GB 15193.4-2003 Salmonella typhimurium/mammalian microsomal enzyme test
Some standard content:
ICS 07.100
National Standard of the People's Republic of China
GB15193.4—2003
Replaces GB15193.4-1994
Salmonella typhimurium/mammals
Microsomal enzyme test
Salmonella typhimurium/mammals microsomalenzyme test (Ames test)
Issued on September 24, 2003
Ministry of Health of the People's Republic of China
Standardization Administration of the People's Republic of China
Implementation on May 1, 2004
GB 15193.4—2003
The entire text of this standard is mandatory.
This standard replaces GB15193.4--1994 "Salmonella typhimurium/mammals microsomal enzyme test". Compared with GB15193.4-1994, this standard has been modified as follows: a) The specific content of the test objects has been added in the "scope": chemical, biological and physical factors involved in the production, processing, storage, transportation and sales of food that may cause harm to health; the test objects include food additives (including nutritional fortifiers), new food resources and their ingredients, new resource foods, irradiated foods, food containers and packaging materials, food tools, equipment, detergents, disinfectants, pesticide residues, veterinary drug residues, microorganisms used in the food industry, etc.; the scope of inapplicability of this standard has been added; b) Add the setting of control group;
c) In "Preparation of culture medium and reagents": change "add 400mL of distilled water" to "add distilled water to 400mL" in the preparation method of 1.5% agar culture medium; the amount of L-histidine added in the preparation method of 0.5mmol/L histidine-biotin solution (for mutagenesis test) is changed from "17.4mg" to "19.5mg";
--In the preparation method of top culture medium, "add 10mL10.5mol/L" to every 100mL of top agar is changed to "add 10mL0. 5 mmol/L\;
—Change the "0.5mol/L" in L-histidine solution and 0.5mol/LD biotin solution (for identification of strains) to "0.5mmol/L", and the concentration of "\0.5mol/L biotin" in the following content shall be changed to "\0.5mmol/L". d) Change the title of the original standard "7 Dosage, solvent and special treatment of test substances" to "Test design and special treatment of test substances", and add the content of control group setting;
e) Delete the content of "Ames test report". Appendix to this standard A is the normative appendix.
From the date of implementation of this standard, GB15193.4-1994 will be abolished at the same time. This standard is proposed and managed by the Ministry of Health of the People's Republic of China. The drafting units of this standard: Institute of Nutrition and Food Safety, Chinese Center for Disease Control and Prevention, Peking Medical University, Zhejiang Medical University. The main drafters of this standard: Dai Yin, Ding Lan, Jin Zhongchu, Guo Shiping. This standard was first issued in 1994 and this is the first revision. 34
1 Scope
Salmonella typhimurium/mammalian microsomal enzyme test This standard specifies the Ames Basic technical requirements for the test. GB 15193.4—2003
This standard is applicable to the evaluation of the mutagenic effects of chemical, biological and physical factors that may cause harm to health during the production, processing, storage, transportation and sales of food. The test objects include food additives (including nutritional fortifiers), new food resources and their ingredients, new resource foods, irradiated foods, food containers and packaging materials, food tools, equipment, detergents, disinfectants, pesticide residues, veterinary drug residues, microorganisms used in the food industry, etc.
This standard is not applicable to test substances with bactericidal and/or bacteriostatic effects, and is not applicable to test substances that hinder the replication system of mammalian cells.
2 Principle
The mutant (i.e., histidine-deficient) strain of Salmonella typhimurium cannot grow on a medium without histidine, but can grow normally on a medium with histidine. However, if a mutagen is present in a medium without histidine, the mutant type of Salmonella can be reverted to the wild type (phenotype), and thus can grow on a medium without histidine. Therefore, the number of colonies formed can be used to check whether the test substance is a mutagen. Some mutagens need to be metabolically activated before the mutant type of Salmonella can be reverted. The metabolic activation system can be prepared by using a S-9 mixture prepared from rat liver homogenate (S-9) induced by polychlorinated biphenyls (PCBs). 3 Instruments
3.1 Common laboratory equipment.
3.2 Low-temperature high-speed centrifuge, low-temperature refrigerator (-80°C) or liquid nitrogen tank, clean workbench, constant temperature incubator, constant temperature water bath, steam pressure cooker, homogenizer, etc.
