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GB 15193.20-2014: National Food Safety Standard -- TK gene mutation test
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National Food Safety Standard -- TK gene mutation test
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Basic data | Standard ID | GB 15193.20-2014 (GB15193.20-2014) | | Description (Translated English) | National Food Safety Standard -- TK gene mutation test | | Sector / Industry | National Standard | | Classification of Chinese Standard | C53 | | Classification of International Standard | 07.100 | | Word Count Estimation | 9,997 | | Date of Issue | 12/24/2014 | | Date of Implementation | 5/1/2015 | | Older Standard (superseded by this standard) | GB 15193.20-2003 | | Regulation (derived from) | Health Planning Commission Bulletin 2014 No. 21 | | Issuing agency(ies) | National Health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 vitro mammalian thymidine kinase (thymidine kinase, TK) gene mutation test basic test methods and technical requirements. This Standard is applicable to the evaluation of mutagenicity test substance. | GB 15193.20-2014: National Food Safety Standard -- TK gene mutation test---This is a DRAFT version for illustration, not a final translation. Full copy of true-PDF in English version (including equations, symbols, images, flow-chart, tables, and figures etc.) will be manually/carefully translated upon your order.
National Food Safety Standard.TK gene mutation test
National Standards of People's Republic of China
National Food Safety Standard
TK in vitro mammalian cell gene mutation 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 15193.20-2003 "TK gene mutation test."
This standard compared with GB 15193.20-2003, the main changes are as follows.
--- Standard name was changed to "national food safety standard in vitro mammalian cell gene mutation assay TK";
--- Modify the scope;
--- Increasing the terms and definitions;
--- Modified test purposes and principles;
--- Increasing the metabolic activation system;
--- Increased THMG and THG selective media method of preparation;
--- Increased CHAT CHT and selective media method of preparation;
--- Increased trifluorothymidine method of preparation;
--- Increased phosphate buffer preparation methods;
--- Modify the test substance dose setting requirements;
--- Modify control settings;
--- Increase alternative cell lines and the corresponding test methods and data processing;
--- Increasing the requirements of the test report;
--- Expanded to explain the requirements of the test.
National Food Safety Standard
TK in vitro mammalian cell gene mutation test
1 Scope
This standard specifies the in vitro mammalian thymidine kinase (thymidinekinase, TK) gene mutation test basic test methods and techniques
Claim.
This standard applies to the evaluation of the test substance mutagenic effect.
2 Terms and definitions
2.1 TK gene
Mammalian thymidine kinase gene. TK human genes located in the distal end of the long arm of chromosome 17; mice are located in the
On the 11th chromosome.
2.2 mutation frequency
In some cell lines, a particular gene mutant cells (colonies) occupy cells (colonies) of the total proportion (unit usually 10-6).
3 test purposes and principles
TK gene mutation test endpoint detection is TK gene mutations. TK gene mutation autosomal gene mutations.
TK thymidine kinase gene products in vivo catalytic generate thymidylate (TMP) reaction from thymidine (TdR). In normal circumstances
Under this reaction it is not necessary for life, because the body mainly from the TMP-deoxy-uridine (dUMP), that is, from thymidylate
dUMP methylation reaction synthesizing enzyme-catalyzed generation TMP. However, if the thymidine analogue was added in cell culture (e.g. trifluorothymidine, i.e.
trifluorothymidine, TFT), the TFT can generate trifluorothymidine acid catalyzed thymidine kinase, and then incorporated into the DNA, causing death
Mutation, so the cells can not survive. If the TK gene mutations that cause defects thymidine kinase, phosphorylation of the TFT can not, nor can be incorporated
DNA, so the mutant cells in a culture medium containing the TFT can grow, that exhibit resistance to TFT. According to the number of colony forming mutations set,
Calculate the mutation frequency and infer mutagenicity test substance. In the TK gene mutation test results can be found in two types of observation significantly different
Colonies, namely large/small colonies (L5178Y cells) or normal growth/slow-growing colonies (TK6 cells), studies have shown that large colonies/Normal
Growth colonies mainly by a point mutation or deletion of a small range of other causes, and small colony/colonies mainly by the slow growth of a wide range of abnormal chromosomes
Change, or deletion of the gene involved in the regulation of cell proliferation caused.
4 instruments and reagents
4.1 Instrument
Commonly used laboratory equipment, a deep freezer (-80 ℃) or liquid nitrogen tank, biological safety cabinet, incubator, inverted microscope, centrifuge.
