Some standard content:
IcS13.300:11.100
National Standard of the People's Republic of China
GB/T21786—2008
Chemicals
Test method for bacterial reverse mutation
Chemicals-Test method of bacterial reverse mutation2008-05-12Promulgated
General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of ChinaStandardization Administration of China
2008-09-01Implementation
GB/T21786—2008
This standard is equivalent to the Organization for Economic Cooperation and Development (OECD) Chemical Testing Guide No. 471 (1997) "Bacterial Reverse Mutation Test (English Version)".
This standard has been edited as follows:
- The scope part has been added;
:- The measurement units have been changed to the legal measurement units of my country: The reference part of OECD has been deleted.
This standard is proposed and managed by the National Technical Committee for Standardization of Hazardous Chemicals Management (SAC/TC251). The responsible drafting unit of this standard is the Institute of Occupational Health and Poison Control, Chinese Center for Disease Control and Prevention. The participating units of this standard are: Beijing Center for Disease Control and Prevention, Ningbo Entry-Exit Inspection and Quarantine Bureau: The main drafters of this standard are: Deng Ying, Mu Qun, Wu Wei, Zhao Chaoying, Long Zaihao. 1
GB/T21786—2008
OECD Introduction
1. Bacterial reverse mutation test The amino acid-requiring Salmonella Typhimuritm and Escherichia coti strains are used to detect point mutations caused by substitution, addition or deletion of one or more base pairs of DNA. The principle of this test is to detect the reverse mutations present in the test strains and restore the ability to synthesize essential amino acids. The bacteria with reverse mutation can grow under the condition of lacking specific essential amino acids, while the parent strain cannot grow and can be detected. When the nutritional deficiency bacteria undergo reverse mutation under the action of the test sample, the ability to synthesize specific amino acids is restored, so they can form colonies on the culture medium without the amino acid. However, those strains that have not mutated and the parent strains of the mutant strains cannot form colonies on the culture medium without the amino acid because they cannot synthesize specific amino acids. 2. Point mutation is the cause of many genetic diseases in humans. In addition, there is sufficient evidence to prove that point mutations in oncogenes and tumor suppressor genes in somatic cells of humans and experimental animals are related to tumor occurrence. The bacterial reverse mutation test has the advantages of being fast, economical and relatively easy to operate. Many test strains also have some characteristics that make them more sensitive to certain types of mutagens. These characteristics include responsive DNA sequences at the site of the reversion mutation, increased permeability of cells to large molecule substances, and deletion (deletion) of the DNA repair system or increased DNA error-prone repair. The test results obtained by specific strains can provide valuable research data for the types of mutations induced by genotoxic substances. A database of bacterial reversion mutation test results containing large molecule-unstructured chemicals has been established, from which the required information can be obtained. At the same time, a complete method has been established for testing chemicals with different physical and chemical properties (such as volatile substances). 3. The bacterial reversion mutation test uses prokaryotic cells, which are different from mammalian cells in terms of absorption, metabolism, chromosome structure, DNA repair process, etc. Moreover, this test is an in vitro test: it generally requires an exogenous metabolic activation system. The exogenous metabolic activation system cannot completely simulate the metabolic conditions in vivo. Therefore, the results of this test cannot provide direct evidence for the mutagenicity and carcinogenicity of the test sample to dairy animals.
4. The bacterial reverse mutation test is usually used as a preliminary screening test for genetic toxicity, and is particularly suitable for detecting the ability of the test sample to induce point mutations. A large amount of research data shows that many chemicals that obtain positive results in this test also have mutagenic activity in other tests. However, there are also examples where some mutagens are negative in this test. This defect of this test may be caused by the special nature of the detection endpoint, different metabolic activation processes, and differences in bioavailability. On the other hand, some factors that increase the sensitivity of this test will cause an overestimation of mutagenic activity. 5. The bacterial reverse mutation test is not suitable for evaluating certain types of chemicals, such as compounds with strong bactericidal effects (such as certain antibiotics) and compounds that are believed or known to have specific interference effects on the replication process of mammalian cells (such as topoisomerase inhibitors, nucleotide analogs, etc.). These substances are more suitable for mammalian mutagenicity tests. 6. Although many chemicals that obtain positive results in this test are carcinogens in mammals, this correlation is not absolute, but is related to the type of chemical. There are also some carcinogens that cannot be detected by this test because these substances cause cancer through other, non-genetic carcinogenic mechanisms or mechanisms that the test strain does not possess (exist). 1 Scope
Bacterial reverse mutation test method for chemicals
GB/T 21786-2008
This standard specifies the scope, terms and definitions, basic test principles, test methods, test data and reports of bacterial reverse mutation test for chemicals.