4 Reagents
Except as specified, the components or reagents of the culture medium should be at least chemically pure and non-mutagenic. Avoid repeated high-temperature treatment and select an appropriate storage temperature and period. For example, the broth should be stored at 4°C for no more than six months. For other details, please refer to the following instructions for each culture medium and solution. 4.1 Nutrient broth medium
Beef extractWww.bzxZ.net
Pancreas (or mixed protein)
Sodium chloride
Dipotassium hydrogen phosphate (K,HPO·3H,O)
Distilled water
Heat to dissolve, adjust pH to 7.4, sterilize at 0.103MPa20min after aliquoting, store in ordinary refrigerator for standby use, and the shelf life shall not exceed half a year. 4.2 Nutrient broth agar medium
a) Crystal violet sensitivity test for genotype identification, ampicillin and tetracycline resistance test, ultraviolet sensitivity test. b) Bacterial activity identification.
Agar powder
Nutrient broth medium
Heat to melt, adjust pH to 7.4, and sterilize at 0.103MPa20min. 35
GB15193.4—2003
4.3 Reagents and preparation of bottom culture medium
4.3.1 Phosphate stock solution
Sodium ammonium hydrogen phosphate (NaNH.HPO·4H,O)
Citric acid (CHO,·H,O)
Dipotassium hydrogen phosphate (K,HPO4)
Magnesium sulfate 1 (MgSO4·7H2O)
Add distilled water to 100mL, sterilize at 0.103MPa for 20min. 4.3.2 40% glucose solution
Glucose
Add distilled water to 100mL, sterilize at 0.055MPa for 20min. 4.3.31.5% agar medium
Agar powder
Add distilled water until
melts and sterilize at 0.103MPa for 20min.
4.3.4 Bottom culture medium (sterile operation)
While hot (80℃), add the following to the sterilized agar medium (400mL): phosphate stock solution
40% glucose solution
Mix thoroughly, and pour into a flat plate when it cools to about 80℃. Culture 25mL per plate (Φ90mm) at 37℃ overnight to remove moisture and check for contamination.
4.4 Composition and preparation of top culture medium
4.4.1 Top agar
Agar powder
Sodium chloride
Add distilled water to
4.4.20.5mmol/L histidine-biotin solution (for mutagenic test) D-biotin (molecular weight 244)
L-histidine (molecular weight 155)
Add distilled water to 250mL.
