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GB 15193.10-2014 English PDF

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GB 15193.10-2014: National Food Safety Standard -- Unscheduled DNA synthesis test
Status: Valid

GB 15193.10: Evolution and historical versions

Standard IDContents [version]USDSTEP2[PDF] delivered inStandard Title (Description)StatusPDF
GB 15193.10-2014English189 Add to Cart 3 days [Need to translate] National Food Safety Standard -- Unscheduled DNA synthesis test Valid GB 15193.10-2014
GB 15193.10-2003English279 Add to Cart 3 days [Need to translate] Unscheduled DNA synthesis test Obsolete GB 15193.10-2003
GB 15193.10-1994English199 Add to Cart 2 days [Need to translate] Unscheduled DNA synthesis test Obsolete GB 15193.10-1994

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Basic data

Standard ID GB 15193.10-2014 (GB15193.10-2014)
Description (Translated English) National Food Safety Standard -- Unscheduled DNA synthesis test
Sector / Industry National Standard
Classification of Chinese Standard C53
Classification of International Standard 07.100
Word Count Estimation 8,859
Date of Issue 12/24/2014
Date of Implementation 5/1/2015
Older Standard (superseded by this standard) GB 15193.10-2003
Regulation (derived from) Health Planning Commission Bulletin 2014 No. 21
Issuing agency(ies) National Health and Family Planning Commission of the People's Republic of China
Summary This Standard specifies in vitro mammalian cell DNA damage repair (unscheduled DNA synthesis) basic test methods and test requirements. This Standard is applicable to the evaluation of mutagenicity test substance and (or) carcinogenic.

GB 15193.10-2014: National Food Safety Standard -- Unscheduled DNA synthesis test

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National Food Safety Standard.Unscheduled DNA synthesis test National Standards of People's Republic of China National Food Safety Standard In vitro mammalian cell DNA damage repair (Unscheduled DNA synthesis) test Issued on.2014-12-24 2015-05-01 implementation People's Republic of China National Health and Family Planning Commission released

Foreword

This standard replaces GB 15193.10-2003 "unscheduled DNA synthesis test." This standard compared with GB 15193.10-2003, the main changes are as follows. --- Standard name was changed to "national food safety standard in vitro mammalian cell DNA damage repair (unscheduled DNA synthesis) test"; --- Modified test purposes and principles; --- Revised test methods; --- Modify the data processing; --- Modify the results of the evaluation. National Food Safety Standard In vitro mammalian cell DNA damage repair (Unscheduled DNA synthesis) test

1 Scope

This standard specifies the in vitro mammalian cell DNA damage repair (unscheduled DNA synthesis) test basic test methods and techniques Claim. This standard applies to the evaluation of the test substance mutagenicity and (or) carcinogenic.

2 Terms and definitions

2.1 unscheduled DNA synthesis When the damaged DNA, DNA repair synthesis mainly in other cell cycle other than S phase, said the unscheduled DNA synthesis.

3 test purposes and principles

Under normal circumstances, DNA synthesis only in the mitotic cycle S on schedule. When chemical or physical agent-induced DNA damage After the cells start unscheduled DNA synthesis procedures to repair DNA damage in the area. Primary mammalian non S Isolation, Culture Cells or continuous cell lines added 3H- thymidine (3H-TDR), autoradiography or liquid scintillation counting technique by DNA radiation (LSC) was detected in cells exposed to the amount of 3H-TDR incorporation of DNA, and may indicate the extent of damaged DNA repair synthesis. In cultured cells, with the lack of semi-essential amino acid arginine medium (ADM) synchronize culture, DNA synthesis initiating Blocked, the cells were synchronized in G1 phase; and inhibit residual semi-conservative DNA replication with drugs (commonly used hydroxyurea), the incorporation by 3H-TDR It can display unscheduled DNA synthesis (UDS).