This standard is not applicable to the detection of the mutagenicity of chemicals (except those with tea fungus effect). 2 Terms and definitions
The following definitions and terms apply to this standard. 2.1
Reverse mutation test
Reverse mutation test
Detects the mutation of amino acid-requiring strains (histidine and tryptophan, respectively) in Xanthomonas typhimurium or Escherichia coli to produce a strain that does not rely on external amino acids. 2.2
Base pair substitution mutagens are substances that can cause changes in DNA bases. In the back mutation test, this change can occur at the primary mutation site, and mutations can also occur at a second site in the bacterial genome.
Frameshift mutagens are substances that can cause the addition or deletion of single or multiple base pairs in DNA, thereby changing the reading frame of RVA. 3 Basic principles of the test
3.1 The bacterial suspension is contacted with the test sample under the condition of adding or not adding an exogenous metabolic activation system. In the flat blood doping test, the above mixture is fully filtered with the top agar and quickly poured onto the bottom agar plate (minimum nutrient agar). In the preincubation test, the bacterial suspension is first mixed with the test sample for preincubation, and then fully mixed with the top agar and quickly poured onto the bottom agar plate (minimum nutrient agar). After 2-3 days of culture, the number of recovered colonies is counted and compared with the number of spontaneously recovered colonies in the solvent control group.
3.2 There are several methods for bacterial reverse mutation test, among which the most commonly used are the blood spike method, preincubation method, shaking culture method and suspension culture method. There are also improved methods for detecting gas dry vapor. 3.3 This standard mainly describes the blood spike method and preincubation method: these two methods can be carried out under the conditions of adding or not adding metabolic activation system. Some chemicals are more effective with the preincubation method, including short-chain aliphatic nitrosamines, divalent metals, aldehydes, azo dyes and diazo compounds, butyric photoalkaloids, allyl compounds and nitro compounds. In the study, it was also found that some categories of chemicals were not always detected with positive results using standard methods such as the blood spike method and preincubation method. This situation should be regarded as a "special case" and it is strongly recommended to use other alternative test procedures for testing. In the literature, alternative methods have been used to identify the mutagenicity of the following "special cases" (with examples of the test procedures used). "Special cases" generally include azo dyes and diazo compounds, gaseous or volatile substances and glucose. Changes to standard methods should be scientifically justified.
4 Test methods
4.1 Test preparation
4.1.1 Bacteria
4.T.1.1 Fresh bacterial cultures should be grown to the late exponential growth period or the early stationary phase (bacterial concentration is 10 cells/mL). Cultures that have grown to the late stationary phase should not be used. The viable titer of the culture used in the test should be high. The viable titer can be determined based on the historical control data of the bacterial growth curve, or by measuring the viable count by drop plating at each test. 4.1.1.2 Recommended culture temperature: 37
4.1.1.3. At least five strains were used, including four Salmonella typhimurium (TAL535; TA1537 or TA97a or TA97TA98 and TA100). The detection results of these bacteria were reliable and had good reproducibility between different laboratories. These four strains had GC base pairs at the initial reversion mutation site, which were known to be unable to detect certain oxidative mutagens, DNA crosslinkers and tantalum substances. For the detection of these substances, the E. coli WP2 strain or the Salmonella typhimurium TA102 strain with AT base pairs at the initial reversion mutation site should be selected. Therefore, the recommended strain combination is as follows: Salmonella Typhimurium TA1535 and Salmonella Typhimurium TA1537 or TA97 or TA97a and Salmonella Typhimurium TA98 and Salmonella Typhimurium TAO and Escherichia coli WP2uvrA or Escherichia coli WP2uvrA (pKM101) or Salmonella Typhimurium A102. It is best to use Salmonella Typhimurium TA1Q2 or an Escherichia coli with DNA repair function to detect DNA cross-links WP2uvrA or Escherichia coli WPuvrA (PKMIO1) as one of the receptive strains. 