4.4.3 Preparation of top culture medium
Heat to melt the top agar, and add 10mL0.5mmol/L histidine-biotin solution to every 100mL of top agar. Mix well, dispense into 100mL Erlenmeyer flasks, and sterilize at 0.103MPa for 20min. When using, melt and dispense into small test tubes, 2mL per tube, and keep warm in a 45℃ water bath. 4.5 Preparation of special reagents and culture media
4.5.10.8% ampicillin solution (for strain identification, aseptic preparation): weigh 40 mg of ampicillin, dilute to 5 mL with 0.02 mol/L sodium hydroxide solution, and store in a refrigerator. 4.5.20.1% crystal violet solution (for strain identification): weigh 100 mg of crystal violet, dissolve in 100 mL of sterile water. 4.5.3L-histidine solution and 0.5 mmol/LD biotin solution (for strain identification): weigh 0.4043 g of L-histidine and 12.2 mg of D-biotin, dissolve in 100 mL of distilled water, sterilize at 0.103 MPa for 20 min, and store in a 4°C refrigerator. 4.5.40.8% tetracycline solution (for tetracycline resistance test and ampicillin-tetracycline plate): weigh 40 mg of tetracycline, dilute to 5 mL with 0.02 mol/L sodium hydroxide solution, and store in a refrigerator.0.2mol/L hydrochloric acid buffer was diluted to 5mL and stored in a 4℃ refrigerator. 1) After other reagents are completely dissolved, slowly add magnesium sulfate and continue to dissolve, otherwise precipitation is easy to precipitate. 36
GB 15193.4—2003
4.5.5 Ampicillin plates (used as master plates for TA97, TA98, and TA100 strains) and ampicillin-tetracycline plates (used as master plates for TA102 strains), each 1000 mL of which consists of the following components: bottom culture medium
phosphate stock solution
40% glucose solution
910 mL
histidine aqueous solution (0.4043 g/100 mL) 10 mL 0.5 mmol/L biotin
0.8% amoxicillin solution
0.8% tetracycline solution
Tetracycline is added only when using TA102 strains resistant to tetracycline. The above components have been sterilized or aseptically prepared. 4.5.6 Histidine-biotin plate (histidine is required for testing), each 1000mL consists of the following components: bottom culture medium
phosphate stock solution
40% glucose solution
914 mL
histidine aqueous solution (0.4043g/100mL) 10mL 0.5mmol/L biotin
The above components have been sterilized separately.
4.5.7 Dimethyl sulfoxide: spectrally pure, sterilized at 0.103MPa for 20min. 4.6 Preparation of S-9 cofactor (mixed liquid reagent) 4.6.1 0.4mol/L magnesium chloride (MgCl2) solution: weigh 3.8g, dilute to 100mL with distilled water. 4.6.2 1.65mol/L potassium chloride (KCl) solution: weigh 12.3g, dilute to 100mL with distilled water. 4.6.3 0.2 mol/L phosphate buffer (pH 7.4), each 500 mL is composed of the following ingredients: disodium hydrogen phosphate (NazHPO4) (14.2 g/500 mL) sodium dihydrogen phosphate (NaH,PO4·H2O) (13.8 g/500 mL) pH adjusted to 7.4, 0.103 MPa for 20 min sterilization or filter bacteria. 440 mL
4.6.4 Coenzyme-Ⅱ (oxidized form) solution: accurately weigh coenzyme-Ⅱ, dissolve in sterile distilled water to prepare a 0.025 mol/L solution, and store at low temperature (below -20°C).
4.6.5 Glucose-6-phosphate sodium salt solution: weigh glucose-6-sulfate sodium salt, dissolve in sterile distilled water to prepare a 0.05 mol/L solution, and store at low temperature (below -20°C).
4.7 Preparation of 10% S-9 mixed solution
Each 10mL consists of the following ingredients, prepared just before use. Phosphate buffer (0.2mol/L, pH7.4) Potassium chloride solution (1.65mol/L)
Magnesium chloride solution (0.4mol/L)
Glucose-6-phosphate sodium salt solution (0.05mol/L) Coenzyme-ear solution (0.025mol/L)
Liver S-9 solution
Mix evenly and place in an ice bath for later use.
