Chemicals—Genetic toxicology—Test method of Saccharomyces cerevisiae mitotic recombination
Some standard content:
ICS 13. 300;11. 100
National Standard of the People's Republic of China
GB/T 27832--2011
Chemicals
Genetic toxicity
Test method of Saccharom yces cerevisiaemitotic recombination
Issued on December 30, 2011
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 according to the rules given in GB/T 1.1-2009. GB/T 27832-—2011
Saccharomyces cerevisiae
This standard is consistent with the technical content of the Organization for Economic Cooperation and Development (OECD) Chemical Test Method No. 81 (1986) Genotoxicity Splitting and Recombination Test (English version). This standard has been edited as follows:Www.bzxZ.net
Added a chapter on thresholds;
-The content of the original "Definition" of ECD481 is used as "2 Terms and Definitions" of this standard;-The content of the original "Required Information" of OECD481 is used as "4.1.1 Basic Information" of this standard;-·-The measurement units are uniformly changed to the legal measurement units of my country. This standard was proposed and approved by the National Technical Committee for Standardization of Hazardous Chemicals (SAC/TC251). Drafting units of this standard: Institute of Occupational Health and Poisoning Control, China Center for Disease Control and Prevention, China Chemical Economic and Technological Development Center, Chaobei Exit-Entry Inspection and Quarantine Bureau.
The main drafters of this standard are Li Chaolin, Wang Xiaobing, Wu Wei and Guo Jian. TTTKANTKACA
1 Scope
Test method for chemical genotoxicity and mitotic recombination of Saccharomyces cerevisiae
GB/T 27832—2011
This standard specifies the terms and definitions, test principles, test methods, test data and reports for the test method for mitotic recombination of Saccharomyces cerevisiae for chemical genotoxicity.
This standard is applicable to the detection of non-specific deoxyribonucleic acid (DNA) damage for the detection of chemical genotoxicity.
2 Terms and definitions
The following terms and definitions apply to this document. 2.1
Mitotic crossing-over usually occurs between genes, but more commonly occurs between genes and their centromeres, which can produce crossover products.
Mitotic gene conversion
Mitotic gene conversion
The unidirectional transfer of sequence information within a genome, usually resulting in non-reciprocal products. 3 Principle of the assay
Mitotic crossing over and mitotic genome conversion can be detected in Saccharomyces cerevisiae. Mitotic crossing over is tested by the production of colonies homozygous for recessive genes or colonies with partially differentiated recessive genes in heterozygous strains, while mitotic gene conversion is determined by the production of prototrophic restorers in a heterozygous strain carrying two different defective alleles for the same gene. The most commonly used strains for detecting mitotic gene conversion are D, (alleles in ade: and tP>, BZ (alleles in arg.), D, (alleles in trPs) and JD: (alleles in hig. and trps). D. and D, used to determine the mitotic gene conversion and reversion of homozygous ilv1-92, can detect mitosis that can produce red or pink homozygous fragments. Both of the above strains have heterologous complementary alleles of ade. 4 Test method
4.1 Test substance
4.1.1 Basic information:
Solid, liquid, vapor or gas test substance; Chemical properties of the test substance:
Test substance purity (impurities):
Solubility properties;
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GB/T 27832---2011
—melting point/boiling point;
—pH value (if applicable):
vapor temperature data (if available).
4.1.2 Dissolved test substances and control substances should be prepared freshly before use, and appropriate solvents should be used when necessary. The final concentration of the solvent should not have a significant effect on cell viability and growth characteristics. 4.2 Test strain selection
The most commonly used double-borrowing strains include D,,D, D, and JI1. Other strains may also be used if appropriate. 4.3 Culture medium
Select an appropriate culture medium to determine cell survival rate and mitotic mutation rate. 4.4 Metabolic activation system
Cells are exposed to the addition of an exogenous mammalian metabolic activation system or a non-human one. The most commonly used metabolic activation system is the liver homogenate microsomal enzyme system obtained from rodents pretreated with enzyme inducers. Other species, tissues, microsomal enzyme systems or methods with metabolic activation functions may also be applicable.
4.5 Exposure concentration
At least 5 concentration groups with sufficiently large concentration intervals should be set. Cytotoxicity and solubility of the test substance should be considered when determining the exposure concentration. The lowest concentration should not affect cell activity. For readily soluble and non-toxic compounds, the highest concentration should be determined according to the specific test conditions. The highest concentration of the avoided chemicals should not cause cell survival. The viability is lower than 5% to 10%. For poorly soluble test substances, appropriate methods should be used to increase their solubility. 4.6 Spontaneous mitotic recombination frequency
The spontaneous mitotic mutation rate of the cultured strain used for subculture should be within the acceptable normal range. 4.7 Plate number
For the prototrophic test and survival test produced by mitotic gene conversion, at least 3 replicate plates should be used for each concentration level; for the recessive homozygous test produced by mitotic exchange, the number of replicate plates should be increased to obtain a sufficient number of bacteria. 4.8 Control
Each test should have a positive control with and without the addition of a metabolic activation system, and a flux control. The following can be used as positive controls:
Methylnitrosourea, ethylnitrosourea, 4-nitrozoline-N-oxide (direct agent)-cyclophosphamide (indirect agent).
4.9 Operation steps
Stationary or growing phase S. cerevisiae cells are usually treated with the filter test method. The first test should be carried out on growing phase cells. 1×10? cells/mL~~5×10" cells/mL are infected with the test substance at 28~37℃ for 18h. In the test requiring metabolic activation, a sufficient amount of mammalian metabolic activation system should be added. After the infection, the cells are centrifuged, washed and inoculated into appropriate culture medium.