4.2 Medium
4.2.1 complete medium
RPMI1640 medium, 10% horse serum (culture flasks) or 20% horse serum (96 well plates) and the amount of antibiotic (Green
Final concentration of neomycin, streptomycin were 100IU/mL and 100μg/mL).
4.2.2 THMG and THG selection medium
THMG medium. 3μg/mL thymidine (thymidine, T) 5μg/mL hypoxanthine (hypoxanthine, H)
0.1μg/mL methotrexate (methotrexate, M) 7.5μg/mL glycine (glycine, G).
THG medium. 3μg/mL thymidine (T) 5μg/mL hypoxanthine (H) 7.5μg/mL glycine (G).
Above a concentration of each reagent at a final concentration in the medium. Practical tests, often according to the method of Table 1 and THG dubbed THMG
100-fold concentration.
Configuration Table 1 T, H, M, G reagent
Reagent relative molecular mass mass/mg and the volume of solvent concentration/(mg/mL)
T 242.2 30 10mLH2O 3
H 136.1 5 1mL1mol/LHCl 5
M 454.5 5 50mLH2O 0.1
G 75.07 75 10mLH2O 7.5
The above-described four kinds of reagents formulated in a 1000-fold concentration, this concentration then were taken of T, H, M, G 5mL each solution (20 mL total), or
T, H, G solution each 5mL (a total of 15mL), were diluted with distilled water to 50mL, dubbed 100X THMG or THG stock solution.
Finally filtered through a membrane filter sterilization, packaging, stored at -20 ℃. 1% of the proportion of complete medium when applying.
4.2.3 CHAT selection medium and CHT
CHAT media. 1 × 10-5mol/L deoxycytidine (cytosinedeoxyriboside, C) 2 × 10-4 mol/L hypoxanthine
(Hypoxnathine, H) 1 × 10-7mol/L aminopterin (aminopterin, A) 1.75 × 10-5mol/L thymidine (thymidine, T).
CHT medium. 1 × 10-5mol/L deoxycytidine (C) 2 × 10-4mol/L hypoxanthine (H) 1.75 × 10-5mol/L Chest
Glycosides (T).
Above a concentration of each reagent at a final concentration in the medium. Practical tests, often according to the method of Table 2 and CHT dubbed the CHAT
100-fold concentration.
Table Configuration 2 C, H, A, T reagent
Reagent relative molecular mass mass/mg and the volume of solvent concentration/(mol/L)
C 227.2 113.6 50mLH2O 1 × 10-2
H 136.1 1361.0 50mL1mol/LHCl 2 × 10-1
A 440.4 2.2 50mLH2O 1 × 10-4
T 242.2 423.85 10mLH2O 1.75 × 10-2
The four kinds of reagents formulated in a 1000-fold concentration, and then were taken of the concentration C, H, A, T 5mL each solution (20 mL total), or C, H, T
Solution of each 5mL (a total of 15mL), were diluted with distilled water to 50mL, dubbed 100X CHAT or CHT stock solution. Finally filter
Membrane filtration sterilization, packaging, stored at -20 ℃. 1% of the proportion of complete medium when applying.
4.3 metabolic activation system
4.3.1 S9 cofactors
4.3.1.1 magnesium potassium solution
1.9g of magnesium chloride and potassium chloride 6.15g add distilled water to 100mL.
4.3.1.2 0.2mol/L phosphate buffer (pH 7.4)
Disodium hydrogen phosphate (Na2HPO4,28.4g/L) 440mL, sodium dihydrogen phosphate (NaH2PO4 · H2O, 27.6g/L) 60mL, pH adjusted
7.4,0.103MPa20min sterilization or to filter bacteria.
4.3.1.3 coenzyme -Ⅱ (oxidized) solution
Under sterile conditions weigh coenzyme -Ⅱ, with sterile distilled water formulated to 0.025mol/L solution. Using now.
4.3.1.4 Glucose-6-phosphate sodium salt solution
Glucose-6-phosphate sodium salt was weighed, dissolved in distilled water formulated as 0.05mol/L, sterilized by filtration. Using now.
4.3.2 Preparation of rat liver S9 fraction
Choose healthy male adult SD or Wistar rats weighing about 150g ~ 200g, about 5 weeks to 6 weeks of age. Using phenobarbital
Flavonoids and β- naphthalene induced by the method of preparation by oral gavage to rats phenobarbital and β- naphthalene flavonoids, doses were 80mg/kg
Weight, continuous 3d, 16h after fasting decapitated animals were sacrificed before fasting 12h.
