Chemicals—Genetic toxicology—Test method of Saccharomyces cerevisiae gene mutation
Some standard content:
ICS13.300;11.100
National Standard of the People's Republic of China
GB/T27831-2011
Chemicals
Genetic toxicity
Test method of Saccharomyces cerevisiae gene mutation
Chemicals-Genetic toxicology-Test method of Saccharomyces cerevisiae gene mutation2011-12-30 Issued
General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of ChinaStandardization Administration of China
Implementation on August 1, 2012
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 Organization for Economic and Development (OECD) Guide for Testing of Chemicals (480-1986) (Genetic Toxicity Testing\English version).
This standard has been modified in the following ways: adding a chapter on scope,
- The "Required Information" part in the original text of 0ECD180 is used as 1.1.1.1 of this standard; the measurement unit is changed to the legal measurement unit of my country. GB/T27831—2011
Gene mutation test of ethanol mother bacteria
This standard is proposed and managed by the National Technical Committee for the Management of Hazardous Chemicals (5AC/TC251). The drafting units of this standard are: Institute of Occupational Health and Poisoning Control, Chinese Center for Disease Control and Prevention, Guangxi Institute of Occupational Disease Prevention and Control, and China Chemical Economic and Technological Development Center. The main drafters of this standard are: Chen Wanhan, Li Chaolin, Wang Xiaobing, Wu Weikai, and Lin Zheng. TTTKAONTKACA
1 Scope
Genetic toxicity of chemicals
Gene mutation test method for Saccharomyces cerevisiae
CB/T 27831-2011
This standard defines the terms and definitions, test principles, test methods, test data and reports for the genetic toxicity test method for Saccharomyces cerevisiae.
This standard is applicable to the detection of genetic mutation of Saccharomyces cerevisiae and forward or reverse mutation (base substitution and frameshift) of eukaryotic microorganism Saccharomyces cerevisiae for the genetic toxicity of chemicals.
2 Terms and definitions
The following terms and definitions apply to this document. 2.1
Basesobstitutionmutagens
Base substitution mutagens
Chemicals that can cause base changes in deoxyribonucleic acid (DNA). In the reverse mutation test, this base change may occur at the primary mutation site or the second mutation site in the genome. 2.2
Frameshift Mutagens
A chemical substance that causes the addition or loss of a single or multiple base pairs in a DNA molecule. 3 Principle of the test
Many haploid and diploid strains of S. cerevisiae can be used to detect the gene mutation products caused by chemical factors. In the haploid strain forward mutation system, the adenine-dependent mutants (ade-1, ade-2) with red colonies are induced by the test substance: they mutate into white mutants that depend on two adenines. In the selection system, canavanine and cycloheximide can be used to induce drug resistance.
The most widely used and proven reverse mutation system includes the haploid strain XV185-11C, which carries the ochre nonsense mutant genes ade2-1, argf-17, lys-1 and trp5-48, which induces specific site mutations or ochre suppressor gene mutations through base substitution to produce reverse mutations. XV185-14C also carries the his1-7 marker, which is a missense mutation gene, mainly a reversion mutation caused by a second site mutation, while the hom3-10 marker is a reversion mutation caused by a frameshift mutagen. The only widely used diploid strain is D7, which is homozygous for ilv1-92. 4 Test methods
4.1 Test preparation
4.1.1 Test substance
4. 1. 1. 1 Basic information:
a) Solid, liquid, vapor or gas test substance: 1
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Chemical properties of the test substance:
c) Purity (impurities) of the test substance:
d) Solubility properties;
e) Melting point/boiling point:
F) pH value (if applicable);
) Vapor pressure data (if available).
4.1.1.2 Dissolved test substances and resistivity controls should be prepared freshly before use, and appropriate solvents should be used when necessary. The final concentration of the solvent should not significantly affect cell viability and growth characteristics. 4.1.2 Test Strains
Haploid strain XV185-11C and -borrower strain 17 are the most widely used strains for gene mutation studies. Other strains may also be used.