4 Reagents and materials

4.1 Reagents Note. Unless otherwise indicated, all the reagents, were of analytical grade, test water is double distilled water or ultrapure water. 4.1.1 cell growth medium Medium Eagle's minimum requirements (Eagle'sMinimalEssentialMedium, EMEM), 10% fetal calf serum, penicillin Su, streptomycin stock solution, final concentration of penicillin is 100 units, streptomycin at a final concentration of 100μg/mL. EMEM medium You can choose a variety of powder medium supply of goods by manufacturers to provide information on the preparation and sterilization. 4 ℃ refrigerator storage. 4.1.2 Synchronization cell culture medium The arginine-free EMEM medium (the ADM), was added calf serum, penicillin, streptomycin with cell density growth medium. 4.1.3 Hanks balanced salt solution (the HBSS) 4.1.3.1 accumulator A. Sodium chloride 160g, potassium chloride 8g, magnesium sulphate (MgSO4 · 7H2O) 2g and magnesium chloride (MgCl2 · 6H2O) 2g dissolved in 800mL distilled water (50 ℃ ~ 60 ℃) in. Another 2.8g anhydrous calcium chloride dissolved in 100mL double-distilled water. The above two After the mixed solution, add water to 1000mL, chloroform was added 2mL, stored at 4 ℃ refrigerator. 4.1.3.2 accumulator B. The disodium hydrogenphosphate (Na2HPO4 · 12H2O) 3.04g (or Na2HPO4 · 2H2O1.2g), potassium dihydrogen phosphate (KH2PO4 · 2H2O) 1.2g (or KH2PO40.95g), 20g of glucose were dissolved in double distilled water 800mL, 100mL added 0.4% phenol red solution [phenol red take 1g, dissolved in 3mL sodium hydroxide (1mol/L) in], until completely dissolved, double distilled water to 250mL, add water to 1000mL, chloroform was added 2mL, stored at 4 ℃ refrigerator. 4.1.3.3 reservoir C. 1.4% sodium bicarbonate in distilled water preparation. Preparation 4.1.3.4 Application Solution. Take 1 part liquid A, B solution 1, 18 parts of water, mixed packed in glass containers; autoclaving, 4 ℃ ice Save me. Before using the solution C was added to adjust the pH to 7.2 to 7.4. 4.1.4 without calcium, Dulbecco's phosphate buffer magnesium pH7.4, take the sodium chloride 8.00g, potassium dihydrogen phosphate (KH2PO4) 0.20g, KCl 0.20g, disodium hydrogen phosphate (Na2HPO4 · 12H2O) 2.89g dissolved and dilute to 1000mL double distilled water. 4.1.5 0.02% ethylene diamine tetraacetate tetrasodium or disodium (EDTA) solution of Take EDTA0.2g dissolved calcium, magnesium phosphate buffer, the volume to 1000mL. Autoclaving after use. 4.1.6 antibiotic stock solution Take clinical injection of penicillin G100 million units and streptomycin 1g powder under aseptic dissolved in 100mL of sterile distilled water To a concentration of 1 million units of penicillin, streptomycin concentration for 10000μg/mL, 100mL each added to the medium when using antibacterial Prime stock solution 1mL. Preparation of components 4.1.7 rat liver microsomes-9 S S-9 mixture may be formulated in the following manner. The disodium hydrogenphosphate (Na2HPO4 · 12H2O) 86.8mg, monopotassium phosphate 7.0mg, Magnesium chloride (MgCl2 · 6H2O) 8.1mg, 6- glucose phosphate (G-6-P) 5.4mg, coenzyme Ⅱ (CoⅡ) (purity 90%) 4mg dissolved ADM was added N-2- hydroxyethyl piperazine--N-2- ethanesulfonic acid (HEPES) solution (1mol/L) 0.2mL rat liver S-9 and components 0.8mL ~ 4mL or 20mL, and adjust the pH with sodium bicarbonate solution to 7.2 to 7.4. 4.1.8 developer and fixer (by autoradiography) 4.1.8.1 KodakD-170 developer Reservoir. Anhydrous sodium sulfite 25g, Potassium bromide 1g, water was added to 200mL. When diluted with water use, into 2-amino-phenol hydrochloride (Amitol) 4.5g, dilute to 1000mL. 4.1.8.2 KodakD-196 developer Water (50 ℃) 500mL, dissolved sequentially metol (sulfuric acid methyl amino phenol) 2g, anhydrous sodium sulfite 72g, hydroquinone 8.8g, Anhydrous sodium carbonate 48g, potassium bromide 4g, dilute to 1000mL. 4.1.8.3 Stop Bath 98% glacial acetic acid 15mL, dilute to 1000mL. 4.1.8.4 KodakF-5 Fixer Water (50 ℃) 100mL, successively dissolved Hyperion 240g, anhydrous sodium sulfite 15g, 28% acetic acid 48mL, boric acid 7.5g, potassium alum 15g, dilute to 1000mL. 4.1.9 perchloric acid (0.25mol/L and 0.5mol/L) 70% perchloric acid 8.57mL, add water to 100mL, concentration of 1mol/L. 4.1.10 scintillation fluid Weigh 2,5-diphenyl oxazole (PPO) 5g, 1,4- bis - [5-phenyl-oxazol-2-yl] - benzene (POPOP) 300mg was dissolved in toluene, the volume To 1000mL. 4.2 Material Recommend the use of disposable cell culture vessels before and microporous membrane, general test equipment should use cell culture techniques requires cleaning package Tie, disinfection and sterilization. On the glass with a rubber stopper and metal utensils available without high-temperature sterilization, the temperature rose to 140 ℃, the holding 2h. Rubber and class Glass with a rubber stopper with the application autoclave 120 ℃, 30min. In addition to solution heat-sterilization filtration applications, the heat available autoclaving Bacteria, usually with 115 ℃, 10min.

