Some standard content:
ICS 13.300;11.100
National Standard of the People's Republic of China
GB/T 21793—2008
Chemicals-Test method of in vitro mammalian cell gene mutation
Issued on 2008-05-12
General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China Administration of Standardization of the People's Republic of China
Digital Enough
Implementation on 2008-09-D1
GB/T21793—2008
This standard is equivalent to the Organization for Economic Cooperation and Development (OECD) Chemical Testing Guide No. 176 (1997) "In vitro mammalian cell gene mutation test" (English version).
This standard is edited as follows:
——The scope part is added;
The measurement unit is changed to the legal measurement unit of my country;-The reference part of OECD is deleted. This standard is proposed and managed by the National Technical Committee for Hazardous Chemicals Management Standardization (SAC/TC251). The responsible drafting unit of this standard is: Occupational Health and Poison Control Institute of China Center for Disease Control and Prevention. The participating drafting units of this standard are: Beijing Center for Disease Control and Prevention, Tianjin Institute of Inspection and Quarantine Science and Technology. The main drafters of this standard are: Wu Wei, Lou Qun, Li Chaolin, Lin Zheng, Zhang Yuan, Li Ningtao. 1
GB/T21793—2008
OECD cited
1. In vitro mammalian gene mutation test can be used to detect gene mutation induced by chemicals. Available cell lines include L5178Y mouse lymphoma CHO cells, AS52 and V79 strains of Chinese hamster cells, and TK6 human lymphoblastoid cells. In these cell lines, the most commonly used genetic endpoints are the detection of thymidine kinase (TK) and hypoxanthine-guanine phosphoribosyltransferase (HPRT) mutations, as well as xanthine-guanine phosphoribosyltransferase (XPRT) benzene mutations. TK, HPRT and XPRT mutation tests detect different genetic event spectra. TK and XPRT are located on autosomes and can detect genetic events that HIPRI (located on chromosome X) cannot detect, such as large fragment deletions.
2. In in vitro mammalian cell gene mutation tests, established cell lines or cell line cultures can be used. Cells should be selected based on their ability to grow in culture and whether the white hair mutation rate is stable. In vitro tests usually require the use of exogenous metabolic activation systems. However, the exogenous metabolic activation system cannot completely simulate the metabolic conditions in mammals, so measures should be taken to avoid the situation where the gene mutation in vivo cannot be reflected. Changes in pH and osmotic pressure concentration or high cytotoxicity of the test sample can cause false positive results, so that the test results cannot reflect the true situation of gene mutation in vivo. 3. This test can be used to screen mammalian mutagens and carcinogens. Many compounds that are positive in this test are mammalian carcinogens, but there is no good correlation between this test and carcinogenicity. The correlation depends on the type of chemical. More and more evidence shows that many carcinogens cannot obtain positive results in technical tests because they act through other, non-genetic mechanisms or because their carcinogenic mechanisms are not easily detected in these cells. 1. National
Chemicals
In vitro mammalian cell
Gene mutation test method
GB/T 21793—2008
This standard specifies the scope, terms and definitions, basic principles of the test, test methods, test data and reports of in vitro mammalian cell gene mutation tests for chemicals.
This standard is applicable to the detection of base mutations in mammalian cells in vitro. 2 Terms and Definitions
The following terms and definitions apply to this standard. 2.1
Forward mutatian
A gene mutation that changes from the prototype (wild type) to a mutant type, which can cause changes and losses in enzyme activity and encoded proteins. 2.2
Base pair substitution mutagens Substances that can cause substitutions of one or several base pairs in DNA 2.3
Frameshift mutagens Substances that can cause the addition or deletion of single or multiple base pairs in a DNA molecule 2.4
Phenotypic expression time Phenotypic expression time The time required for new mutant cells to deplete the unchanged gene product. 2.5
Mntant frequency
Mutation frequency
The ratio of the number of mutant cells observed to the number of surviving cells. 2. 6
Relative total growth numberrelative lotal growthThe number of cells increased during the whole process compared with the number of cells in the negative control group, which is equal to the product of the number of cells in suspension growth and the relative colony formation efficiency.