4.1,3 Culture Media
Use appropriate culture media to determine cell viability and mutant counts. 4.1, 4 Metabolic Activation
Cells are exposed with or without the addition of an exogenous mammalian metabolic activation system. The most commonly used metabolic activation system is the liver plasma microsomal enzyme system obtained from rodents pretreated with enzyme inducers. Other species, tissues, microsomal enzyme systems or methods that have metabolic activation functions are also suitable. 4.2 Test Conditions
4.2.1 Exposure Concentration
At least five well-spaced concentration groups should be set up, and cytotoxicity and the solubility of the test substance should be considered when determining the exposure concentration. The lowest concentration that does not affect cell viability. The highest test concentration of toxic chemicals should not make the cell survival rate lower than 5%~1%. Appropriate methods should be used to increase the solubility of poorly soluble test substances. For substances that are easy to fall into water and non-toxic, the highest concentration should be determined according to the specific situation. 4.2.2 Spontaneous mutation rate
The spontaneous mutation rate of subculture should be within the recognized normal range. 4.2.3 Number of parallels
At least 3 parallel blood samples should be used for each test toxic concentration to detect gene mutations and cell viability. When using low mutation rate strains such as hom3-10 as a marker, the number of parallels should be increased to meet the requirements of statistical analysis. 4.2.4 Control
Each test should have a positive control with and without the addition of a metabolic activation system, and a solvent control. The following can be used as positive controls!
a) Methylnitrosoamine, ethylnitrosoamine, 4-nitrozoline-N-oxide (direct agents); b) N-nitrosodimethylamine, cyclophosphamide (indirect agents); c) Acridine mutagen (Acridine Mutagen, ICR-170) (direct acting frameshift mutagen). 4.3 Experimental operation
Yeast treatment is usually carried out in vivo using cells in the stationary phase or growth phase. The initial test uses cells in the growth phase, 2
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1×10°cells/mL~5×10°cells/mL cells are continuously exposed to the test substance for 18 hours at a temperature of 28℃～37℃, and need to be cultured with shaking. For tests requiring metabolic activation, sufficient metabolic activators should be added during the test. At the end of the test, the cells are centrifuged, eluted and inoculated in a suitable culture medium. After culturing at 28-30℃ in the dark for 4 days, calculate the number of surviving blood cells and the number of gene mutations induced. If the first test is negative, repeat it once using cells in the stable period. If the first test is positive, verify it with a separate experiment of 100 appropriate tests.
5 Test data and report
5.1 Result processing
List the test data in a table, including the number of colonies, the number of mutant colonies, and the survival rate of mutants. The data should be processed by appropriate statistical methods. 5.2 Result evaluation
Evaluation criteria for positive results:wwW.bzxz.Net
a) The increase in the number of mutants and the mutation rate is dose-related and statistically significant. b) The test substance has at least one concentration point to detect mutagenicity, and the reproducibility is good and statistically significant. c
If the case rate caused by a test substance has neither a statistically significant dose-related relationship nor a reproducible and statistically significant positive reaction is detected at any concentration point, the test substance will be considered to be non-mutagenic in this test system.
In the assessment, a comprehensive consideration should be made in combination with biological and statistical significance. 5.3 The test report should include the following: a) The strain used; test conditions: - cells in the stationary phase or growth phase; - composition of the culture medium; - culture temperature and duration; - metabolic activation system. ) Treatment conditions: - infection concentration: - infection operation and duration; - infection concentration; - positive and negative controls. d) Number of colonies and mutant colonies: - survival rate and mutation rate; - dose-response relationship (if any): - statistical evaluation of the data! e) Discussion of the results; f) Interpretation of the results.
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参考文献
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TTKaNRYKAcAElsevier Scientific, Amsterdam, 1977:119-134[8_ F, K. Zimmermann, VM Maycr and JM Parry, J.Appt. T oxicot. 1982,2,1-10[9] FK Zimmermann, RC yon Borstel,E, S, von Halle,J. M, Parry,D. Siebert,G. Zetterberg.R, Barale and N, Loprieno,Testing of chemicals for genetic activity with Saccharam yces cereurisiae,inRepart to the US Environrental Protcetion Agency Gene-Tox Program, Mutation Res. , 1gB4,133:199-244
TTKaNRYKAcAElsevier Scientific, Amsterdam, 1977:119-134[8_ F, K. Zimmermann, VM Maycr and JM Parry, J.Appt. T oxicot. 1982,2,1-10[9] FK Zimmermann, RC yon Borstel,E, S, von Halle,J. M, Parry,D. Siebert,G. Zetterberg.R, Barale and N, Loprieno,Testing of chemicals for genetic activity with Saccharam yces cereurisiae,inRepart to the US Environrental Protcetion Agency Gene-Tox Program, Mutation Res. , 1gB4,133:199-244
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