4.1.1.4 The culture products of bacterial species should be preserved and labeled according to established production procedures. Each time a culture of a frozen stock is revived, an amino acid requirement test (S. Typhimurium requires histidine and E. coli requires tryptophan) should be performed. Likewise, other phenotypic characterizations should be performed, including the presence of R factor recombinants [i.e., resistance to cyanine in TA98, TA100, and TA97a or TA97, VP2, VIA, WP2, UVIA (pKM101) strains and resistance to alanine and tetracycline in TA102] and characteristic mutations such as the rIa mutation in C. enterica serovar Typhimurium that renders it sensitive to crystal violet, the uvIA mutation in E. coli, and the presence of serovars in S. enterica serovar Typhimurium that are sensitive to crystal violet (e.g., S. Typhimurium is extrasensitive). These strains will produce spontaneous reversions. The number of spontaneous reversions obtained by plate counting should be within the historical range of the control data reported in the literature. 4.1.2 Culture Media Use an appropriate minimal nutrient agar (containing Gell-Bonner minimal medium E and required sugars) or a top agar containing histidine and biotin or tryptophan to count the number of cell divisions. 4.1.3 Metabolic Activation The bacteria should be contacted with the test sample in the presence or absence of an appropriate metabolic activation system. The most commonly used activation system is the post-mitochondrial fraction (S9) prepared from rodent liver with added cofactors after enzyme inducer treatment. The enzyme inducers used include Aruclor 1251 or phenobarbital and beta-ferol. The concentration of the post-mitochondrial fraction used is 5% to 30% (volume fraction) of the 59 mixture. The metabolic activation system and its application conditions should be selected according to the type of the test sample. In some cases, more than one concentration of S9 can be used. For azo dyes or diazo compounds, a reductive metabolic activation system should be selected. 4.1.4 Test samples/preparation
Before treating the strain, the solid test sample should be dissolved or suspended in a suitable latent or excipient, and can be appropriately diluted if necessary. The liquid test sample can be directly added to the assay system or appropriately diluted before treatment. The test sample should be freshly prepared, otherwise data should be provided to prove its storage stability.
4.2 Test conditions
4.2.1 Solvents/excipients
The solvent/excipient should not react chemically with the test sample and have no effect on the survival of the bacteria and the activity of S9. If the commonly used solvent or excipient is not selected, data should be provided to provide appropriate reasons. If water can be used as a solvent or excipient, water should be considered as the solvent or excipient first. If the test sample is unstable to water, an organic solvent or excipient that does not contain water should be selected. 4.2.2 Toxic dose
4.2.2.1 The maximum concentration of the test sample to be used shall be determined based on the toxicity of the test sample to bacteria and its solubility in the final mixture. It is recommended to conduct a preliminary test to determine the toxicity and solubility of the test sample. The reduction in the number of revertant colonies, the reduction in the volume of the background bacterial moss, or the reduction in the survival rate of bacteria after treatment can be used as indicators of cell detachment. The presence of a metabolic activation system can change the toxicity of the test sample. Under actual test conditions, the final mixture is judged to be insoluble if a visible precipitate appears. For non-cytotoxic soluble test samples, the recommended maximum test concentration should be 5 mg/mL or 5 μL/mL. For non-cytotoxic test products whose solubility does not reach 5 mg/mL or 5 μL/mL, precipitation should occur at one or more concentrations in the final treatment mixture. For test samples that show cytotoxicity at levels below 5 mg/mL or 5 μL/mL, the highest concentration should reach the concentration at which cytotoxicity occurs. Precipitation should not affect the result count. 4.2.2.2 At least 5 test concentrations should be selected for analysis. At the beginning of the test, the dose interval is generally half a logarithm (e.g. V10), and a smaller dose interval can also be used when studying the dose-response relationship. 4.2.2.3 If the test sample contains a substance that may have a mutagenic effect, the test concentration may exceed 5 mg/mL or 5 μL/mL. 4.2.3.1 Each test should be accompanied by positive and negative (drug or excipient) controls, which should be set up with or without the metabolic activation system. When the metabolic activation system is added, the concentration of the positive selected for each test should be able to demonstrate the validity of the test. 4.2.3.2 To test the metabolic activation system used, appropriate positives should be selected according to the type of test strain. The following chemicals are suitable positives for metabolic activation tests: 9,0-dimethylanthracene [CAS No. 781-43] 7,12-dimethylbenzene Congo red Congo red 2-dinethylbenzanthracene [CAS No. 57-97-6] [CAS Na] pyrene 573-58-07 573-58-07 573-58-07 573-58-07 573-57 ... 7
monohydrate】 [GAsNo.