4.8 Preparation of activation system (S-9 and S-9 mixed solution) 6.0mL
Use mammals such as rats, treated with inducers, and take liver tissue to prepare homogenate, centrifuge at 9000g, the supernatant is the S-9 component, and it is combined with auxiliary components in an appropriate proportion to form an S-9 mixed solution, which is used as the metabolic activation system in the experiment. 4.8.1 Induction and preparation of rat liver S-9
4.8.1.1 Healthy male adult SD or Wistar rats were selected, weighing about 150g and aged about 5 to 6 weeks. Polychlorinated biphenyls (Aro-37
GB 15193.4—2003
clor1254 or domestic PCB-pentachloro) were dissolved in corn oil at a concentration of 200mg/mL, and injected intraperitoneally once at 500mg/kg body weight by aseptic operation. The animals were decapitated 5 days later, the liver was removed and weighed, and the liver was continuously rinsed several times with fresh ice-cold 0.15mol/L potassium chloride solution to remove hemoglobin that can inhibit the activity of microsomal enzymes. Add 3 mL of 0.1 mol/L potassium chloride solution to each gram of liver (wet weight), move the beaker into an ice bath, chop the liver with sterilized scissors, and make liver homogenate in a glass homogenizer (less than 4,000 r/min, reciprocating for 1 min to 2 min) or a tissue homogenizer (20,000 r/min, 1 min). The above operations must be performed in a sterile and locally cold environment. 4.8.1.2 Centrifuge the prepared liver homogenate at 9,000 g for 10 min in a low-temperature (0°C to 4°C) high-speed centrifuge, aspirate the supernatant as the S-9 component, and dispense it into sterile cryovials or bottles, about 2 mL per bottle. It is best to freeze it with liquid nitrogen or on ice and then store it at 80°C. 4.8.1.3 After S-9 is prepared, it is sterile and the protein content is determined (Lowry method). The protein content per milliliter should not exceed 40 mg. After the biological activity of indirect carcinogens (mutagens) is qualified, it is stored in deep low temperature or frozen and dried. The shelf life shall not exceed one year. 4.8.2 Preparation of S-9 mixed solution
It is composed of S-9 solution and auxiliary factors (S-9 mixed solution reagent). The auxiliary factors are formulated according to Ames (1983) and stored at low temperature (below -20°C). The mixed solution is freshly prepared aseptically before use, or filtered and sterilized. Generally, it is prepared into a 10% mixed solution at a ratio of 1:9. Use 0.5 mL S-9 mixed solution (containing 20 μL to 50 μL S-9) per dish to determine its biological activity against known positive carcinogens (mutagens) to determine the most suitable amount, or use the general dosage, that is, 0.5 mL S-9 mixed solution (containing 50 μL S-9) per dish. The activity and dosage of S-9 should be stated in the report. 5 Strains and their identification and preservation
5.1 Test strains
Four mutant strains of Salmonella typhimurium, TA97, TA98, TA100, and TA102, were used. TA97 and TA98 can detect various frameshift mutagens; TA100 can detect mutagens that cause base pair substitution; TA102 can detect certain mutagens that other test strains cannot detect or rarely detect, such as formaldehyde, various hydrogen peroxide compounds, and cross-linking agents such as mitomycin C. Generally, when used to test the mutagenicity of the test substance, it must pass the test of four of the strains. If necessary, any strain of TA1535, TA1537 or TA104 can be added. 5.2 Identification of strains
The characteristics of the strains should be consistent with the Ames test standard (see Table A.1). Some characteristics of mutant bacteria are easy to lose or mutate. The genotype of the strain should be identified in the following situations:
After receiving the cultured strain;
b) When preparing a new set of frozen preservation strains or freeze-dried strains; When the number of spontaneous back mutations per dish is not within the normal range; c
When losing sensitivity to standard mutagens; d)
When using the master plate for subculture;
f) Before use.
The identification method is as follows:
5.2.1 Enrichment culture
Inoculate the stock culture in 5mL of nutrient broth medium and culture at 37℃ with shaking (100 times/min) for 10h or static culture for 16h for use.