5 Test methods

5.1 test substance When the test subjects was freshly prepared. First test substance dissolved in a suitable solvent (distilled water, dimethyl sulfoxide, acetone, etc.), to prepare a desired Concentration, when added to the test medium to a final concentration of 1% of solvent, does not affect cell viability. Control 5.2 Each test should be set both with and without metabolic activation system, both positive and negative case (vehicle) control. When using rat hepatocytes tested positive controls available 7,12 DMBA (7,12-DMBA, 7,12-dimethyl- benzathracene) and 2-acetylaminofluorene (2-AAF, 2-acetylaminofluorene). If a cell line, in the absence of metabolic activation system affection Under conditions, autoradiography and LSC can be detected with 4-nitroquinoline (4-NQO, 4-nitroquinolineoxide) as a positive control Thereof; and in the case with metabolic activation system, dimethylnitrosamine then with N- (N-dimethylnitrosamine) as a positive control. 5.3 Exposure concentrations The highest concentration of the test substance exposure obtained by the choice of pre-test. Concentration of the test was multiple choice pre-test to determine the concentration of drug should be covered Suitable range of reactions. The highest concentration of the test substance should have some exposure cytotoxicity. Relatively insoluble in water test substance should be able to reach its The maximum solubility of the test. For water soluble test substance shall be determined according to the range of values exposed to the test substance concentration cell viability. 5.4 metabolic activation Unless primary hepatocytes, cell lines built in drug metabolizing enzyme activation activity is generally low, and therefore subject to a number of metabolic enzymes live Of it shows the DNA damage chemicals can be added to the test system in rat liver microsomes composed of enzymes and cofactors In vitro activation system (S-9 mixture). 5.5 Cell Culture 5.5.1 Cell Selection Primary cultured cells (eg rat liver cells), human lymphocytes or cell lines have been built (such as human amnion FL cell lines, human diploid fibroblasts Dimensional cells, Hela cells) can be used for this test. UDS response of human cells is greater than rodent cells. Most human use fine Cell fibroblasts, peripheral blood lymphocytes, monocytes and Hela cells. 5.5.2 Culture Conditions The choice of appropriate growth medium, CO2 concentration, temperature and humidity to maintain cell growth. Cell lines should regularly check for mycoplasma Pollution. 5.5.3 cell passaging, maintenance and storage Grown monolayer cells, the medium was removed. After washing with Hanks balanced salt solution (HBSS), or 0.1% trypsin with 0.02TA The enzyme solution (in no Ca2, Mg2 of phosphate buffer) at 37 ℃ under treatment for several minutes the cells shrink, increased cell gap. Then HBSS washed once by adding an appropriate amount of the medium, repeated pipetting, so take off from the glass surface of the cell and dispersed in the medium. Take a cell suspension Drops in blood count plus the pool, counting the number of cells in 4 large grid of calculated cell concentration in the suspension (4 large grid of cells/4 × 104 ie Contained per ml of cells). The cell suspension was diluted with growth medium to 0.5 × 105 universal measure word/mL ~ 1 × 105 universal measure word/mL. The above cells The suspension seeded in culture flasks (30mL flasks can be vaccinated can be vaccinated flasks 3mL.100mL 10mL). 3 parts of each vaccination, Grow into a confluent monolayer after taking the above method wherein the bottle then passaged received 3 bottles. Another two bottles prove successful discarded after passage or for tests Use, it will ensure the continuation of holding cells in the test. Conditional press law the cells were stored in liquid nitrogen. If you do not have a long time, do not To maintain in the laboratory. After removing the need for through proliferation when used in the test. The proliferating cells to the required number, press the cell suspension into the legal system Solution (in a growth medium used). Cell concentration of 1 × 106 a /mL~1.5×106 a/mL. In an ice bath, was gradually added to the cell suspension The total amount of 10% solution of sterile dimethylsulfoxide. The cell suspension was then divided glass containers in a clean, dry sterilized or cryopreservation dedicated Small plastic tubes, each 1mL. After sealing, at 4 ℃ in 2h ~ 3h, then moved to an indoor ice conventional refrigerator of 4h ~ 5h, then moved Low temperature freezer -30 ℃ ~ -20 ℃ within overnight. The next morning moved biological ampoule with liquid nitrogen storage. When required, the ampoules or small Plastic pipe saw (play) open, remove medium containing dimethyl sulfoxide, adding an appropriate amount of growth medium used, and adjust the cell concentration to 10 × 104/mL ~ 15 × 104 universal measure word/mL. Minutes in cell culture flasks, 37 ℃ 1h after culture, a culture change, it will be non-viable cells Removed until after a long pass into the proliferation of sub-confluent monolayers maintained in the laboratory. 