50-18-0(CA Sno. 6055-19-2))
Linoanthracene[CASNe.613-13-82-aminototal enzyme
2-nitrobenzoic acid can be used as an indicator for evaluating the activity of S9 mixtures. If it is selected as a positive substance for S9, another mutagenic substance that requires high microsomal metabolic activation should be selected to confirm its activity, such as benzo[beta]-[beta]-[beta]-[beta]-[beta]-[beta]-[beta]-[beta]-[beta]-[beta]-[beta]-[beta]-[beta]-[beta]-[beta]-[beta]-[beta]-[beta]-[beta]-[beta]-[beta]-[beta]-[beta]-[beta]-[beta]- 4.2.3.3 For the test of activation system without generation, the positive control substances specific to each strain are shown in Table 1. Chemical name and CAS number SNo.26628-22-81 Sodium dinitrogen B8ic 2-nitrafluc ASNo.607-57-81 9-aminoacdine ASNo ICR191 [CASn0, 17C70-] Isobutylene hydroperoxide cuniene droren Table of specific control substances for each strain Ta535#TA100 45-9 or xide ICASNo. 80-15-9 J
Mitomycin C nitoroyin CrCAS0-0N-Z-N-nitro N-nitrosoguanidine N ethyl N nitru N-nilrsoguartidine [CASNa, 70-25-7 di- or 4-nitrozoline 1-oxide nitroquinoline1-oxide [CASno. 56-57-5 ]
furfuryifuramide (AF-2) [C.ASNo.3588-53-7] TA1537TA97 and TA97
WP2urA and TA102
WP2.WP2uvIA and WP2uVIA (PKMI01) Plasmid-containing strains
4.2.3.4 Other suitable positive substances can also be used. If possible, consider using positive controls related to chemical classes. 4.2.3.5 Negative control In each test, except for adding only the quencher or carrier to the test medium, the rest of the treatment should be the same as that of each treatment group. In addition, if there is no historical data to prove that the agent or carrier used has no toxicity or mutagenicity, a blank control group (no treatment) should also be set up.
4.3 Test steps
4.3.1 Test sample treatment
4.3.1.1 Flat III spike method: When metabolic activation is not required, usually 0.05mL or 0.1mL of the test sample solution, 0.1mL of fresh bacterial culture fluid (containing 108 bacteria) and 0.5mL of sterile buffer are mixed with 2.0mL of top agar. If a metabolic activation system is required, 0.5 mL of metabolic activation mixture containing appropriate mitochondrial components (about 5% to 30% of the total volume of metabolic activation mixture) is usually mixed with bacteria and test substance/test solution and top agar. After the above mixture is fully mixed, it is poured on the minimum nutrient agar. After the top agar solidifies, it is placed in the incubator for culture. 4.3.1.2 Pre-incubation method: pre-incubate the test substance/test solution with the test strain (containing about 10° bacteria) and sterile buffer or metabolic activation system (0.5 mL) at 30℃~37℃ for 20 min or longer, then mix with the top agar, and then pour it on the minimum nutrient agar. Usually 0.05 mL or 0.1 mL of test substance/test solution, 0.1 ml of bacterial solution, 0.5 mL of S9 mixture or sterile buffer, and 2.0 mL of top agar are mixed. During the pre-incubation, the test tube should be placed in a shaker to inflate. 4.3.1.3 In order to evaluate the variability of the results, three parallel plates should be made for each dose level. If there is a scientific reason, two parallel plates can also be made. The occasional loss of data from one plate does not affect the reliability of the results. 4.3.1.4 Gaseous or volatile substances should be tested in an appropriate manner, such as in a closed culture medium. 4.3.2 Culture
All plates in the test should be cultured at 37℃ for 48h~72h: After the incubation, count the number of colonies with reverse mutations in each plate. 5 Test data and report
5.1 Data processing
5.1.1 The number of colonies with reverse mutations in each plate should be listed. The number of colonies with reverse mutations in each negative control (solvent control, sometimes blank control) and positive control should also be recorded. 5.1.2 The number of reverse mutant colonies per blood, the average number of reverse mutant colonies and the standard recommendation for each group of test samples, negative controls and positive controls should be listed.