Animals were sacrificed after the liver was removed, weighed fresh ice-cold solution of potassium chloride (0.15mol/L) continuous flushing liver several times to remove
It can inhibit the activity of microsomal hemoglobin. Per gram of liver (wet weight) was added potassium chloride solution (0.1mol/L) 3mL, along with an ice bath and transferred to a beaker
The liver minced with sterile scissors in glass homogenizer (less than 4000r/min, 1min ~ 2min) or tissue homogenizer (less than
20000r/min, 1min) is made in the liver homogenates. The above operation should pay attention to the local cold and sterile environment.
The liver homogenates prepared at low temperature (0 ℃ ~ 4 ℃) high-speed centrifuge at 9000g centrifugation 10min, the supernatant was aspirated S9 fraction,
Aliquot in sterile freezer tubes, each tube about 2mL, frozen with liquid nitrogen or dry ice rear -80 ℃ cryopreservation.
After S9 fraction prepared by the sterility test, measuring protein content (Lowry method) per milliliter protein content of not more than 40mg is appropriate, and by
Indirect mutagens qualified to identify its biological activity after storage at -80 ℃ low temperature or freeze dry, a period not exceeding one year.
4.3.3 10% S9 mixture
Usually by the S9 fraction and cofactors press 1.9 mixture consisting of 10% S9, sterile, now with the existing or filter sterilized. 10% S9
10mL mixture prepared as follows.
6.0mL phosphate buffer
Magnesium potassium solution 0.4mL
Glucose-6-phosphate sodium salt solution 1.0mL
Coenzyme -Ⅱ solution 1.6mL
Liver S9 fraction 1.0mL
Mix, set in an ice bath until ready to use.
S9 mixture concentration is generally 1% to 10%, the actual concentration determined by individual laboratories, but its activity needs identified,
It should be able to significantly activate the positive control, and cells without significant toxicity.
4.4 phosphate buffer (phosphatebuefferdsaline, PBS)
The 8.0gNaCl, 0.20gKCl, 2.74gNa2HPO4 · 7H2O, 0.20gKH2PO4 dissolved in distilled water and dilute to
1000mL, pH7.2 ~ 7.4.
4.5 preparation of trifluorothymidine (trifluorothymidine, TFT) of
Take TFT30mg, was dissolved in PBS was added to 10mL, dubbed the stock solution 3mg/mL of. The application by 1 ‰ volume Bijiarupei
Support group.
5 Test methods
5.1 and cell culture conditions
tk/- genotype of mouse lymphoma L5178Y-3.7.2C or TK6 human lymphoblastoid cells.
Both cell were 5% carbon dioxide, 37 ℃, saturated humidity condition to make a conventional suspension culture.
To avoid the influence of culture and passage during the spontaneous mutation of cells on the test results, before the official test, should be cleared of spontaneous mutation
tk -/- genotype cells. the way is.
a) For L5178Y cells were treated with medium using THMG 24h, to 800r/min ~ 1000r/min centrifugal speed
4min ~ 6min, after washing without methotrexate in the culture medium THG 2d;
b) For TK6 cells treated with medium use CHAT 48h, to 800r/min ~ 1000r/min speed centrifugal 4min ~
6min, washed and cultured in aminopterin-free medium of CHT 3d.
5.2 test substance
5.2.1 formulated test substance
Test substance prior to use now with the existing, it shall be verified under specific storage conditions do not affect its stability.
5.2.2 test substance dose setting
Shall be at least 4 for analysis of the concentration of 3 ~. For the cytotoxicity of the test substance, the test should be based on the results of pre-cytotoxic,
In the RS or RSG 20% to 80% are within a three to four-dose (concentration) level, and should consider the test substance solubility, pH
And osmolarity affected. The method is. take good cell growth, adjusting a density of 5 × 105/mL, 1% by volume by adding different
Concentration of the test substance, 37 ℃ shaking process 3h (L5178Y cells) or 4h (TK6 cells), cells were washed by centrifugation, for 2d (L5178Y
Cell) or 3d (TK6 cells) expression of culture, counting each day and calculate the relative density of the cell suspension growth (RSG). Or a fine after taking the above treatment
Cell suspension, for dilution to 108 cells/mL, inoculated 96 (per well 0.2mL, an average of 1.6 cells/well), each dose Species
Cultivation of a ~ two plates, 37 ℃, 5% carbon dioxide, saturated humidity 12d, counting each plate has a number of holes colony growth is calculated
Relative survival (RS).
