GB/T 27819-2011 Test Method for In Vitro Mammalian Cell Transformation of Chemicals GB/T27819-2011 |tt||Standard compression package decompression password: www.bzxz.net
This standard specifies the test principles, test methods, test data and reports for in vitro mammalian cell transformation tests of chemicals. This standard can be used as an alternative test method for the carcinogenicity of chemicals.
This standard was drafted in accordance with the rules given in GB/T1.1-2009.
This standard is consistent with the technical content of the European Commission Regulation 440/2008: Test methods for Regulation No. 1907/2006 of the European Parliament and of the Council on Registration, Evaluation, Authorization and Restriction of Chemicals (REACH) Regulation Method B.21 (2008) "In Vitro Mammalian Cell Transformation Test" (English version).
This standard has been modified in the following structural and editorial aspects:
———A chapter on scope has been added;
———The measurement units have been changed to the legal measurement units of China.
This standard was proposed and managed by the National Technical Committee for Standardization of Hazardous Chemicals Management (SAC/TC251).
The drafting units of this standard are: Institute of Occupational Health and Poisoning Control, Chinese Center for Disease Control and Prevention, Beijing Center for Disease Control and Prevention, and China Chemical Economic and Technological Development Center.
The main drafters of this standard are: Mu Xiaoqun, Li Chaolin, Wang Xiaobing, Xiong Xiaoyan, Zhao Sujuan, and Wu Weiai.

IcS13.300:11.100
National Standard of the People's Republic of China
GB/T27819—2011
Chemicals
Test method of in vitro mammalian cell transformationChemicals-Test method of in vitro mammalian cell transformation2011-12-30Issued
General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of ChinaStandardization Administration of the People's Republic of China
2012-08-01Implementation
This standard is based on the rules given in GB/T1.1—2009. GB/T 278192011
This standard is consistent with the technical content of Commission Regulation 440/2008: Test methods for Regulation No. 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorization and Restriction of Chemicals (REACH) Regulation Method B.21 (2008] In vitro mammalian cell transformation test (English version)
This standard has been modified as follows: the scope chapter has been added;
The measurement unit has been changed to the legal measurement unit of my country. . This standard is proposed and managed by the National Technical Committee for Standardization of Hazardous Chemicals Management (SAC/TC251). Drafting units of this standard: Occupational Health and Poisoning Control Institute of China Center for Disease Control and Prevention, Beijing Center for Disease Control and Prevention, China Chemical Economic and Technological Development Center.
Main drafters of this standard: Mu Xiaoqun, Li Chaolin, Wang Xiaobing, Xiong Xiaoyan, Zhao Sumu, Wu Wei. TTTKANTKACA
1 Scope
Test method for in vitro mammalian cell transformation of chemicals GB/T 27819—2011
This standard specifies the test principles, test methods, test data and reports for in vitro mammalian cell transformation tests of chemicals. This standard can be used as an alternative test method for the carcinogenic effect of chemicals. 2 Test principles
Mammalian cell culture test systems can be used to detect phenotypic changes caused by chemicals, which are related to their malignant transformation in the in vivo system. Widely used cell lines include C3H10T1/2, 33, SHE, and rat Fischet cells, which mainly observe the morphology of cells, the shape of transformation foci in agar or changes in anchorage independence. This system is also used to detect other physiological and morphological changes after cells are exposed to chemicals, but it is not widely used. At present, no in vitro test point has been proven to have a clear relationship with the mechanism of tumor occurrence. Some test systems can detect carcinogens. The degree of cytotoxicity can be reflected by measuring the effect of the test substance on the colony formation ability (clone formation rate) or the growth rate of cultured cells, so the cytotoxicity can be evaluated by calculating the correlation between exposure and toxicity. However, some tests require extended culture time. The culture medium is replaced from time to time, therefore, sometimes the cytotoxicity data cannot be used to calculate the transformation rate. 3 Test methods
3.1 Test preparation
3.1.1 Cells
Many cell lines and primary cells can be used as test cells, and appropriate cell types should be selected according to the selected transformation test method. Before the test, the researcher should ensure that the test cells will produce corresponding phenotypic changes after contact with carcinogens, and the laboratory should also have records showing that such phenotypic changes are effective and reliable.
3.1.2 Culture medium (liquid)||tt ||The culture medium and experimental conditions used should be suitable for cell transformation tests. 3.1.3 Test substances
Before cells are exposed to the test substances, the test substances should be mixed with the culture medium and evenly mixed. They can also be dissolved or suspended in an appropriate solvent. The final concentration of the solvent in the culture medium should not affect cell activity, growth rate or transformation. 3.1.4 Metabolic activation
Cells should be exposed to the test substances in the presence and absence of a suitable metabolic activation system. When the cell type used has its own metabolic transformation ability, it should be considered whether its activation characteristics are suitable for the chemical properties of the test substance. 3.2 Test conditions
3.2.1 Negative controls and positive controls
In each test, a direct-acting compound and a compound that requires metabolic activation should be selected as positive controls. In addition, a negative (solvent) control should be set up.
Compounds that can be used as positive control:
a) Direct action:
1) Ethyl methanesulfonate; 2) β-propiolactonc. 1) Indirect action:
1) 2-Acetylaminofluorcne! 2) 4-Dimethylaminoazobenzene; 3) 7,12-dimethylbenzanthracene. If necessary, a similar compound to the test substance should be set as a positive control. 3.2.2 Toxic intensity
Multiple concentration points of the test substance should be set. These designed concentrations should produce concentration-dependent toxic effects, that is, the highest concentration group should reduce the survival rate of the cells, while the survival rate of the cells in the lowest concentration group should be basically consistent with the negative control. For test substances with low solubility in water, appropriate measures should be taken to reach saturation concentration. The maximum concentration of low-toxic test substances soluble in water is determined according to specific circumstances. 3.3 Test steps bzxz.net
Determine the appropriate exposure time according to the selected test system. If the exposure time is prolonged, the culture medium needs to be changed, and the test substance concentration should be readjusted during the replacement (if necessary, a freshly prepared metabolic activation mixture should be added). For cells that have no metabolic activation ability, the test substance should be exposed to the test substance under the conditions of the presence or absence of the metabolic activation system. After the exposure, the test substance should be washed away and cultured under conditions suitable for cell phenotypic transformation and the transformation incidence should be observed. All results are completed in an independent experiment. 4 Test data
All test data should be listed in a table, and indicators should be determined according to the selected method, such as blood count, positive blood count, number of transformed cells, etc. Survival is best expressed as the percentage of surviving cells, and the transformation rate is expressed as the number of transformed cells per a certain number of surviving cells. Appropriate statistical methods should be used to statistically process the data. 5 Experimental report
Whenever possible, the report should include the following: the type of cells used, the number of cells cultured, and the cell culture method; experimental conditions: test substance concentration, solvent used, incubation time, duration and number of test substance treatments, cell density during treatment, type of exogenous metabolic activation system used, negative and positive controls, description of the phenotype to be tested, the selection system selected (when applicable), and the reasons for the dose design;
Methods used to identify and count viable and transformed cells: statistical analysis:
Discussion of results;
Interpretation of results.
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