GB/T 27820-2011 Test method for sister chromatid
exchange in mammalian cells in vitro for chemicals GB/T27820-2011 |tt||Standard compression package decompression password: www.bzxz.net This standard specifies the terms and definitions, test principles, test methods, test data and reports for the test method for sister chromatid exchange in mammalian cells in vitro for chemicals. This standard is applicable to the test method for sister chromatid exchange in mammalian cells in vitro for 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 United Nations Organization for Economic Cooperation and Development (OECD) Chemical Testing Guide No. 479 (1986) "In vitro mammalian cell
sister chromatid exchange test" (English version).
This standard has been modified in the following structural and editorial aspects:
———A chapter on scope has been added;
———The "Introduction" in the original OECD 479 is used as the "Introduction" of this standard;
———The "Required Information" in the original OECD 479 is used as "4.1.1" of this standard;
———The measurement units are uniformly changed to the legal measurement units of China.
This standard is proposed and managed by the National Technical Committee for Standardization of Dangerous 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, Liaoning Provincial Institute of Occupational Disease Prevention and Control, China Chemical Economic and Technological Development Center, Jiangsu Entry-Exit Inspection and Quarantine Bureau.
The main drafters of this standard are: Qu Bo, Lin Zheng, Li Xuefei, Bai Yu, Yang Ting, Tang Lijun.

ICS t3. 300; 11, 100
National Standard of the People's Republic of China
GB/T27820—2011
Chemicals-Test method of in vitro mammalian cells sister chromatid exchange2011-11-30 Issued
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
Implementation on August 1, 2012
This standard was drafted in accordance with the rules given in GB/T 1.1—2009. GB/T278202011
This standard is consistent with the technical content of the United Nations Organization for Economic Cooperation and Development (OECD) Chemical Testing Guide No. 479 (1986) "In vitro mammalian cell sister chromatid exchange test" (English version). This standard has been modified as follows: Scope has been added, and the contents of the "Introduction" in the original text of ECD479 have been used as the "Introduction" of this standard; the contents of the "Required information" in the original text of ECD479 have been used as the "4.1.1" of this standard. The accounting units have been uniformly changed to the legal accounting units of my country. This standard was proposed and managed by the National Technical Committee for Standardization of Hazardous Chemicals Management (5AC/TC251). The drafting units of this standard are: Institute of Occupational Health and Poisoning Control, Chinese Center for Disease Control and Prevention, Liaoning Provincial Institute of Occupational Disease Prevention and Control, China Chemical Economic and Technological Development Center, Jiangsu Exit-Entry Inspection and Quarantine Bureau. The main drafters of this standard are: Qu Bei, Lin Zheng, Li Xuefei. Yu, Yang Ting, Tang Lijun. TTTKANYKACA
CB/T 27820—2011
The in vitro sister chromatid exchange (SCE) test is a rapid test for the exchange of DNA between sister chromatids of chromosomes during replication. SCE represents the phase exchange of DVA replication products at homologous sites on sister chromatids. Although its molecular mechanism is still unclear, it is speculated that the exchange process may include two steps: DNA breakage and reconnection. Detection of SCEs requires a method to label different chromosomes. For example, a method can be used to incorporate bromadeoxyuridine (BrdU) into chromosomal DNA and replicate two cell cycles. SCEs can be performed in mammalian or non-mammalian systems. TT KAAN KACA
1Scope
Chemicals In vitro mammalian cell sister chromatid exchange test method
GB/T 27820—2011
This standard specifies the terms and definitions, test principles, test methods, test data and reports for the in vitro mammalian cell sister chromatid exchange test of chemicals.
This standard is applicable to the in vitro mammalian cell sister chromatid exchange test of chemicals. 2 Terms and Definitions
The following terms and definitions apply to this document. 2.1
Sister chromatid exchange, SCE The mutual exchange of two chromatids within a single chromosome during cell division and replication . This exchange can be seen in the metaphase of cell division and may require enzymatic transfer and ligation of the DNA double helix. 3 Experimental Principles
Mammalian cells are cultured in the presence of the test agent, bromodeoxyuridine (BrdU), for two consecutive cell cycles with and without the addition of an exogenous metabolic activation system. Spindle inhibitors (such as colchicine) are added to converge the dividing cells and stop at the metaphase-like stage. The cells are harvested and chromosomes are prepared. 4 Experimental Methods
4.1 Preparation
4. 1.1 Test Substances
The physical state, purity, solubility, melting point/boiling point, pH value (if applicable), vapor pressure (if any) and other information of the test substance should be provided. The medium containing the test substance should be prepared before cell infection or the test substance should be dissolved in a suitable medium. The test substance should be freshly prepared. The concentration of the medium should not significantly affect cell viability, growth rate and SCE rate. 4.1.2 Cells and culture methods
Primary cultured cells (such as human lymphocytes) or established cell lines (such as Chinese hamster ovary cells or lung cells) can be used. The cell line should be free of mycoplasma contamination and have a stable karyotype. Use appropriate culture medium and culture conditions (such as temperature, culture dish, CO, concentration and humidity). 4.1.3 Metabolic activation
The cells are infected with the test substance in the presence or absence of an appropriate mammalian metabolic activation system. Commonly used metabolic activation systems include an activation system prepared by inducing mammalian liver homogenate microsomal enzyme system with enzyme inducers and adding auxiliary factors, and primary cultured mammalian liver cells.