relative suspension growlh
Relative suspension growth
The number of cells increased during the whole expression period compared with the negative control group, 2.8
Eviahility
Cell viability
The ability of cells inoculated in selective medium to form colonies after the expression period. 2.9
Cell survival rate
surival
The efficiency of cells inoculated in selective medium to form colonies after the treatment, which is expressed as the ratio of the survival rate to the number of cells in the control group. 3 Basic principles of the test 3.1 Since TK+ cells are mutated to TK-/- cells lacking TK, the mutants are resistant to the cytotoxic effects of the fluorothymidine analogue thymidine (TFT). Cells rich in TK are sensitive to TFT, which leads to inhibition of cell metabolism and further differentiation of cells. Therefore, mutant cells can grow rapidly in the presence of TFT, while positive cells cannot grow and proliferate due to the presence of TK. Similarly, cells lacking HPRT or XPRT can be selected because they are resistant to 6-thioguanine (6-TG) and 8-azaguanine (8-AG), respectively. If the test substance is a base analog or a compound related in structure to the selector, the characteristics of the test substance should be carefully considered when conducting any mammalian cell benzene mutation test to determine whether it is suitable for this test. For example, the possible selective toxicity of the test sample to mutant cells and non-mutant cells should be studied. In other words, when testing compounds related in structure to the selector, the characteristics of the selection system/selector must be verified. 3.2 With or without the addition of a metabolic activation system, cells in suspension or monolayer culture are exposed to the test substance for a certain period of time, and then the cells are subcultured to determine cytotoxicity and to express their phenotype before selecting mutant cells: Cytotoxicity is generally measured by measuring the relative colony formation efficiency (viability) or the relative total growth number of colonies after the cells are treated. After the treated cells are kept in the culture medium for a sufficient time, the induced mutant phenotype is expressed to a near observable level according to the characteristics of the selected cells and the mutation site. The mutation rate is determined by inoculating cells for a known number of days in a medium containing a selection agent to detect the number of mutant cells, or by detecting the colony formation efficiency (viability) in a medium without a selection agent, and incubating for an appropriate time to count the cell colonies. The rate is calculated using the formula based on the number of mutant colonies in the selective culture medium and the number of colonies in the non-selective culture medium. 4 Test Methods
4.1 Test Preparation
4.1.1 Cells
4.1.1.1 There are a variety of cell types available for this test, including L5178Y subpopulation, CHO, AS52, V79 or TK6 cells. The cell types used in this test should have clear sensitivity to chemical mutagens, high colony formation efficiency and stable white hair mutation rate. The cells should be tested for mycoplasma infection, and cannot be used if they are already infected. 4.1.1.2 The sensitivity and detection capacity (power) should be determined in advance when setting up the test. The drug dose group should be able to reverse the specific parameters
The number of cells used, the number of cultured cells/cells and the minimum number of cells that survive after treatment and the minimum number of cells used in each test stage should be determined based on the spontaneous mutation rate. The general principle is that the number of cells used is at least 10 times the reciprocal of the spontaneous mutation rate. However, the cell system used for the application should have appropriate quality data to ensure that the stable cell number of the test results is at least 10% of the cells. Laboratory reliability evaluation
4.1.2 Culture medium and culture conditions
The culture medium and incubation conditions (culture medium, temperature, CO volume fraction and concentration) should be selected according to the selection system and cell type used in the test. It is particularly important that the selected culture conditions should ensure that the cells are in the optimal growth state during the expression period and that the mutant cells have the ability to form colonies. 4.1.3 Culture preparation
The cells obtained from the bacterial culture are inoculated into the culture medium and incubated at 37 °C. The mutant cells in the test cells need to be cleared in advance.
4.1.4 Metabolic activation
The cell line should be contacted with the test sample in the presence or absence of an exogenous mammalian metabolic activation system. The most commonly used activation system is the post-mitochondrial fraction (S.) with helpers prepared from the liver of rodents treated with enzyme inducers. The enzyme inducers used include Aroclor 1254 or a combination of phenobarbital and β-naphthoflavone. S, is usually used in the culture medium in a final volume fraction range of 1% to -10%. The metabolic activation system and its application conditions should be selected according to the characteristics of the test sample. In some cases, more than one S, concentration may be used. With the continuous development of metabolic activation systems, genetically engineered cell lines that can express special active enzymes have been constructed, which can provide cells with endogenous metabolic activation capabilities. However, the cell lines used should be selected according to scientific methods (for example, analyzing the correlation between cell chromatin P450 coenzymes and test substance metabolism). 4.1.5 Test substance/preparation
Solid test samples should be dissolved or suspended in a suitable solvent or excipient, and can be appropriately diluted if necessary before treating cells. Soluble test samples can be directly added to the assay system or appropriately diluted before treatment. Freshly prepared test substances should be used. Otherwise, data should be available to prove that the solution is stable in storage.