5.2.2 Identification of histidine deficiency
5.2.2.1 Heat and melt two bottles of bottom culture medium (one bottle without histidine, the other with histidine). For the one without histidine, add 0.6mL of 0.5mmol molecular D biotin per 100mL of bottom culture medium; for the one with histidine, add 1mL of L-histidine (0.4043g per 100mL) and 0.6mL of 0.5mmol molecular D biotin per 100mL of bottom culture medium. Cool to about 50℃ and pour two plates each. 5.2.2.2 Inoculation: Take one plate with histidine and one plate without histidine, take one platinum fungus liquid in the order of strain number, streak (straight line) on the surface of the culture medium and culture at 37℃ for 48h. 5.2.2.3 Result determination: Each of the four strains of bacteria grows a bacterial film on the surface of the histidine culture medium plate, and there is no bacterial film on the non-histidine culture medium plate except for the spontaneous reversion colonies, indicating that the tested strain is indeed histidine-deficient. 5.2.3 Identification of lipopolysaccharide barrier defect
5.2.3.1 Heat and melt the nutrient broth agar medium. GB15193.4—2003
5.2.3.2 Inoculation: Take 0.1mL of bacterial solution and transfer it to the plate, quickly pour an appropriate amount of nutrient broth agar medium (cooled to about 50℃) into the plate, mix well, and lay it flat to solidify. Place a piece of sterile filter paper in the center of the solidified culture medium plate, use a pipette to add 10μL of 0.1% crystal violet solution on the filter paper, and culture at 37℃ for 24h. Make a plate for each strain. 5.2.3.3 Result determination: A transparent inhibition zone appears around the paper for positive cases, indicating the presence of rfa (dark and thick) mutation. This change allows certain macromolecules to enter the bacteria and inhibit their growth. TA97, TA98, 1A100 and TA102 all have inhibition zones, while wild-type Salmonella typhimurium has no inhibition zone. 5.2.4 Identification of R factor
5.2.4.1 Heat and melt the nutrient broth agar medium, cool it to about 50℃, pour an appropriate amount into a plate, lay it flat to solidify, use a pipette to suck 10μL of 0.8% ampicillin, and apply a strip along the center line on the surface of the solidified medium. After the ampicillin solution is inoculated, use an inoculation loop to cross the ampicillin strip and inoculate the strain to be identified, and inoculate a strain that does not really have the R factor as a control for ampicillin resistance (several strains can be identified simultaneously in one plate), and culture at 37℃ for 24h. 5.2.4.2 Result determination: After 24 hours of culture, the four strains still grow uninhibited around the ampicillin band, that is, they have an anti-ampicillin effect, proving that they all carry the R factor. 5.2.5 Identification of tetracycline resistance
5.2.5.1 Use a pipette to take 5μL~~10uL of 0.8% tetracycline solution and 0.8% ampicillin solution, and apply a strip along the center line on the surface of the nutrient broth agar medium. After the tetracycline and ampicillin solutions are ten, use an inoculation loop to cross the tetracycline and ampicillin bands to inoculate TA102 and a strain with the R factor (as a control for tetracycline resistance), and culture at 37℃ for 24 hours. 5.2.5.2 Result determination: The growth of TA102 strain is not inhibited, and the control strain has a growth inhibition zone, indicating that TA102 strain has an anti-tetracycline effect.
5.2.6 Identification of uvrB repair defective type
5.2.6.1 Use an inoculation loop to streak the required strain on the surface of the nutrient broth agar medium. 5.2.6.2 Cover half of the inoculated medium with black paper, irradiate for 8 seconds at a distance of 33 cm from a 15W ultraviolet sterilization lamp, and culture at 37℃ for 24 hours. 5.2.6.3 Result judgment: The three strains (TA97, TA98, TA100) sensitive to ultraviolet rays only grow in the half that has not been irradiated, and the strain TA102 with wild-type excision repair enzyme can still grow. 5.2.7 Determination of spontaneous reversion rate
5.2.7.1 Prepare 8 bottom culture medium plates. 5.2.7.2 Melt 8 tubes of top culture medium, 2mL each, and keep warm in a 45℃ water bath. 5.2.7.3 Add 0.1 mL of the bacterial solution of the test strain to be identified to each tube of top culture medium, in duplicate, shake gently, and quickly pour the contents of the test tube into the solidified bottom culture medium plate, rotate the plate to evenly distribute the top culture medium, lay it flat to solidify, and culture it at 37℃ for 48 hours to count the number of colonies.