5.1.3 No confirmation test is required for clear positive results. Suspicious results are best clarified by further testing with improved test conditions. Negative results need to be determined based on their individual circumstances. If it is considered that negative results do not require confirmation tests, the reasons should be stated. In subsequent tests, the test parameters should be improved to expand the range of evaluation conditions. The improved parameters include concentration intervals, treatment methods (average blood ginseng or solution pre-incubation, etc.) and metabolic activation conditions.
5.2 Evaluation and interpretation of results
5.2.1 There are several criteria for judging positive results. For example, the increase in the number of reverse mutant colonies in each group of test samples shows a dose-benefit-response relationship, or at least one strain shows a reproducible increase in the number of reverse mutant colonies per blood at one or several dose levels with or without the metabolic activation system. The biological significance of the results should be considered first. Statistical methods can be used to help evaluate test results, but they cannot be used as the only factor to determine a positive reaction:
5.2.2 Test samples that do not meet the above standards are considered to be non-mutagenic in this test. 5.2.3 Although most tests can give clear positive or negative results, it does not exclude that a very small number of tests cannot make a clear judgment on the activity of the test samples. 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 a bacterial reverse mutation test indicates that the test sample can cause point mutations induced by base substitutions or frameshift mutations in the genome of the Salmonella typhimurium or Escherichia coli used. A repeatable concentration-response relationship is of great significance. A negative result indicates that the test sample does not cause mutations in the test strain under the conditions of this test. 5.3 Test Report
The test report should include the following information:
5. 3.1 Test Sample
a) Name and identification code such as CAS number (if known);
Physical properties and purity;
Physicochemical properties relevant to the conduct of the test;
Stability of the test sample (if known).
Solvents/Excipients
Justification for the selection of solvents/excipients:
Solubility and stability of the test sample in the solvent/excipient (if known). Bacteria
Strain used:
Number of cells injected per blood sample;
Strain characteristics.
Test conditionsbzxz.net
CB/T21786—2008
Amount of test sample added to each test tube (mg/mL or μg/mL), basis for the selection of the reagent, number of test tubes at each level; selected culture medium;
Type and composition of metabolic activation system, including the criteria for judging acceptance: treatment process.
Sensitive performance;
Precipitation phenomenon;
Bacterial count of each test tube;
Average number of mutant colonies per tube and standard deviation, dose-response relationship (if possible)
Statistical analysis (if any):
Contemporaneous negative (agent/excipient) and positive control data (including data range, mean and standard deviation) Historical negative (solvent//excipient) and positive control data (including range, mean, standard deviation). Discussion of results.
Conclusion.
GB/T 21786-2008
People's Republic of China
National Standard
Bacterial Reversion Test Method
GB/T 21786—2002
Published and distributed by Zhongmin Standard Publishing House
No. 1.6, Sanlihebei Street, Fuxingmenwai, Beijing
Postal Code: 100045
Website spc.net.cn
Tel: 6252394668517548
Printed by China Standard Publishing House, Anhuangdao Printing Factory, distributed by Xinhua Bookstores in various places
Format 880×1230 1/16
Printing sheet 0.75 Words 13,000
First edition July 2008
First printing July 2008
Book number: 1550661-32187 Price 14.00 yuan Replaced by our publishing center
If there is any printing error
Copyright reserved
Infringement will be investigated
Report phone: (010)68533533
80029821 /2 Culture
Place all the samples in the test at 37℃ and culture for 48h~72h: count the number of reverted mutant colonies in each sample after the culture is completed. 5 Test data and report
5.1 Data processing
5.1.1 The number of reverted mutant colonies in each sample should be listed. The number of reverted mutant colonies in each sample of the negative control (solvent control, sometimes blank control) and positive control group should also be recorded. 5.1.2 The number of reverted mutant colonies in each sample of each group, negative control and positive control, the average number of reverted mutant colonies and the standard should be listed.