4.2 Test conditions
4.2. Solvents/excipients
GB/1 21793—2008
Solvents/excipients should not react chemically with the test samples and have no effect on cell survival and S, activity. If the commonly used solvents or excipients are not used, data should be available to support their suitability. It is recommended to use water as a solvent or excipient whenever possible. If the test sample is unstable to water, organic solvents or excipients that do not contain water should be considered first. Water can be removed by molecular sieves: 4.2.2 Contact concentration
4. 2. 2. 1 When determining the maximum concentration, the cytotoxicity of the test substance, its solubility in the test system, and changes in pH or osmotic pressure should be considered.
In the formal test, the reproducibility of cells needs to be measured with or without the presence of a metabolic activation system, using cell integrity and growth as indicators, such as cell colony formation efficiency (viability) or relative total growth number. Determining cell integrity and cell solubility in the preliminary test is conducive to the design of the formal test. 4.2.2.3 At least four commercially available test concentrations should be selected. If there is cytotoxicity, the concentration design should cover the range from maximum toxicity to minimum or no toxicity, and the normal concentration interval should be between 2 and 10. If the highest concentration can produce cytotoxicity, then the cell viability (relative colony formation number) or relative total growth number should be controlled within the range of 10% to 20% (but not less than 10). For the test samples without cytotoxicity or with low cytotoxicity, the highest concentration should reach 5mg/mL, 5μL/mL.
Relatively indifferent
Test samples should be made to reach the solubility limit under the culture conditions as much as possible
The end of cell exposure should be observed
Evaluating the solubility at the beginning and end of the treatment can obtain valuable information, because there are substances insoluble in the test system.
The final
precipitates such as cells, S and serum should not interfere
4.2.3 Controls
The solubility of the test sample may change during the exposure process. Insolubility can be observed by eye. Each test should be set up with positive and negative (solvent or excipient) controls under the conditions of the activation system and without. 4.2.3. 1
When using metabolic activation, the positive substance selected should be a mutagenic substance that requires activation to have a mutagenic effect. 4.2.3.2 The following chemicals can be used as positive controls. See Table
Metabolic activation conditions
No need for exogenous metabolism
Activation system
Require exogenous metabolism
Enzymatic system
Chemicals that can be used as positive controls
Chemicals and their CAS numbers Www.bzxZ.net
Ethylmethanesulfonate LCAS No. 62-50-07
AS No/759-7:F91
Methyl methanesulfonate meLhylmethanesulfomat CAsAo.66 27-3
Ethyl methanesulfonate ethylmethanequlfomele [CAS Na, 62 50 C] ethylnitrosoumer [CAS Ne.759-73-9] 3-methylcholanthren CAS No. 56-19-5] N-nitrogadinethylamine [CAS Na. f2-75-9] 7,12-dimethylbenzanlhre&cne [CAS No. 57-97-6 cyclaphusphamide (monohydrate) LCAS Na.50-18-0 (6055-19-2)]
military (a) well benz (a) pyrere LCAS No, 50-32-8. 3-mcthylcholanthrene, CAS No. 56-15-5] N-nitrosudirnethylamine (high concentration S 4.2.3.3 Other suitable positive control reference substances may also be used. For example, if a certain experiment has historical data on 5-bromo-2°-deoxyuridine [CAS No. 59-14-3], it can also be used as a negative control reference substance. If possible, compounds related to the test substance in chemical structure may also be considered as positive controls.
4.2.3.4 In each experiment, the negative control shall be treated alternately with each treatment group except for the addition of solvents or excipients to the test medium. In addition, if there is no historical data to prove that the solvents or excipients used are non-toxic or mutagenic, a blank control group without any treatment shall also be set up.
4. 3 Experimental steps
4.3.1 Treatment of test samples
4.3.1.1 In the presence or absence of a metabolic activation system, allow the proliferating cells to contact the test samples for an appropriate period of time (usually 3h to 6h is effective). The contact time can also be extended to one or more cell cycles. 4.3.1.2 Each concentration of the test sample can be tested using only one culture dish/bottle, or parallel samples can be made. If only one culture dish/bottle is used, the number of concentration groups should be increased to ensure that there are enough culture dishes/bottles for analysis (e.g., at least 8 concentrations available for analysis). Parallel samples of negative controls (solvents) should also be set up.