5.2.7.4 Result determination: The spontaneous reversion rate of each strain should fall within the normal range listed in Table A.1. 5.3 Preservation of strains
Qualified strains should be stored at deep low temperature (such as -80℃), or added with 9% spectral grade DMSO as a cryoprotectant and stored under liquid nitrogen conditions (-196℃), or freeze-dried to make dry powder and stored at 4℃. Except for liquid nitrogen conditions, the storage period is generally not more than 2 years. The master plate should be discarded after being stored at 4℃ for more than two months, and the TA102 master plate should be discarded after two weeks. 6 Experimental design and special treatment of test substances
6.1 Dose design
The principle for determining the maximum dose of the test substance is the toxicity of the test substance to the test strain and the solubility of the test substance. For pure chemical substances, the general minimum dose is 0.2ug per microliter, the maximum dose is 5mg, or the solubility allows, or the saturation concentration, or the minimum toxic concentration to bacteria. For a finalized product with very low toxicity and a large intake, the maximum possible dose can be adopted according to its solubility and toxicity to bacteria. Each test substance shall be set with 4 (including 4) or more doses at the maximum allowable dose, and the interval between each dose shall not exceed 5 times, and three microliters shall be made for each dose. If the above principles are not followed, the reasons for the selected dose shall be explained. 6.2 Solvent
The solvent can be water, dimethyl sulfoxide (not more than 0.4mL per microliter), or other solvents (below the toxic dose). No matter what solvent is selected, it should not be mutagenic.
6.3 Setting up control groups
The test should have positive control group, solvent control group and untreated control, including both adding S-9 and not adding S-9. The positive control should be selected according to the type of strain, and you can refer to Appendix A or other relevant information. 6.4 Special treatment of test substances
If special test substances are treated unconventionally, they should be explained in the report. The following situations can be treated as follows: 6.4.1 Test substances containing histidine: If the histidine content measured in the food can induce an increase in the reverse mutation rate, a histidine parallel control group can be added; or the test sample can be pre-treated by filtering and eluting with XAD-ⅡI resin column. 6.4.2 Food packaging materials and their product ingredients: According to the composition of the materials or products, they can be treated by screening extraction, evaporation residue and other technical treatments.
6.4.3 Volatile test substances: Vacuum dryer treatment and other methods can be used. 6.4.4 Natural plant materials: crude or pure products can be prepared by phytochemical methods. 7 Test methods
It can be divided into plate incorporation method, pre-culture plate incorporation method and spot test method, which are described as follows: 7.1 Plate incorporation method
7.1.1 Bacterial enrichment culture: Take 5mL of nutrient broth culture medium and add it to a sterile small triangular bottle or sterile test tube. Inoculate the main plate or frozen strain culture into the nutrient broth culture medium, and culture it at 37℃ with shaking (100 times/min) for 10 hours until the logarithmic growth phase. The number of viable bacteria per milliliter should not be less than 1×10°~2×10°. The culture bottle can be wrapped with black paper to prevent light from irradiating the bacteria. 7.1.2 Several plates of bottom culture medium.
