of potassium chloride, and 2.89g of disodium hydrogen phosphate (Na2HPO4·12H,O) and dissolve them in 1000ml of double distilled water.
4.1.6 0.02% disodium or tetrasodium ethylenediaminetetraacetate (EDTA) solution. Take 0.2g of EDTA and dissolve it in phosphate buffer without calcium and magnesium to make it 1000ml. Use after high pressure sterilization. 4.1.7 Antibiotic stock solution
Take 1 million units of penicillin G for clinical injection and 1g of streptomycin powder for injection, dissolve them in 100mL of sterile distilled water under aseptic operation, so that the concentrations of penicillin and streptomycin in the solution are 10,000 units and 10,000μg/mL respectively. When using, add 1mL of antibiotic stock solution to every 100mL of culture medium.
4.1.8 Preparation of rat liver microsome S-9 component: follow GB15193.4. The S-9 mixed solution can be prepared as follows: dissolve 86.8 mg of disodium hydrogen phosphate (NazHPO·12HzO), 7.0 mg of potassium dihydrogen phosphate, 8.1 mg of magnesium chloride (MgCl2*6HzO), 5.4 mg of 6-phosphoglucose (G-6-P), and 4 mg of coenzyme I (COⅡ) (purity 90%) in ADM, and add 1 mol/I. N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES) solution 0.2 mL and rat liver S-9 component 0.8 mL～4 mL or 20 mL, and adjust the pH to 7.2～7.4 with sodium bicarbonate solution. 4.1.9 Developer and fixer (for autoradiography) 4. 1.9.1 Kodak D-170 developer
Storage solution: 25g anhydrous sodium sulfite, 1g potassium bromide, add water to 200mL. When using, dilute with water to 1000mL and dissolve Amitol (2-aminophenol hydrochloride) 4.5g.
4. 1.9.2 Kodak D-196 developer
500mL water (50℃), dissolve 2g of metol (p-aminophenol sulfate), 72g of anhydrous sodium sulfite, 8.8g of hydroquinone, 48g of anhydrous sodium carbonate, 4 of potassium bromide in sequence, add water to the total amount of 1000mL4.1.9.3 Stop solution: 15mL of 98% glacial acetic acid, add water to 1000mL. 4.1.9.4 Kodak F-5 fixer
100mL water (50℃), dissolve 240g of hypochlorite, 15g of anhydrous sodium sulfite, 48mL of 28% acetic acid, 7.5g of boric acid, 15g of potassium alum in sequence, add water to 1000mL.
4.1.10 0.25mol/L and 0.5mol/L perchloric acid: 8.57mL of 70% perchloric acid (sale product), add water to 100mL to make 1mol/L. 4.1.11 Scintillation fluid: weigh 5g of 2,5-diphenyloxazole (PPO) and 300mg of 1,4-bis-[5-phenyloxazolyl-2]-benzene (POPOP) and dissolve them in toluene to make 1000mL.
4.2 Equipment
Using disposable cell culture vessels and microporous filter membranes can avoid tedious washing work and improve the quality of experiments. 4.2.1 Glass: Rinse the dirt in tap water, boil in soap or detergent solution for 5 minutes, cool slightly, wash repeatedly in hot soapy water, and rinse with tap water. After drying, soak in cleaning solution overnight. After taking out, rinse repeatedly with tap water 12 times. Rinse with distilled water for 72
GB15193.10—2003
2 times, and soak in distilled water for 24 hours. After taking out, rinse with distilled water for 2 more times, and dry. Wrap the mouth of the glass bottle with paper, and plug the cotton plug into the proximal tube of the pipette and dropper and wrap it with paper. 4.2.2 Rubber: After rinsing in tap water, boil in soapy water. If it is newly purchased, boil it with 4% sodium hydroxide for 10 minutes, and then boil it with 4% hydrochloric acid for 10 minutes. Rinse with running tap water for more than 2 hours. Boil in distilled water twice again, rinse with distilled water, soak in distilled water for 24 hours, dry, wrap with cellophane, and place in a container. 4.2.36 Glass filter (for sterilization): The newly purchased filter is immersed in hot sulfuric acid containing a small amount of sodium nitrite for 24 hours. After filtering with distilled water, it is filtered with sodium hydroxide solution until neutral, and then filtered with double distilled water twice. After drying, it is equipped with a rubber stopper and wrapped. Immediately after use, the filter is filtered with distilled water once, and then filtered with 1% sodium nitrite sulfuric acid solution (sulfuric acid solution concentration 0.5 mol/L), and then immersed in the sulfuric acid solution (note that both sides of the filter plate must be immersed in the acid). After rinsing with clean water, it is filtered with distilled water for 3 times, and after drying, it is equipped with a stopper and wrapped. 4.2.4 Sterilization of equipment: Glassware and metal utensils without rubber stoppers can be sterilized with ten heat sterilizers, and the temperature is raised to 140℃ and maintained for 2 hours. Rubber and glassware with rubber stoppers should be sterilized by autoclave at 120℃ for 30 minutes. In addition to the heat-resistant solution, which should be sterilized by suction filtration, the heat-resistant solution can be sterilized by autoclave, generally at 115℃ for 10 minutes.