For extremely low cytotoxicity of the test substance, the concentration should be set up to 5mg/mL, 5μL/mL or 0.01mol/L. For relatively insoluble
Substance solution, which should be set to achieve the highest concentration does not affect the cell culture may be added to the maximum concentration.
5.2.3 Control settings
Under normal circumstances, each one trial, in the presence of metabolic activation system and the absence shall be provided with positive and negative (vehicle) on
Control group.
When using a metabolic activation system, the positive control was requested metabolic activation should be used, and can cause mutation colonies typical substances that can make
With 3-methyl-MCA (3-methylcholanthrene), cyclophosphamide (cyclophosphamide, CP) and the like. In the absence of metabolic activation system,
Positive controls may be used methyl methanesulfonate (methylmethanesulfonate, MMS), mitomycin C (mitomycinC,
MMC), ethyl methane sulfonate (ethylmethanesulfonate, EMS) and the like. It may also be used other suitable positive controls.
Solvent should be non-mutagenic not react chemically with the test substance, does not affect cell survival and S9 activity. The preferred solvent of distilled water, such as
Non-aqueous solvent (optionally dimethylsulfoxide, acetone, ethanol, etc.), need additional vehicle control.
5.3 Processing
Take good cell growth, adjusting a density of 5 × 105/mL, at 1% by volume of added test substance (required metabolic activation, while adding
The final concentration of 1% to 10% of a mixture of S9), 37 [deg.] C shaking treatment 3h (L5178Y cells) or 4h (TK6 cells) to
Speed centrifugal 800r/min ~ 1000r/min for 4min ~ 6min, the supernatant with PBS or serum-free medium cells were washed
2 times, cells were resuspended in RPMI1640 medium containing 10% horse serum solution, and adjust the cell density of 2 × 105/mL.
5.4 PE0 (0d plate plating efficiency) Determination
Take appropriate cell suspension for dilution to 108 cells/mL, inoculated 96 (per well 0.2mL, an average of 1.6 cells /
Kong), one species per dose ~ 2 plates, cultured 12d at 37 ℃, 5% carbon dioxide and saturated humidity, counting each plate there are colonies
Growth of the number of holes.
5.5 Expression
Take 5.3 resulting cell suspension for 2d (L5178Y cells) or 3d (TK6 cells) expression of culture, the cell density counted every day and keep
Density of 106/mL or less, calculated relative survival (RSG).
5.6 PE2 (L5178Y cells) or PE3 (TK6 cells) measured
After the end of the expression of the culture, take appropriate cell suspension was measured PE2/PE3 by 5.4 methods.
5.7 mutation frequency (MF) Determination
5.7.1 L5178Y cells
After L5178Y cells cultured 2d, take appropriate cell suspension, adjusting the cell density of 1 × 104/mL, was added TFT (final concentration
3μg/mL), mix inoculated 96 (per well 0.2mL, an average of 2000 cells/well), each dose for 2 to 4 plates,
Cultured 12d at 37 ℃, 5% carbon dioxide and saturated humidity conditions, mutations count the number of colonies growing hole. Mutant colonies by large colonies
(LargeColony, LC. diameter ≥1/4 aperture, low density) and small colonies (smalcolony, SC. diameter < 1/4 aperture, high density) points
Do not count. After minimal colonies can be cultured 3d counting.
5.7.2 TK6 cells
After TK6 cells cultured 3d, take appropriate cell suspension, adjusting the cell density to 1.5 × 105/mL, was added TFT (final concentration
3μg/mL), mixed, seeded 96 well plates (0.2mL added per well, i.e., an average 30,000 cells/well), each dose for 2 ~ 4 plates,
Cultured 12d at 37 ℃, 5% carbon dioxide, saturated humidity, counting the normal growth of mutant colonies (normal-growthcolony, NC).
Then an appropriate amount of each hole and then an additional TFT, cultured 12d, count slowly grow into new growth mutant colonies (slow-growthcolony, SC).
6 Data processing and evaluation of results
6.1 Data Processing
6.1.1 plating efficiency (PE0, PE2/PE3)
Plating efficiency (%) is calculated, see equation (1).
PE = -
ln (EW/TW)
1.6 × 100%
(1)
Where.
EW --- no colony growth of the number of holes;
TW --- Total number of holes;
1.6 --- inoculated cells per well.
6.1.2 Relative survival (RS)
Relative survival rate (%) is calculated, see formula (2).
RS =
PE treatment
PE negative/vehicle control × 100%
(2)
Note. When using a non-aqueous solvent vehicle, compared with the vehicle control.