7.1.3 Melt the top culture medium and dispense it into sterile small test tubes, 2mL per tube, and keep it warm in a 45℃ water bath. 7.1.4 Add 0.1 mL of fresh enrichment solution of the test strain to the insulated top culture medium in sequence and mix well; add 0.05 mL to 0.2 mL of the test substance (generally add 0.1 mL. If activation is required, add 0.5 mL of 10% S-9 mixed solution), mix well again, and quickly pour it onto the bottom culture medium. Rotate the plate to make the top culture medium evenly distributed on the bottom, lay it flat to solidify, and culture it at 37℃ for 48 hours to observe the results. 7.1.5 Also make positive control, solvent control and untreated control. The positive control does not add the test substance, but only adds the standard mutagen (see A.2.2, A.2.3 in Appendix A); the solvent control adds all reagents except the test substance and the standard mutagen, such as the solvent dimethyl sulfoxide (spectrally pure or analytically pure); the untreated control only adds the bacterial solution to the culture medium; other methods are the same as above. 7.2 Pre-culture plate incorporation method
Pre-culture can achieve better results for some test substances. Therefore, it can be determined whether to perform pre-culture according to the situation. Before adding the top agar, the following pre-culture steps should be performed: In the test, the test substance (add 10% S-9 mixed solution when activation is required) and the bacterial solution are cultured at 37℃ for 20 minutes, or at 30℃ for 30 minutes, and then 2mL of top agar is added. The rest is the same as the above-mentioned plate incorporation method. 7.3 Spot test method
7.3.1 is the same as the incorporation method 7.1.1.
7.3.2 is the same as the incorporation method 7.1.2.
7.3.3 is the same as the incorporation method 7.1.3.
7.3.4 Add 0.1mL of the test strain enrichment solution (add 0.5mL of 10% S-9 mixed solution when necessary) to the top culture medium kept warm in a water bath, mix well, and quickly pour it onto the bottom culture medium. Rotate the plate to evenly distribute the top culture medium on the bottom. After solidification, take a sterile filter paper disc (6mm in diameter) and carefully place it on the appropriate position of the solidified top culture medium. Use a pipette to take an appropriate amount of the test substance (e.g. 10μL) and spot it on the paper disc, or add a small amount of solid test substance crystals to the paper disc or agar surface, and incubate at 37℃ for 48h to observe the results. 7.3.5 Also make positive control, solvent control and untreated control. Replace the addition of the test substance with the addition of a standard mutagenic substance (see A.2.1, 40
A.2.3 in Appendix A) or a solvent (e.g. dimethyl sulfoxide), and the other steps are the same as above. 8. Determination of results
8.1 Determination of results of the incorporation method
GB15193.4—2003
The result is determined by directly counting the number of reverted colonies grown on the culture medium. If, under good background growth conditions, the number of reverted colonies in the test substance group increases by more than one time (i.e., the number of reverted colonies is equal to or greater than 2 times the number of untreated controls), and there is a dose-response relationship or at least one test point has a repeatable and statistically significant positive reaction, the test substance can be considered to be positive for mutagenicity. 8.2. Determination of the spot test method
If more densely populated mutagenic colonies grow around the test article spot paper, and there is a clear difference compared with the untreated control, it can be preliminarily determined that the test article is positive for mutagenicity, but it should be confirmed by the spike test. 9. Result report
When reporting, positive results should be tested at least three times, and negative results should be tested at least twice before a judgment can be made on the test article. The mutagenicity of the test article should be confirmed by the plate spike test. The test conditions should be indicated in the report, and all the result information should be attached. If there is any doubt about the result, it needs to be statistically analyzed. The same test article must include the results of activation and non-activation. The dosage unit is micrograms per plate, with special exceptions. 41