5.1.3 No confirmation test is required for clear positive results. Suspicious results are best clarified by further testing with improved test conditions. Negative results need to be determined based on their individual circumstances. If it is considered that a negative result does not need to be confirmed, the reason should be stated. In subsequent tests, the test parameters should be improved to expand the range of evaluation conditions. The improved parameters include concentration interval, treatment method (pre-incubation of sample or solution, etc.) and metabolic activation conditions.
5.2 Evaluation and interpretation of results
5.2.1 There are several criteria for determining positive results. For example, the increase in the number of reverse mutant colonies in each group of the test samples shows a dose-benefit-response relationship, or at least one strain shows a reproducible increase in the number of reverse mutant colonies per blood at one or several dose levels under the conditions of the presence or absence of a metabolic activation system. The biological significance of the results should be considered first. Statistical methods can be used to help evaluate the test results, but they cannot be used as the only factor for determining a positive response:
5.2.2 Test samples that do not meet the above standards are considered to be non-mutagenic in this test. 5.2.3 Although most tests can give clear positive or negative results, it does not exclude that a very small number of tests cannot make a clear judgment on the activity of the test samples. For example, no matter how many times the test is repeated, the results are still ambiguous or suspicious. 5.2.4 A positive result of the bacterial reverse mutation test indicates that the test sample can cause base substitution or frameshift mutation-induced point mutations in the genome of the Salmonella typhimurium or Escherichia coli used. Reproducible concentration-response relationships are of great significance. A negative result indicates that the test sample does not cause mutation of the test strain under the test conditions. 5.3 Test report
The test report should include the following information:
5. 3.1 Test sample
a) Name and identification code such as CAS number (if known); b)
Physical properties and purity;
Physical and chemical properties relevant to the conduct of the test; d)
Stability of the test sample (if known).
Solvents/excipients
Justification for the selection of solvents/excipients:
Solubility and stability of the test sample in the solvent/excipient (if known). Bacteria
Strain used:
Number of cells injected per blood sample;
Strain characteristics.
Test conditions
CB/T21786—2008
Amount of test sample added to each test tube (mg/mL or μg/mL), basis for the selection of the reagent, number of test tubes at each level; selected culture medium;
Type and composition of metabolic activation system, including the criteria for judging acceptance: treatment process.
Sensitive performance;
Precipitation phenomenon;
Bacterial count of each test tube;
Average number of mutant colonies per tube and standard deviation, dose-response relationship (if possible)
Statistical analysis (if any):
Contemporaneous negative (agent/excipient) and positive control data (including data range, mean and standard deviation) Historical negative (solvent//excipient) and positive control data (including range, mean, standard deviation). Discussion of results.
Conclusion.
GB/T 21786-2008
People's Republic of China
National Standard
Bacterial Reversion Test Method
GB/T 21786—2002
Published and distributed by Zhongmin Standard Publishing House
No. 1.6, Sanlihebei Street, Fuxingmenwai, Beijing
Postal Code: 100045
Website spc.net.cn
Tel: 6252394668517548
Printed by China Standard Publishing House, Anhuangdao Printing Factory, distributed by Xinhua Bookstores in various places
Format 880×1230 1/16
Printing sheet 0.75 Words 13,000
First edition July 2008
First printing July 2008
Book number: 1550661-32187 Price 14.00 yuan Replaced by our publishing center
If there is any printing error
Copyright reserved
Infringement will be investigated
Report phone: (010)68533533
80029821 /2 Culture
Place all the samples in the test at 37℃ and culture for 48h~72h: count the number of reverted mutant colonies in each sample after the culture is completed. 5 Test data and report
5.1 Data processing
5.1.1 The number of reverted mutant colonies in each sample should be listed. The number of reverted mutant colonies in each sample of the negative control (solvent control, sometimes blank control) and positive control group should also be recorded. 5.1.2 The number of reverted mutant colonies in each sample of each group, negative control and positive control, the average number of reverted mutant colonies and the standard should be listed.
5.1.3 No confirmation test is required for clear positive results. Suspicious results are best clarified by further testing with improved test conditions. Negative results need to be determined based on their individual circumstances. If it is considered that a negative result does not need to be confirmed, the reason should be stated. In subsequent tests, the test parameters should be improved to expand the range of evaluation conditions. The improved parameters include concentration interval, treatment method (pre-incubation of sample or solution, etc.) and metabolic activation conditions.