5 Operation steps
5.1 Cell passage, maintenance and storage
There are many types of mammalian cells used in experimental studies on the induction of UDS by chemical carcinogens in cultured cells in vitro. The UDS reaction of human cells is greater than that of rodent cells. Many people use human cells in chemical carcinogenicity tests. This has a desirable advantage, that is, when evaluating the hazard of a chemical to humans, the risk of inference errors caused by species differences can be reduced. The most commonly used human cells are fibroblasts, peripheral blood lymphocytes, monocytes and Hela cells. The human amniotic membrane cell FI. strain is used, which is an epithelial cell line and is rich in inducible drug-metabolizing enzymes. The cells grown into a monolayer are removed from the culture medium. After washing with Hanks balanced salt solution (HBSS), treat with 0.02% EDTA or 0.1% trypsin solution (in phosphate buffer without Ca2+ and Mg2+) at 37°C for several minutes to cause the cells to shrink and increase the intercellular space. Wash once with HBSS, add an appropriate amount of culture medium, and repeatedly blow and aspirate to remove the cells from the glass surface and disperse them in the culture medium. Take a drop of cell suspension and add it to the blood cell counting pool. Count the number of cells in the four large grids and calculate the cell concentration in the suspension (the number of cells in the four large grids/4×104 is the number of cells per milliliter). Dilute the cell suspension growth medium to 0.5×10°/mL~1×10°/mL. Inoculate the above cell suspension in a culture bottle (30mL culture bottle can be inoculated with 3mL. 100mL culture bottle can be inoculated with 10mL). Inoculate 3 copies each time. After growing into a fused monolayer, take one of the bottles and inoculate 3 bottles according to the above method. The other two bottles are discarded or used for experiments after proving that the subculture is successful, so as to ensure that the cells are maintained in the experiment. If conditions permit, the cells can be stored in liquid nitrogen as follows. If not used for a long time, they do not need to be maintained in the laboratory. When needed, take them out and proliferate for use in experiments. After the cells have proliferated to the required number, prepare a cell suspension (in the growth medium) according to the above method. The cell concentration is 1×104/mL to 1.5×104/mL. In an ice bath, gradually add 10% of the total amount of the cell suspension with sterilized dimethyl sulfoxide. Then divide the cell suspension into clean and dry sterilized or cell freezing special plastic tubes, 1mL per portion. After sealing, place it at 4℃ for 2h~3h, then move it to the ice room of an ordinary refrigerator for 4h~5h, and then move it to a low-temperature refrigerator at -30℃~-20℃ overnight. The next morning, move the cells to a biological liquid nitrogen storage container. When needed, saw (open) the cells or small plastic tubes, remove the culture medium containing dimethyl sulfoxide, add an appropriate amount of growth medium, and adjust the cell concentration to 10×104/mL~15×104/mL. Divide and seed in cell culture bottles, culture at 37℃ for 1h, change the culture medium once, remove the inactive cells, and grow into a confluent monolayer and then divide and proliferate in the laboratory. 5.2 UDS autoradiographic display method
After the cells proliferate to the required number, prepare a single cell suspension according to the above method, and suspend the cells in the growth medium (EMEM85 calf serum 15). The concentration is 0.5×105/mL to 1×105/ml. Inoculate the above cell suspension in a culture bottle with a small cover slip (18mm×6mm), culture at 37℃ for 1 to 3 days, and let the cells grow to an appropriate density on the cover slip. The number of culture bottles inoculated depends on the number of samples and the selected dose level. Make 2 to 3 samples for each dose, and prepare 4 to 6 samples for solvent control and positive control of known carcinogens. After the cells proliferated, they were cultured synchronously for 3 to 4 days in a synchronous culture medium (ADM supplemented with 1% calf serum). On the afternoon of the day before the experiment, hydroxyurea (HU) solution dissolved in ADM was added to make the final concentration of HU in the culture medium 73
GB15193.10—2003
is 10mmol/L. Continue to incubate at 37℃ for 16h, then place the coverslip with cells in a synchronous culture medium containing different concentrations of the test product, HU (concentration is 10mmol/L) and 3H-thymidine (5uCi/mL～10μCi/mL, 30Ci/mmol). Incubate at 37℃ for 5h. Use solvents and known carcinogens as controls. The test product and known carcinogen solutions are freshly prepared during the experiment. First dissolve them in appropriate solvents (distilled water, dimethyl sulfoxide, acetone, etc.) to prepare a stock solution of 100 times the highest test concentration. Then dilute them with solvents by 10 times in sequence to prepare test product solutions of different concentrations. Add various concentrations of test product solutions to the culture medium so that the final concentration of the solvent is 1%. Positive control group and negative control group should be set up at the same time, including two conditions with S-9 and without S-9.