6.1.3 Relative suspension growth (RSG)
Calculate the relative suspension growth (%) see formula (3).
RSG =
Expression of cell proliferation in multiples treated during
Cell proliferation in multiples during the negative/vehicle control group was × 100%
(3)
Note. When using a non-aqueous solvent vehicle, compared with the vehicle control.
6.1.4 relative total growth (relativetotalgrowth, RTG)
Calculating the relative total growth (%) see formula (4).
RTG = RSG × RSn × 100% (4)
Where.
RSn --- Day 2 (L5178Y cells) or three days (TK6 cells) relative survival.
6.1.5 mutation frequency (MF)
Mutation frequency calculation see formula (5).
MF (× 10-6) = -
ln (EW/TW)/N
PE2/3
(5)
Where.
EW --- no colony growth of the number of holes;
TW --- Total number of holes;
N --- per seeded cells (L5178Y cells 2000, TK6 cells 30,000);
PE2/3 --- Day 2 (L5178Y cells) or three days (TK6 cell) of plating efficiency.
In addition, L5178Y cells, large colonies can be calculated separately mutation frequency (L-MF), small colonies mutation frequency (S-MF) and total mutation
Frequency (T-MF). For TK6 cells can calculate normal colony mutation frequency (N-MF), slowly growing colony mutation frequency
(S-MF) and total mutation frequency (T-MF).
6.1.6 small colony mutants percentage (smalcolonymutation, SCM) or slow the growth of mutant colonies percentage (slowly-growth
colonymutation, SCM)
Small percentage of mutant colonies or slow the growth of mutant colonies percentage calculation see formula (6).
SCM =
S-MF
T-MF × 100%
(6)
6.2 Evaluation Results
6.2.1 Test conditions established
L5178Y cells used in the test spontaneous mutation frequency should be between 50 × 10-6 ~ 200 × 10-6; spontaneous mutation frequency TK6 cells
Rate should be between 1.5 × 10-6 ~ 5.5 × 10-6, while spontaneous mutation frequency should be within the range of laboratory history. Negative/vehicle control
The PE0 between 60% ~ 140%, PE2/PE3 value of between 70% to 130%. Positive control and negative T-MF/vehicle control
There are significant differences, or negative/vehicle control more than 3 times.
6.2.2 The test substance is determined positive and negative results
6.2.2.1 The results of positive determination. More than one dose of the test substance (concentration) of T-MF group was significantly higher than the negative/vehicle control or female
More sexual/vehicle control three times, and a dose - response trend, it is judged as positive. However, if only in relative survival rate of less than 20% of the high dose
By the amount of positives cases, the result is judged as "suspicious."
Determination 6.2.2.2 negative results. In the case of increase relative survival rate of no less than 20% mutation frequency, it can be judged to be negative.
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, identification data, CAS number (if known), purity, and with the physical and chemical properties of the test related to the test substance
Stability.
7.6 vehicle and vehicle. vehicle or carrier selected on the basis, solubility and stability of the test substance in a solvent and carrier.
7.7 cell lines. name, origin and their characteristics.
7.8 Test conditions. solvent, doses, metabolic activation system, the positive control, and other steps.
7.9 Test Results. Each dose (concentration) expression during cell culture density, plating efficiency 0d, the first 2 days or 3 days flat
Board plating efficiency, relative survival, relative suspension growth and relative total growth, total mutation frequency [gives a large colony mutation frequency, if necessary, a small collection
Off the mutation frequency and small colony mutants mutation percentage (L5178Y cells), or normal colony mutation frequency of slow growth and mutation frequency
Of slow growth mutant percentage (TK6 cells)], statistical results, whether the dose - response trend, concurrent vehicle control and yang
The results of this laboratory control and vehicle control and the positive control results historic range.
7.10 Test conclusion.
Explanation 8 trial
TK gene mutation assay with high sensitivity can be detected including point mutations, large deletions, recombination, aneuploidy and other large range
Multiple genetic alterations, including genomic alterations, prolonged treatment can also check out a certain breaking agents, and polyploid induction spindle poisons like. but
In vitro tests do not fully simulate the in vivo mammalian metabolic conditions, therefore, the test results can not be directly extrapolated to mammals. Positive results
Indicates that the test sample may be used to cause gene mutations in mammalian cells under the test conditions; negative results showed that the sample under the test conditions
Product does not cause gene mutations in mammalian cells used. Evaluation of biological significance and must be considered statistically significant.
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