4.3.1.3 Gaseous or volatile substances should be tested in an appropriate manner, such as in a closed culture vessel. 4. 3.2 Detection of viability, survival and mutation rate 4.3.2.1 At the end of the test substance exposure, the cells are washed and cultured to detect the viability and express the mutant phenotype. The detection band of cell tractability is expressed as the relative colony formation efficiency (viability) or the relative total growth number measured after the infection. 4.3.2.2 The optimal phenotypic expression of the newly induced mutants at each mutation site requires a minimum time (HPRT and XPRT site mutations require at least 6d to 8d, TK at least 2d). The cells are grown in culture medium containing and without selection agents to respectively determine the number of mutants and colony formation efficiency. The detection of viability (using T to calculate the mutation efficiency) can be carried out from the end of the expression period by detecting cells inoculated in non-selective culture medium. 4.3.2.3 If the test substance is positive in the L5178YTK+/- test, the colony size should be determined for at least one test sample concentration (highest positive concentration) and for negative and positive controls. If the test sample is negative in the L5178YTK+ test, the colony size should be determined for negative and positive controls. In TK-/- tests using the TK6 strain, the colony size should also be determined. 5 Test Data and Reporting
5.1 Handling of Results
5.1.1 Data should include cytotoxicity and viability, colony counts, and mutation rates for the treated and control groups. If the 15178Y TK test result is positive, the number of large and small colonies should be counted for at least one test sample concentration (highest positive concentration) and for negative and positive controls. The molecular and cytogenetic characteristics of large and small mutant colonies have been studied in detail. In the TK-- test, the colony count criteria are normal growing colonies (large colonies) and slow growing colonies (small colonies). The cause of small colonies is that mutant cells suffer severe genetic damage, resulting in prolonged doubling period and slow cell number growth. Typical damage ranges from the loss of entire genes to typical chromosomal aberrations visible in the cell nucleus. The induction of small mutant colonies is related to chemical-induced significant chromosomal aberrations. The growth rate of inflammatory cells with less damage is similar to that of parental cells and can form larger colonies. 5.1.2 Survival capacity (relative colony formation efficiency) or relative total growth rate data should be provided. The mutation rate can be expressed as the proportion of mutant cells in the number of surviving cells.
5.1.3 Test data for each culture should be provided. In addition, all data should be listed in tabular form. 5.1.4 Confirmatory tests are particularly required for clear positive results. Results with unclear significance are best clarified by further testing with improved test conditions.
Negative results should be determined based on specific circumstances. If it is considered that negative results require confirmatory tests, the basis should be provided. For suspicious or negative results, the test parameters should be improved in subsequent tests to expand the scope of the test cases. The parameters that can be modified include the insertion interval, metabolic activation conditions, etc.
5.2 Evaluation and interpretation of results
GB/T21793--2008
5.2.1 There are multiple criteria for judging positive results. If there is a dose-response relationship, or the mutation rate increases, the results can be repeated. The biological significance of the results should be considered first. Statistical methods can be used to help evaluate the test results, but statistical significance cannot be used as the only factor for judging positive reactions.
5.2.2 Test samples that do not meet the above criteria are considered to be mutagenic in this test system. 5.2.3 Although most tests can give clear positive or negative results, it does not rule out rare cases where a clear judgment cannot be made on the activity of the test substance. For example, no matter how many times the test is repeated, the results are still ambiguous or suspicious. 5.2.4 A positive result in the in vitro mammalian cell gene mutation test indicates that the test sample can cause gene mutations in the mammalian cells used and has a reproducible concentration-response relationship. A negative result indicates that under the test conditions, the test sample does not cause gene mutations in the mammalian cells used.
5.3 Test report
The test report should include the following information:
5.3.1 Sample
a) Name and identification code such as CA number (if known); b) Physical properties and purity;
c) Physicochemical properties related to the implementation of the test; d) Stability of the test sample.
5.3.2 Excipients
a) Reasons for selecting solvents/excipients;
h) Solubility and stability of the test sample in the solvent/excipient (if known). 5.3.3 Cells
) Cell type and source:
b) Number of cell culture tubes/bottle:
c) Number of cell passages (if applicable): d) Maintenance method of cell culture (if applicable): No evidence of mycoplasma.