After incubation, wash thoroughly with HBSS, treat with 1% sodium citrate solution for 10 minutes, and then fix with ethanol-glacial acetic acid (3:1) overnight (4℃). After drying in the air, use a small amount of neutral gum to stick the cover slip on the slide, with the side with cells facing up, and bake at 45℃ for 24h
In the dark room, transfer an appropriate amount of nuclear-4 latex into the glass blood used for immersion, place it in a 40℃ water bath to melt it, and at the same time take an equal amount of distilled water in a measuring cylinder and place it in the water bath to heat it. After the latex melts, pour the hot distilled water into the latex solution, continue to heat it in the water bath, and stir it gently with a glass rod, wait for 10 min~20 min to allow the bubbles to escape. During the above preparation process, place the slide to be processed for autoradiography on the platform of the water bath to preheat. After preparation, immerse the slide vertically in the 1:1 diluted latex solution for about 5 seconds, slowly lift the slide out, and wipe the latex on the back of the slide with gauze or lens paper. Move the slide coated with latex into a temperature box with a temperature of 29℃ and a certain humidity (4h) to wait for the latex to drip. Then place it in an exposure box with a built-in bag of appropriate desiccant (color-changing silica gel). The exposure box is wrapped with black light-proof paper and plastic paper and placed in a refrigerator at 4℃ for 10 days. After exposure, move the slide to a slide rack made of organic glass, develop it in D-170 or D-196 developer at a liquid temperature of 19℃ for 5 minutes, rinse it in stop solution for 2 minutes, fix it in F-5 fixer for 6 minutes~10 minutes, and then rinse it with water for several hours.
Cells can be stained with orcein (2%) glacial acetic acid solution before latex smearing or with HE or Giemsa dye after development. After the slides are dehydrated and made transparent, they are sealed with coverslips. Under an oil stain microscope, count the number of developed silver grains on the cell nuclei of each sample, and count the number of background silver grains of equivalent area at the same time, and subtract them from the number of silver grains on the nuclei. Count at least 50 cell nuclei on each slide, and calculate the mean and statistical value of the number of silver grains/nucleus in the control samples, samples treated with various concentrations of test products, and positive carcinogen control samples. 5.3 UDS liquid scintillation counting display method
Suspend the experimental cells in the growth medium at a cell concentration of 0.5×105/mL, inoculate the cells in liquid scintillation counting bottles, 1 mL per bottle, and add lC-thymidine to a final concentration of 0.01μCi/mL (50mCi/mmol). The cells were cultured at 37°C for 48 hours to proliferate and pre-label. The culture medium was removed and washed with HBSS, and then replaced with synchronization medium containing 1C-thymidine (0.01μCi/mL). Synchronous culture was carried out at 37°C for 2 to 4 days. After the synchronous culture was completed, the culture medium was removed in the afternoon of the day before the experiment, and after being fully washed with HBSS, synchronization medium containing 10mmol/L hydroxyurea (HU) was added. The cells were incubated at 37°C for 16 hours. UDS induction was displayed by autoradiography. The cells were exposed to different concentrations of the test articles in synchronization medium containing HU and 1H-thymidine (5μCi/mL, 30Ci/mmol) for 5 hours. After the incubation, the culture medium and test articles were removed. Wash twice with cold saline, then treat twice with ice-cold 0.25mol/L perchloric acid solution, 2 minutes each time, to fix the cells and remove acid-soluble components, then treat with ethanol for 10 minutes to remove fat-soluble components and unincorporated markers, dry and hydrolyze with 0.5mol/L~1mol/L perchloric acid 0.5mL in a 75℃80℃ incubator for 40 minutes to release the incorporated markers. After cooling, add 3.5mL of ethylene glycol ether and 5mL of scintillation fluid (PpO0.5%, POPOP0.03%, toluene as solvent), shake to make a homogeneous phase, and measure the radioactivity of 14C and \H in each sample with a liquid scintillation counter. At least 6 culture bottles are made for each group (including the control group). The \H radioactivity in the specimen reflects the amount of \H-thymidine incorporated into UDS; and the 4C radioactivity reflects the number of experimental cells or their DNA content. Therefore, the ratio of \H and 1C radioactivity (\H/14C) is the reflection value of the UDS level per unit mass of DNA or per unit number of cells. If the \H and 14C ratio of the solvent control is taken as 100% (1.00), the change in each sample relative to the control can be calculated, and the relevant statistics in samples of various test concentrations can be calculated. 5.4 Metabolic activity of the test substance