5.6 Procedure NOTE. According to the laboratory, the DNA selectable by autoradiography or liquid scintillation techniques chromogenic assay (LSC) method for the determination of exposed cells revealed The incorporation of 3H-TdR. 5.6.1 UDS method of autoradiography The proliferating cells to the desired quantity, the above process a single cell suspension. Ge concentration of 0.5 × 105/mL ~ 1 × 105 Ge/mL. The above cell suspension in the home of a small cover sheet (18mm × 6mm) of the culture flasks, 37 ℃ culture 1d ~ 3d, the cells on the cover sheet Grown to an appropriate density. The number of inoculated culture bottle according to the number of the test substance, selected dosage level may be. Each dose for 2 ~ 3 samples Tablets, and other equipment 4 to 6 samples for vehicle control and the positive control of known carcinogens. Cell proliferation after the culture, the media synchronization (ADM supplemented with 1% fetal bovine serum) for synchronous culture 3d ~ 4d. In the afternoon the day before the test, dissolved in ADM of hydroxyurea (HU) Solution, so that in the medium HU final concentration of 10mmol/L. Incubated for 16h at 37 ℃, then the cells have long said cover sheet Placed, containing different concentrations of the test substance, HU [c (HU) = 10mmol/L] and 3H- thymidine (5μCi/mL ~ 10μCi/mL, 30Ci/mmol) of synchronization with the medium. 37 ℃ incubated for 5h. After incubation, the HBSS sufficiently washed with 1% sodium citrate solution treated 10min, followed by ethanol (64-17-5) - acetic acid (3.1) Fixed (4 ℃) overnight, after air drying, with a small amount of neutral gum, cement cover sheet on the slide, having cell side toward On, 45 ℃ baking 24h. In a dark room, the amount of transferred nuclear -4 latex impregnated with the glassware, the melt in 40 ℃ water bath, while taking an equal amount of distilled Water in a graduated cylinder is also disposed in the water bath, the latex until melted, distilled water was poured onto the hot latex was continued heated in a water bath, and treated with Glass rod stirring gently, wait 10min ~ 20min, air bubbles escape. The slides were prepared to make the development process from the water bath and placed on the platform Preheating. The slides were immersed vertically to 1.1 dilution of the latex bath for about 5s, slowly raise the slide and latex on the back of the slide with gauze or rub Wipe mirror paper, which has been coated with a latex of the slide moved to a temperature of 29 ℃ and a certain humidity of the incubator (4h) to be latex dry operation is paused. Then placed in Exposure setting appropriate amount of desiccant (silica gel discoloration) bags of the box. Exposure box outsourcing dark black paper and plastic sheet, placed in 4 ℃ refrigerator exposure 10d. After the end of exposure, the slides were transferred to the slide rack made of plexiglass, a liquid temperature of 19 ℃ of the D-170 or D-196 color solution Developing 5min, rinsed stop bath in 2min, the fixing 6min ~ 10min in the F-5 fixing solution, and then rinsed with water for several hours. Cells can be glacial acetic acid solution or after the developer with HE staining or Giemsa dye latex smears before land Yi Hong (2%). The glass After dehydration transparent sheet with a cover sheet sealing. In grunge microscope, counting each sample nuclei developed silver grains while silver background counting grains of the same size, and calculates both Difference. Each slide counted at least 50 nuclei calculated in the control group, each test substance group and the silver grains in the positive control group the mean and standard Quasi poor statistics. To distinguish between UDS and semi-conservative DNA replication normal, medium, low serum medium may be lacking or arginine in culture Hydroxyurea group added ways to reduce and inhibit the normal semi-conservative replication of DNA. 5.6.2 UDS liquid scintillation technique chromogenic assay The test cells were suspended in the growth medium used, cell concentration of 0.5 × 105 universal measure word/mL, the cells were seeded in liquid scintillation counting bottles The bottle 1mL, and 14C-thymidine was added to a final concentration of 0.01μCi/mL (50mCi/mmol). 37 ℃ cultured 48h make Cell proliferation and pre-labeled, and the medium was washed with HBSS, change containing 14C- thymidine (0.01μCi/mL) of the synchronization training Support group, performed at 37 ℃ in synchronized cultured 2d ~ 4d, on the day before the test in the afternoon to the medium, HBSS thoroughly washed after use, containing added Hydroxyurea [c (HU) = 10mmol/L] synchronize with the medium, 37 ℃ incubated 16h, UDS induced with radiographic display method, fine In synchronous cells with medium containing HU and 3H- thymidine (5μCi/mL, 30Ci/mmol) with various concentrations of the test substance is then Touch 5h, after the end of incubation, the medium was removed and the test substance. Washed twice with cold saline, followed by ice-cold perchloric acid [7601-90-3, c (HClO4) = 0.25mol/L] was treated twice, each time 2min, then ethanol treatment 10min, to 0.5mL dry perchlorate [C (HClO4) = 0.5mol/L ~ 1mol/L] in a thermostat of 75 ℃ ~ 80 ℃ hydrolyzed 40min. After cooling, ether was added ethylene glycol 3.5mL and scintillation fluid (PPO0.5%, POPOP0.03%, toluene as solvent) 5mL, shaking uniform liquid scintillation counter measurement Each sample the radioactivity of 3H and 14C. Each group (including controls) do at least six flasks. Specimens 3H radioactivity that is reflected in the UDS 3H- thymidine incorporation nucleosides; and 14C radioactivity reflects the test cell The number or amount of DNA, thus 3H and 14C radioactivity ratio of (3H/14C) is the number of units or cells per unit mass in DNA UDS Level. If the negative control group 3H/14C as 100% (1.00) to calculate the amount of change of each test substance group and negative control group and the test Mean and standard deviation statistics for each test concentration was set.