5.2 Evaluation and interpretation of results
5.2.1 There are several criteria for determining positive results. For example, the increase in the number of reverse mutant colonies in each group of the test samples shows a dose-benefit-response relationship, or at least one strain shows a reproducible increase in the number of reverse mutant colonies per blood at one or several dose levels under the conditions of the presence or absence of a metabolic activation system. The biological significance of the results should be considered first. Statistical methods can be used to help evaluate the test results, but they cannot be used as the only factor for determining a positive response:
5.2.2 Test samples that do not meet the above standards are considered to be non-mutagenic in this test. 5.2.3 Although most tests can give clear positive or negative results, it does not exclude that a very small number of tests cannot make a clear judgment on the activity of the test samples. For example, no matter how many times the test is repeated, the results are still ambiguous or suspicious. 5.2.4 A positive result of the bacterial reverse mutation test indicates that the test sample can cause base substitution or frameshift mutation-induced point mutations in the genome of the Salmonella typhimurium or Escherichia coli used. Reproducible concentration-response relationships are of great significance. A negative result indicates that the test sample does not cause mutation of the test strain under the test conditions. 5.3 Test report
The test report should include the following information:
5. 3.1 Test sample
a) Name and identification code such as CAS number (if known); b)
Physical properties and purity;
Physical and chemical properties relevant to the conduct of the test; d)
Stability of the test sample (if known).
Solvents/excipients
Justification for the selection of solvents/excipients:
Solubility and stability of the test sample in the solvent/excipient (if known). Bacteria
Strain used:
Number of cells injected per blood sample;
Strain characteristics.
Test conditions
CB/T21786—2008
Amount of test sample added to each test tube (mg/mL or μg/mL), basis for the selection of the reagent, number of test tubes at each level; selected culture medium;
Type and composition of metabolic activation system, including the criteria for judging acceptance: treatment process.
Sensitive performance;
Precipitation phenomenon;
Bacterial count of each test tube;
Average number of mutant colonies per tube and standard deviation, dose-response relationship (if possible)
Statistical analysis (if any):
Contemporaneous negative (agent/excipient) and positive control data (including data range, mean and standard deviation) Historical negative (solvent//excipient) and positive control data (including range, mean, standard deviation). Discussion of results.
Conclusion.
GB/T 21786-2008
People's Republic of China
National Standard
Bacterial Reversion Test Method
GB/T 21786—2002
Published and distributed by Zhongmin Standard Publishing House
No. 1.6, Sanlihebei Street, Fuxingmenwai, Beijing
Postal Code: 100045
Website spc.net.cn
Tel: 6252394668517548
Printed by China Standard Publishing House, Anhuangdao Printing Factory, distributed by Xinhua Bookstores in various places
Format 880×1230 1/16
Printing sheet 0.75 Words 13,000
First edition July 2008
First printing July 2008
Book number: 1550661-32187 Price 14.00 yuan Replaced by our publishing center
If there is any printing error
Copyright reserved
Infringement will be investigated
Report phone: (010)68533533
80029821 /No. 6
Postal code: 100045
Website: spc.net.cn
Tel: 6252394668517548
Printed by China Biaowei Publishing House, Anhuangdao Printing Factory, distributed by Xinhua Bookstores in various places
Book size: 880×1230 1/16
Printing sheet 0.75 Words 13,000
First edition July 2008
First printing July 2008
Book number: 1550661-32187 Price 14.00 yuan Replaced by our publishing center
If there is any printing error
Copyright reserved
Infringement will be investigated
Report phone: (010)68533533
80029821 /No. 6
Postal code: 100045
Website: spc.net.cn
Tel: 6252394668517548
Printed by China Biaowei Publishing House, Anhuangdao Printing Factory, distributed by Xinhua Bookstores in various places
Book size: 880×1230 1/16
Printing sheet 0.75 Words 13,000
First edition July 2008
First printing July 2008
Book number: 1550661-32187 Price 14.00 yuan Replaced by our publishing center
If there is any printing error
Copyright reserved
Infringement will be investigated
Report phone: (010)68533533
80029821 /
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