4.2 Test conditions
4.2.1 Infection concentration
Use at least three test substance concentrations with sufficiently large inhibitory differences. The highest concentration should be able to cause obvious toxic effects, but at the same time, it should ensure that there are enough cells for replication. For relatively water-insoluble test substances, appropriate methods should be selected to improve solubility and use their maximum drop test. For water-soluble and non-cell-thin test substances, the maximum concentration depends on the situation. 4.2.2 Number of cultures
Each test point should have at least two replicate culture specimens. 4.2.3 Control group
Each dose group should have a positive control of direct cleavage agent and indirect cleavage agent. Solvent control should also be set. The following substances can be selected as positive controls:
Ethyl methanesulfonate (EMS) Mitomycin C (nitomycin C) (direct cleavage agent): - Cyclophosphamide (cyclophosphamide) (indirect cleavage agent): 4.3 Operation method
4.3.1 Preparation of experimental cell culture
For established cells, they can be obtained from cultured cells (by trypsin digestion or blowing), inoculated into culture blood at an appropriate density, and cultured at 37°C. For monolayer cultured cells, the inoculation density should be such that the cells do not fuse when harvested. Floating culture cells can also be used, such as Heparan cells isolated from human blood, which can be cultured at 37°C after appropriate treatment. 4.3.2 Infection
The cells should be exposed to the test substance during the exponential growth phase of the cells. The action time is 1 h to 2 h, but sometimes it may need to be extended to two complete cell cycles. In some cases, infection in serum-free culture medium is more effective. Cells should be infected with and without the metabolic activation system. At the end of the infection, the test substance is washed off. Brd is added to the new culture medium and the cells are cultured for two complete cell replication cycles. It is also possible to culture the cells in a culture medium containing both the test substance and BrdU for two complete cell cycles. Human blood lymphocytes should be infected when they are in a semi-synchronous state. The cells should be analyzed in the second mitotic phase after the start of the infection to ensure that the cells are exposed to the test substance during the most sensitive cell cycle: all cultured blood cells with BrdU added should be exposed to as little light as possible before cell harvesting to reduce the incorporation of BrdU. Photolysis of DNA:
4.3.3 Harvesting cells
1h~~4h before harvesting cells, treat cells with spindle inhibitors (such as colchicine), harvest and treat cells in each dose group, and prepare chromosomes.
4.3.4 Chromosome preparation and staining
Use standard cell culture techniques to prepare chromosome specimens. Various techniques can be used to stain to show SCEs, such as fluorescence plus Giemsa staining.
4.3.5 Chromosome analysis
Generally, at least 25 metaphase cells with well-dispersed chromosomes are analyzed for each culture, but the number of analyzed cells will also depend on the number of cells analyzed.
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GB/T27820—-2011Www.bzxZ.net
The spontaneous exchange rate varies according to the group. The slides should be numbered before analysis. For human blood lymphocytes, only metaphase cells with 46 centromeres are analyzed: for established cell lines, only metaphase cells with ±2 centromeres are analyzed. Whether the exchange is with centromeres or not is counted as one SCE. The results of the experiment need to be verified by independent experiments. 5 Experimental data and report
5.1 Data processing
The results are presented in tabular form. The number of SCEs, chromosomes and the average number of SCEs per chromosome of each metaphase cell in the poisoned group and the control group should be listed. Use appropriate statistical methods for statistical processing. 5.2 Result evaluation
Criteria for positive results: The average number of SCEs in each metaphase cell increases significantly with the increase of the poisoning dose; or at least at one detection point, SCEs have a repeatable and statistically significant increase. Criteria for negative results: No statistically significant correlation is observed between the average number of SCEs in a single cell and the poisoning concentration, and there is no repeatable and statistically significant increase in SCEs at a certain detection point. 5.3 Experimental Report
The experimental report shall include the following contents:
The cells used and the cell culture method;
The test conditions including culture medium distribution, concentration, test substance concentration, perfusion, culture temperature, exposure time, hammertoe inhibitor, spindle inhibitor concentration and action time, type of mammalian metabolic activation system used, positive and negative controls, BrdU concentration;
The number of cultured cells for each test point;
Detailed technical description of slide preparation;
List the number of metaphase cells analyzed (for each culture); List the average number of SCEs per cell and chromosome (for each culture): - Standard for SCEs counting;
Principles for setting the exposure concentration:
- Dose-response relationship (if possible);
Statistical evaluation
...Discussion of the results,
- Interpretation of the results. || tt | -Tox Program.MtationRes.198187:17-62
[2_ P. Perry,L. Henderson and D. Kirkland, Sister Chranatid Exchange in Cultured Cells,inRe-port of the UKEMS Subcommittee on Guidelincs for Mutagenicity Testing,Part I ,l984:89-122[3P.E, Perry and EJThomsan,The Methodology of Sister Chrvmatid Exchange,in Hand-bookuf Mutagenicity Test Procedures,Znd Edition (edited by BJKilbey,M,I.egator,W.NirhnlsandC,Ramel),Elscvier Scientific+Amstcrdam,1984,495-530[4] PE Perry, Chemical Mutagens and Sister Chromatid Exchangc,in Chemical Mutagens,Vol, 6(edited by FJde Serre s and A, Hollaender), Plenum Publishing Co., Ncw York, iy80: l-39 [5l S. Takehisa, Induction of Sister Chromatid Exchange by Chemical Agents, in Sister Chro-matid Eachange (edited by S. Wolfetal.), John Wiley & Sons, New York, 1982.87-147
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