5.3.4 Conditions
Justification for selection of cell concentration and number of cells cultured, including cytotoxicity data and solubility limits (if applicable); medium composition, CO, volume fraction;
test sample concentration:
excipients and test sample volumes added: incubation temperature;
incubation time;
duration of treatment;
cell density at treatment;
type and composition of metabolic activation system, including acceptable markers; positive and negative controls;
duration of expression (including cell inoculum number, passaging and inoculation procedures and culture media added, if applicable); k)
d) selective agents:
m) criteria for designating the test as positive, definitive or equivocal; n) method for counting viable and mutant cells; o) definition of colony size and type designation (including criteria for so-called small and large colonies, if applicable). 12
GB/T 21793—2008
Cytotoxicity performance;
Precipitation phenomenon;
pH value and permeability data during contact with the test sample (if possible); channel size (at least including data of negative and positive control groups); statement of the laboratory's ability to detect small mutation accumulation in the L5178YTK+/- system (if applicable); dose-response relationship (if possible):
Statistical analysis (if any);
Negative (solvent/excipient) and positive control data of the same period; historical negative (solvent/excipient) and positive control data, including range, mean, standard deviation; mutation rate.
Results and Discussion
GB/T21793-2008
People's Republic of China
National Standard
Chemicals in vitro mammalian cells
Gene mutation test method
GB/T 21793—2008
Published and distributed by China Standards Publishing House
No. 16 Sanwanghe North Street, Fuxingmenwai, Beijing
Postal Code: 100045
Website spc. net. cn
Tel: 6852394668517548
Printed by China Standards Publishing House, Anhuangdao! Distributed by Xinhua Bookstore in Yinkedi
Format 880×1231/16
Printing sheet 0.75 Word count 14,000 words
First edition in July 2008, the first printing in July 2008, Book number: 155066·1-32190, Price 14.00 yuan, Replaced by our publishing center
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8002561211 There are multiple criteria for determining positive results. For example, there is a dose-response relationship, or the mutation rate increases, and the results are repeatable. The biological significance of the results should be considered first. Statistical methods can be used to help evaluate the test results, but statistical significance cannot be used as the only factor in determining a positive reaction.
5.2.2 Test samples that do not meet the above criteria are considered to be mutagenic in this test system. 5.2.3 Although most tests can give clear positive or negative results, it does not rule out rare cases where a clear judgment on the activity of the test substance cannot be made. For example, no matter how many times the test is repeated, the results are still ambiguous or suspicious. 5.2.4 A positive result in an in vitro mammalian cell gene mutation test indicates that the test sample can cause gene mutations in the mammalian cells used and has a repeatable concentration-response relationship. A negative result indicates that under the conditions of this test, the test sample does not cause gene mutations in the mammalian cells used.
5.3 Test Report
The test report should include the following information:
5.3.1 Samples
a) Name and identification code such as CA number (if known); b) Physical properties and purity;
c) Physicochemical properties relevant to the conduct of the test: d) Stability of the test sample.
5.3.2 Excipients
a) Reasons for selecting solvents/excipients:
h) Solubility and stability of the test sample in solvents/excipients (if known). 5.3.3 Cells
|) Cell type and source:
b) Number of cell cultures/bottles:
c) Number of cell passages (if applicable): d) Maintenance methods for cell cultures (if applicable): No evidence of mycoplasma.
5.3.4 Conditions
Justification for selection of cell concentration and number of cells cultured, including cytotoxicity data and solubility limits (if applicable); medium composition, CO, volume fraction;
test sample concentration:
excipients and test sample volumes added: incubation temperature;
incubation time;
duration of treatment;
cell density at treatment;
type and composition of metabolic activation system, including acceptable markers; positive and negative controls;
duration of expression (including cell inoculum number, passaging and inoculation procedures and culture media added, if applicable); k)
d) selective agents:
m) criteria for designating the test as positive, definitive or equivocal; n) method for counting viable and mutant cells; o) definition of colony size and type designation (including criteria for so-called small and large colonies, if applicable). 12
GB/T 21793—2008
Cytotoxicity performance;
Precipitation phenomenon;
pH value and permeability data during contact with the test sample (if possible); channel size (at least including data of negative and positive control groups); statement of the laboratory's ability to detect small mutation accumulation in the L5178YTK+/- system (if applicable); dose-response relationship (if possible):
Statistical analysis (if any);
Negative (solvent/excipient) and positive control data of the same period; historical negative (solvent/excipient) and positive control data, including range, mean, standard deviation; mutation rate.