The activity of the drug metabolism activation enzyme system in the established cell line is generally very low. Therefore, for some chemical substances that require enzymatic metabolic activation to show their DNA74
GB 15193.10—2003
damaging effects, an in vitro activation system (S-9 mixed solution) composed of rat liver microsomal enzyme system and auxiliary factors can be added to the test system. Magnesium chloride (MgCl2·6H2O), disodium hydrogen phosphate, potassium dihydrogen phosphate, 6-phosphoglucose (G-6-P), NADP (CoⅡ) and rat liver S-9 component can be dissolved or added to the culture medium used when the test substance is in contact with the test substance. The proportion of S-9 component varies depending on the lipophilicity of the test substance. A low percentage of S-9 can be used for a lipophilic substance with a strong lipophilicity, and conversely, the proportion of S-9 component used in the activation system can be higher. HEPES (10 μmol/mL) was added, and the pH was adjusted to 7.2-7.4 with sodium bicarbonate solution. 6
Data processing
The "t-test" can be used to test whether there is a significant difference between the contact of each test product and the solvent control to make a judgment. 7 Result judgment
The number of cells treated with the test group\H-TdR doping increases with the increase of dose and is statistically significant, or the minimum dose group has a positive reaction and is statistically significant compared with the control group, which can be judged as positive for this test. The number of cells treated with the test substance group\H-TdR doping does not increase with the increase of dose, and there is no statistical difference between any dose group and the control group. It is considered that the test substance does not cause UDS in this test system. When judging the results, biological significance and statistical significance should be considered comprehensively. 7501μCi/mL) of synchronization medium, synchronized culture was carried out at 37°C for 2-4 days. After the synchronization culture was completed, the medium was removed in the afternoon of the day before the experiment, and after being fully washed with HBSS, synchronization medium containing 10mmol/L hydroxyurea (HU) was added, and incubated at 37°C for 16h. UDS induction was displayed by autoradiography. The cells were exposed to different concentrations of the test articles in synchronization medium containing HU and \H-thymidine (5μCi/mL, 30Ci/mmol) for 5h. After the incubation, the medium and test articles were removed. Wash twice with cold saline, then treat twice with ice-cold 0.25mol/L perchloric acid solution, 2 minutes each time, to fix the cells and remove acid-soluble components, then treat with ethanol for 10 minutes to remove fat-soluble components and unincorporated markers, dry and hydrolyze with 0.5mol/L~1mol/L perchloric acid 0.5mL in a 75℃80℃ incubator for 40 minutes to release the incorporated markers. After cooling, add 3.5mL of ethylene glycol ether and 5mL of scintillation fluid (PpO0.5%, POPOP0.03%, toluene as solvent), shake to make a homogeneous phase, and measure the radioactivity of 14C and \H in each sample with a liquid scintillation counter. At least 6 culture bottles are made for each group (including the control group). The \H radioactivity in the specimen reflects the amount of \H-thymidine incorporated into UDS; and the 4C radioactivity reflects the number of experimental cells or their DNA content. Therefore, the ratio of \H and 1C radioactivity (\H/14C) is the reflection value of the UDS level per unit mass of DNA or per unit number of cells. If the \H and 14C ratio of the solvent control is taken as 100% (1.00), the change in each sample relative to the control can be calculated, and the relevant statistics in samples of various test concentrations can be calculated. 5.4 Metabolic activity of the test substance
The activity of the drug metabolism activation enzyme system in the established cell line is generally very low. Therefore, for some chemical substances that require enzymatic metabolic activation to show their DNA74
GB 15193.10—2003
damaging effects, an in vitro activation system (S-9 mixed solution) composed of rat liver microsomal enzyme system and auxiliary factors can be added to the test system. Magnesium chloride (MgCl2·6H2O), disodium hydrogen phosphate, potassium dihydrogen phosphate, 6-phosphoglucose (G-6-P), NADP (CoⅡ) and rat liver S-9 component can be dissolved or added to the culture medium used when the test substance is in contact with the test substance. The proportion of S-9 component varies depending on the lipophilicity of the test substance. A low percentage of S-9 can be used for a lipophilic substance with a strong lipophilicity, and conversely, the proportion of S-9 component used in the activation system can be higher. HEPES (10 μmol/mL) was added, and the pH was adjusted to 7.2-7.4 with sodium bicarbonate solution. 6