6 Data processing and evaluation of results

6.1 Data Processing Selection of appropriate statistical methods such as "t test" to determine between the test substance group and the vehicle control group was statistically significant difference in the presence or absence of data into For analysis and evaluation. 6.2 Evaluation Results Cell 3H-TdR incorporation test substance group number increases with dose, and compared with the negative control group was statistically significant, or at least In one test point to get repeatable and statistically significant increase in the amount of incorporation, it may be determined that the test was positive.

7 Test report

7.1 Name of test, the test unit name and contact details, report number. 7.2 Test Requester name and contact information, sample acceptance date. 7.3 Test start and end dates, test project manager, technical director of the test unit, date of issue. 7.4 test summary. 7.5 test substance. name, cell lines, CAS number (if known), purity, and this trial-related physical test substance and chemical properties and stability Qualitative and so on. 7.6 Solvent. solvent selection basis, solubility and stability of the test substance in the vehicle. 7.7 cell lines. name, source, concentration and culture conditions (including the composition of the medium, temperature, CO2 concentration and incubation time). 7.8 Test conditions. dosage, metabolic activation system, cell lines, mutagens, and other steps. 7.9 Test Results. The test substances on cell lines toxicity, if a dose - response relationship, the statistical results, the vehicle control simultaneously and yang Control of the mean and standard deviation. 7.10 Conclusion. Under the experimental conditions of the test was whether the mutagenic effect. Explanation 8 trial Cell 3H-TdR incorporation test substance group number does not change with dose increases, each dose group and the control group were not statistically significant, the That the test substance does not cause UDS in this experimental system. When the result of the determination, and the biological significance should be considered statistically significant.

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