Results and Discussion
GB/T21793-2008
People's Republic of China
National Standard
Chemicals in vitro mammalian cells
Gene mutation test method
GB/T 21793—2008
Published and distributed by China Standards Publishing House
No. 16 Sanwanghe North Street, Fuxingmenwai, Beijing
Postal Code: 100045
Website spc. net. cn
Tel: 6852394668517548
Printed by China Standards Publishing House, Anhuangdao! Distributed by Xinhua Bookstore in Yinkedi
Format 880×1231/16
Printing sheet 0.75 Word count 14,000 words
First edition in July 2008, the first printing in July 2008, Book number: 155066·1-32190, Price 14.00 yuan, Replaced by our publishing center
If there is any printing error
Copyright infringement will be investigated
Report telephone: (010)68533533
8002561211 There are multiple criteria for determining positive results. For example, there is a dose-response relationship, or the mutation rate increases, and the results are repeatable. The biological significance of the results should be considered first. Statistical methods can be used to help evaluate the test results, but statistical significance cannot be used as the only factor in determining a positive reaction.
5.2.2 Test samples that do not meet the above criteria are considered to be mutagenic in this test system. 5.2.3 Although most tests can give clear positive or negative results, it does not rule out rare cases where a clear judgment on the activity of the test substance cannot be made. For example, no matter how many times the test is repeated, the results are still ambiguous or suspicious. 5.2.4 A positive result in an in vitro mammalian cell gene mutation test indicates that the test sample can cause gene mutations in the mammalian cells used and has a repeatable concentration-response relationship. A negative result indicates that under the conditions of this test, the test sample does not cause gene mutations in the mammalian cells used.
5.3 Test Report
The test report should include the following information:
5.3.1 Samples
a) Name and identification code such as CA number (if known); b) Physical properties and purity;
c) Physicochemical properties relevant to the conduct of the test: d) Stability of the test sample.
5.3.2 Excipients
a) Reasons for selecting solvents/excipients:
h) Solubility and stability of the test sample in solvents/excipients (if known). 5.3.3 Cells
|) Cell type and source:
b) Number of cell cultures/bottles:
c) Number of cell passages (if applicable): d) Maintenance methods for cell cultures (if applicable): No evidence of mycoplasma.
5.3.4 Conditions
Justification for selection of cell concentration and number of cells cultured, including cytotoxicity data and solubility limits (if applicable); medium composition, CO, volume fraction;
test sample concentration:
excipients and test sample volumes added: incubation temperature;
incubation time;
duration of treatment;
cell density at treatment;
type and composition of metabolic activation system, including acceptable markers; positive and negative controls;
duration of expression (including cell inoculum number, passaging and inoculation procedures and culture media added, if applicable); k)
d) selective agents:
m) criteria for designating the test as positive, definitive or equivocal; n) method for counting viable and mutant cells; o) definition of colony size and type designation (including criteria for so-called small and large colonies, if applicable). 12
GB/T 21793—2008
Cytotoxicity performance;
Precipitation phenomenon;
pH value and permeability data during contact with the test sample (if possible); channel size (at least including data of negative and positive control groups); statement of the laboratory's ability to detect small mutation accumulation in the L5178YTK+/- system (if applicable); dose-response relationship (if possible):
Statistical analysis (if any);
Negative (solvent/excipient) and positive control data of the same period; historical negative (solvent/excipient) and positive control data, including range, mean, standard deviation; mutation rate.
Results and Discussion
GB/T21793-2008
People's Republic of China
National Standard
Chemicals in vitro mammalian cells
Gene mutation test method
GB/T 21793—2008
Published and distributed by China Standards Publishing House
No. 16 Sanwanghe North Street, Fuxingmenwai, Beijing
Postal Code: 100045
Website spc. net. cn
Tel: 6852394668517548
Printed by China Standards Publishing House, Anhuangdao! Distributed by Xinhua Bookstore in Yinkedi
Format 880×1231/16
Printing sheet 0.75 Word count 14,000 words
First edition in July 2008, the first printing in July 2008, Book number: 155066·1-32190, Price 14.00 yuan, Replaced by our publishing center
If there is any printing error
Copyright infringement will be investigated
Report telephone: (010)68533533
80025612100 yuan will be replaced by our issuing center
If there is any printing error
Copyright infringement will be investigated
Report phone number: (010)68533533
80025612100 yuan will be replaced by our issuing center
If there is any printing error
Copyright infringement will be investigated
Report phone number: (010)68533533
800256121
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