Data processing
The "t-test" can be used to test whether there is a significant difference between the contact of each test product and the solvent control to make a judgment. 7 Result judgment
The number of cells treated with the test group\H-TdR doping increases with the increase of dose and is statistically significant, or the minimum dose group has a positive reaction and is statistically significant compared with the control group, which can be judged as positive for this test. The number of cells treated with the test substance group\H-TdR doping does not increase with the increase of dose, and there is no statistical difference between any dose group and the control group. It is considered that the test substance does not cause UDS in this test system. When judging the results, biological significance and statistical significance should be considered comprehensively. 7501μCi/mL) of synchronization medium, synchronized culture was carried out at 37°C for 2-4 days. After the synchronization culture was completed, the medium was removed in the afternoon of the day before the experiment, and after being fully washed with HBSS, synchronization medium containing 10mmol/L hydroxyurea (HU) was added, and incubated at 37°C for 16h. UDS induction was displayed by autoradiography. The cells were exposed to different concentrations of the test articles in synchronization medium containing HU and \H-thymidine (5μCi/mL, 30Ci/mmol) for 5h. After the incubation, the medium and test articles were removed. Wash twice with cold saline, then treat twice with ice-cold 0.25mol/L perchloric acid solution, 2 minutes each time, to fix the cells and remove acid-soluble components, then treat with ethanol for 10 minutes to remove fat-soluble components and unincorporated markers, dry and hydrolyze with 0.5mol/L~1mol/L perchloric acid 0.5mL in a 75℃80℃ incubator for 40 minutes to release the incorporated markers. After cooling, add 3.5mL of ethylene glycol ether and 5mL of scintillation fluid (PpO0.5%, POPOP0.03%, toluene as solvent), shake to make a homogeneous phase, and measure the radioactivity of 14C and \H in each sample with a liquid scintillation counter. At least 6 culture bottles are made for each group (including the control group). The \H radioactivity in the specimen reflects the amount of \H-thymidine incorporated into UDS; and the 4C radioactivity reflects the number of experimental cells or their DNA content. Therefore, the ratio of \H and 1C radioactivity (\H/14C) is the reflection value of the UDS level per unit mass of DNA or per unit number of cells. If the \H and 14C ratio of the solvent control is taken as 100% (1.00), the change in each sample relative to the control can be calculated, and the relevant statistics in samples of various test concentrations can be calculated. 5.4 Metabolic activity of the test substance
The activity of the drug metabolism activation enzyme system in the established cell line is generally very low. Therefore, for some chemical substances that require enzymatic metabolic activation to show their DNA74
GB 15193.10—2003
damaging effects, an in vitro activation system (S-9 mixed solution) composed of rat liver microsomal enzyme system and auxiliary factors can be added to the test system. Magnesium chloride (MgCl2·6H2O), disodium hydrogen phosphate, potassium dihydrogen phosphate, 6-phosphoglucose (G-6-P), NADP (CoⅡ) and rat liver S-9 component can be dissolved or added to the culture medium used when the test substance is in contact with the test substance. The proportion of S-9 component varies depending on the lipophilicity of the test substance. A low percentage of S-9 can be used for a lipophilic substance with a strong lipophilicity, and conversely, the proportion of S-9 component used in the activation system can be higher. HEPES (10 μmol/mL) was added, and the pH was adjusted to 7.2-7.4 with sodium bicarbonate solution. 6
Data processing
The "t-test" can be used to test whether there is a significant difference between the contact of each test product and the solvent control to make a judgment. 7 Result judgment
The number of cells treated with the test group\H-TdR doping increases with the increase of dose and is statistically significant, or the minimum dose group has a positive reaction and is statistically significant compared with the control group, which can be judged as positive for this test. The number of cells treated with the test substance group\H-TdR doping does not increase with the increase of dose, and there is no statistical difference between any dose group and the control group. It is considered that the test substance does not cause UDS in this test system. When judging the results, biological significance and statistical significance should be considered comprehensively. 75
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