GB15193.4—2003
Appendix A
(Normative Appendix)
Standards for identification of biological characteristics of strains, standard diagnostic mutagens, test records and report formatsA.1
Standards for identification of biological characteristics of strains, standard diagnostic mutagens, test records and report formatsThe results of the standards for identification of biological characteristics of strains are shown in Table A.1. Table A.1
Histidine
“+” indicates the need
Histidine
Lipopolysaccharide
Barrier defect
“+” indicates inhibition
Test results and expression methods of standard mutagens A.2. 1
R factor
Anti-tetracycline
(Anti-ampicillin)
“+” indicates the presence
R factor
The test results of standard mutagens in spot tests are shown in Table A.2. Table A.2
Daunorubicin
Sodium azide
ICR-191
Mitomycin C
2,4,7-Trinitrobenzene
4-Nitro-phospho-phenylenediamine
4-Nitroquinoline-N-oxide
Methyl methanesulfonate
Dichlorothiazide
2-Acetamidobenzoate
Aflatoxin Bl
Methyl nitrosoguanidine
Dose/(μg/tablet)
2. 0(μL)
Note: The symbol of the number of reverting colonies per dish (excluding spontaneous reverting): 500;++++
Repair defect
“+” indicates four “+” indicates no repair
Cyclin resistance
Repair ability
-20100;++
100~200;+
Spontaneous reverting
Number of colonies
90~~180
120~200
240~~320
-200~～
>500. Daunorubicin and sodium azide were dissolved in water, and all other compounds were dissolved in DMSO. 2-AF was activated with PCB-induced rat S-9 (20μL/blood). Daunorubicin produces the lowest effect in the spot test and should be tested for plate incorporation (see Table A.3). ICR-191-
-2-methylchloro-6-chloro-9-{3-(2-chloroethyl)aminopropylamine]acridine·2-hydrochloride;inh-growth inhibition caused by mutagenic toxicity. A.2.2 The test results of diagnostic mutagens in plate incorporation are shown in Table A.3. Table A.3
Mutagens
Robustin
Sodium azide
ICR-191
Streptomycin
Mitomycin C
2,4,7-Trinitrocopper
4-Nitro-phospho-phenylenediamine
4-Nitrothioline-N-oxide
Methyl methanesulfonate
Dichlorothiazide
2-Acetylaminoglucose
Benzo(a)pyrene
4 g/blood
1. 0(μL)
GB15193.4—2003
Note: The values listed represent the his ten reverse mutation colony values, taken from the linear part of the dose response, the control value has been subtracted, and PCB-induced rat liver S-9 (20 μL/Ⅲ) activated 2-AF and benzo(a) flower. inh: streptomycin has no mutagenicity detected within the non-toxic range (less than 0.25ug), and the number of reverse mutation colonies caused by each 0.005 μg in TA100 is less than 70; mitomycin is lethal to uvrB strains. Standard mutagens recommended for spot tests and spiked plates are shown in Table A, 4. A. 2. 3
Dixon
2-aminolychee
Dixon
2-aminolychee
Dixon
2-aminolychee
Dixon
2-aminolychee
Sodium azide
2-aminolychee
Sodium azide
2-aminolychee
Dixon
Dixon4—2003
Note: The values listed represent the his ten reverse mutation colony values, taken from the linear part of the dose response, the control value has been subtracted, and PCB-induced rat liver S-9 (20 μL/Ⅲ) activated 2-AF and benzo(a) flower. inh: streptomycin has no mutagenicity detected within the non-toxic range (less than 0.25ug), and the number of reverse mutation colonies caused by each 0.005 μg in TA100 is less than 70; mitomycin is lethal to uvrB strains. Standard mutagens recommended for spot tests and spiked plates are shown in Table A, 4. A. 2. 3
Dixon
2-aminolychee
Dixon
2-aminolychee
Dixon
2-aminolychee
Dixon
2-aminolychee
Sodium azide
2-aminolychee
Sodium azide
2-aminolychee
Dixon
Dixon4—2003
Note: The values listed represent the his ten reverse mutation colony values, taken from the linear part of the dose response, the control value has been subtracted, and PCB-induced rat liver S-9 (20 μL/Ⅲ) activated 2-AF and benzo(a) flower. inh: streptomycin has no mutagenicity detected within the non-toxic range (less than 0.25ug), and the number of reverse mutation colonies caused by each 0.005 μg in TA100 is less than 70; mitomycin is lethal to uvrB strains. Standard mutagens recommended for spot tests and spiked plates are shown in Table A, 4. A. 2. 3
Dixon
2-aminolychee
Dixon
2-aminolychee
Dixon
2-aminolychee
Dixon
2-aminolychee
Sodium azide
2-aminolychee
Sodium azide
2-aminolychee
Dixon
Dixon
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