After culturing at 28℃~30℃ in the dark for 4d~7d, the cell survival rate and mitotic recombination induction rate in the plate are calculated. For mitotic exchange detection, the plates producing red and pink homozygous droplets should be stored at 4℃ for another day before counting. ~2d to produce appropriate color-containing colonies. If the result of the first test is negative, a second test should be carried out using cells in the stable phase. If the result of the first test is positive, a separate repeated test should be carried out for verification. Test data and report
5.1 Result processing
Record the number of colonies, number of mutants, survival rate and recombination rate in a table. Evaluate the test data using appropriate methods. 5.2
Result evaluation
GB/T 27832—2011
One of the criteria for judging positive results is that there is a statistically significant dose-response relationship between the increase in the number of mutants and the mutation rate, and the other criterion is that at least one dose group of the test substance can detect a repeatable statistically significant positive reaction. If the test results of the test substance do not have a statistically significant dose-response relationship, and no repeatable statistically significant positive reactions are detected in all dose groups, it is considered that the substance does not produce DNA recombination in this test system. When evaluating the results, both biological and statistical significance should be considered. 5.3 The report
should include the following contents;
一 The type of bacterial probe used in the test:
Test conditions: stable or growing period membrane wine yeast cells, culture medium composition, culture volume and culture time, metabolic activation system; 一 Infection conditions: infection concentration, infection procedure and time, treatment temperature, setting of positive and negative controls; 一 The number of colonies, the number of mutant colonies, the survival rate and recombination rate, dose-response relationship (if any), and statistical evaluation of the data. 5.4 Result evaluation
·Result discussion:
Drilling nest interpretation. | |tt | G, R, Fink and R, Lowenstcin.J. B acteriol. ,1969,100:1126-1127[4_ D.Kelly and JM Parry,Mutation Res. 1983,108:147-159L5] B, A, Kunz, B, J. Barcley and RH Haynes,Mutation Res. ,1980,73 ,215-20[6] KK Mortimer and TR Manncy, in Chemical Mutagens, Principles and Methodg for their Deteetion,Vol.
[71(edited by A.Hollaender),Flenum Press,New York,1971:289-310[8_M. SS Murthy,Mutation Res. ,1979,64.J-17[9] EM Parry and J M Parry, The assay of genotoxicity of chemicals using the budding ycastS accharomyces cerevisiae,in Mutagenicity tcsting,a practical approach(edited by S. Venitt and JMParry),IRL Press0xlord,1985:119-148[lo] D, C. Sharp and J. M Parry,in Evaluation ol Short-Term Tests for Carcinogens(edited by F.J. de Serres and J. Ashby), Elsevier/North Halland, New York, 198l: 502-626 [11] FK Zimmermann, R. Kern and H. Rosenberger, Mutation Res., 1975, 28, 381-388L12] FK Zimmermann, in Handbook of Mutagenieily Tesl Proccdures, 2nd cdition (edited by B, J. Kilbey, M, Leg ator, W. Nichols, and C. Ramel), Elsevier Scientific, Amsterdam, l984: 215-238[13] FK Zimnernann and I. Scheel, in Evaluation of Short-Term Tests for Carcinogens (edited by F. J. de Serres and J. Ashby), Elsevier/North Holland, New York, 198l:48l-490_14] FK Zimmcrmann, VM Meyer and JM Parry,J. Appl. T oxicol, ,1982,2:1-10[15J F. K, Zimmermann, R C. von Borstel, ES, von Halle+J. M Parry.D Sicbert, G. Zetterberg,R. Barale and N. Lopriena, Testing of chemicals far genetic activity with Saccharomyces ccrevisiaet report of the US Environmental Protection Agency Gene-Tox Program, Mutation Res. , 1981, 133 :199-244
TTKANYKACANichols, and C. Ramel), Elsevier Scientific, Amsterdam, l984: 215-238[13] FK Zimnernann and I. Scheel, in Evaluation of Short-Term Tests for Carcinogens (edited by F. J. de Serres and J. Ashby) ,Elsevier/North Holland,New York,198l:48l-490_14] FK Zimmcrmann, VM Meyer and JM Parry,J. Appl. T oxicol, ,1982,2:1-10[15J F. K, Zimmermann, R C. von Borstel, ES, von Halle+J. M Parry.D Sicbert, G. Zetterberg,R. Barale and N. Lopriena, Testing of chemicals far genetic activity with Saccharomyces ccrevisiaet report of the US Environmental Protection Agency Gene-Tox Program, Mutation Res. , 1981, 133 :199-244
TTKANYKACANichols, and C. Ramel), Elsevier Scientific, Amsterdam, l984: 215-238[13] FK Zimnernann and I. Scheel, in Evaluation of Short-Term Tests for Carcinogens (edited by F. J. de Serres and J. Ashby) ,Elsevier/North Holland,New York,198l:48l-490_14] FK Zimmcrmann, VM Meyer and JM Parry,J. Appl. T oxicol, ,1982,2:1-10[15J F. K, Zimmermann, R C. von Borstel, ES, von Halle+J. M Parry.D Sicbert, G. Zetterberg,R. Barale and N. Lopriena, Testing of chemicals far genetic activity with Saccharomyces ccrevisiaet report of the US Environmental Protection Agency Gene-Tox Program, Mutation Res. , 1981, 133 :199-244
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