GB 15193.4-2014National food safety standard - Bacterial reversion mutation test (English PDF)

2.1 Bacterial reversion mutation test

An in vitro test for detecting gene mutations using auxotrophic mutant strains as indicator organisms. Commonly used strains include histidine auxotroph Salmonella typhimurium and tryptophan auxotroph Escherichia coli.

2.2 Base substitution gene mutation

A certain base on the DNA polynucleotide chain is replaced by another base, causing abnormalities in the DNA base sequence.

2.3 Frameshift gene mutation

When one or several bases (except 3 and multiples of 3) are inserted or deleted in the

DNA base sequence, according to the rules of continuous reading of the triplet code, all the codon composition after this site will be changed, and the polypeptide chain that guides the synthesis will also be completely changed.

4.2.3.2 Phosphate stock solution (Vogel-Bonner minimal medium E, 50 times)

Sodium ammonium hydrogen phosphate (NaNH4HPO4·4H2O) 17.5 g

Citric acid (C6H8O7·H2O) 10.0 g

Dipotassium hydrogen phosphate (K2HPO4) 50.0 g

Magnesium sulfate (MgSO4·7H2O) 1.0 g

Add distilled water to 100 mL to dissolve, and sterilize at 0.103 MPa for 20 minutes.

Note: After the other reagents are completely dissolved, slowly put the magnesium sulfate into it to continue dissolving, otherwise it will easily precipitate.

4.2.3.3 40% glucose solution

Add distilled water to 40.0 g of glucose to 100 mL, and sterilize at 0.055 MPa for 20 minutes.

4.2.3.4 1.5% agar medium

Add 6.0 g of agar powder into a 400 mL conical flask; add distilled water to 400 mL;

after melting, sterilize at 0.103 MPa for 20 min.

4.2.4 Top culture medium

Heat to melt the top agar, add 10 mL of histidine-biotin solution (0.5 mmol/L) to every

100 mL of top agar. Mix well; divide into 4 flasks; sterilize at 0.103 MPa for 20 min.

Melt and divide into small test tubes, 2 mL per tube; keep warm in a 45 °C water bath.

Prepare top agar and histidine-biotin solution (0.5 mmol/L) as follows.

4.2.4.1 Top agar

Add distilled water to 3.0 g of agar powder and 2.5 g of sodium chloride to 500 mL;

sterilize at 0.103 MPa for 20 min.

4.2.4.2 Histidine-biotin solution (0.5 mmol/L) (for mutagenesis test)

Add 250 mL of distilled water to 30.5 mg of D-biotin (relative molecular mass 244) and 19.4 mg of L-histidine (relative molecular mass 155); sterilize at 0.103 MPa for 20 min.

4.2.5 Special reagents and culture media

4.2.5.1 0.8% ampicillin solution (for strain identification, prepared aseptically)

Use sodium hydroxide solution (0.02 mol/L) to dilute 40 mg of ampicillin to 5 mL;

store it in a refrigerator at 4 °C for later use.

4.2.5.2 0.1% crystal violet solution (for strain identification)

Dissolve 100 mg of crystal violet in sterile water to 100 mL.

4.2.5.3 L-histidine solution and D-biotin solution (0.5 mmol/L) (for strain identification)

Respectively dissolve 0.404 3 g of L-histidine and 12.2 mg of D-biotin in distilled water to 100 mL; sterilize at 0.103 MPa for 20 min; store in a 4 °C refrigerator for later use.

4.2.5.4 0.8% tetracycline solution (for tetracycline resistance test and ampicillin- tetracycline plate)

Use hydrochloric acid buffer (0.02 mol/L) to dilute 40 mg of tetracycline to 5 mL; store in a 4 °C refrigerator.

4.2.5.5 Ampicillin plate (used as the master plate for TA97, TA98, and TA100 strains) and ampicillin-tetracycline plate (used as the master plate for TA102 strain)

Each 1 000 mL consists of the following ingredients:

Bottom culture medium 980 mL

Histidine aqueous solution (0.404 3 g/100 mL) 10 mL

Biotin (0.5 mmol/L) 6 mL

0.8% ampicillin solution 3.15 mL

0.8% tetracycline solution 0.25 mL

Tetracycline is added only when using tetracycline-resistant TA102. Each ingredient has been individually sterilized or prepared aseptically.

4.2.5.6 Histidine-biotin plate (histidine needs to be used for testing)

Each 1 000 mL consists of the following ingredients:

Bottom culture medium 984 mL

Histidine aqueous solution (0.404 3 g/100 mL) 10 mL

Biotin (0.5 mmol/L) 6 mL

Each ingredient has been sterilized individually.

oral gavage at a dose of 80 mg/kg for 3 consecutive days without fasting. Kill the animals by decapitation after 16 h of fasting. Other operations are the same as PCB induction.

After killing the animals, remove the liver, weigh, and continuously flush with fresh ice-cold potassium chloride solution (0.15 mol/L) several times to remove hemoglobin that inhibits microsomal enzyme activity. Add 3 mL of potassium chloride solution (0.1 mol/L) per gram of liver (wet weight); move it together with the beaker into an ice bath;

use sterile scissors to cut the liver into pieces; place in a glass homogenizer (less than 4

000 r/min, 1 min ~ 2 min) or tissue homogenizer (less than 20 000 r/min, 1 min) to make liver homogenate. The above operations require attention to sterility and local cold environment.

Centrifuge the prepared liver homogenate in a low-temperature (0 °C ~ 4 °C) high- speed centrifuge at 9 000g for 10 minutes. Aspirate the supernatant into the S9 component and distribute it into sterile freezing tubes or ampoules. Each ampoule is about 2 ml. Quick freeze in liquid nitrogen or dry ice and then store at 80 °C.

After the S9 component is prepared, subject it to a sterility inspection and measure the protein content (Lowry method). The protein content per milliliter should not exceed

40 mg. After qualifying the biological activity by indirect carcinogen (mutagen) identification, store it at a deep low temperature or freeze-dry, and the shelf life shall not exceed 1 year.

4.3.3 Preparation of 10% S9 mixture

The 10% S9 mixture is generally composed of S9 components and cofactors in a ratio of 1:9. The concentration can also be prepared to 30% (different test substances require different S9 concentrations). Freshly prepare aseptically or filter for sterilization before use. 10 mL of 10% S9 mixture is prepared as follows:

Phosphate buffer 6.0 mL

Magnesium potassium solution 0.4 mL

Glucose-6-phosphate sodium salt solution 1.0 mL

Coenzyme-II solution 1.6 mL

Liver S9 fraction 1.0 mL

Mix well and place in ice bath until ready to use.

Use 0.5 mL of S9 mixture per plate (containing 20 µL ~ 50 µL of S9) to measure its biological activity against known positive carcinogens (mutagens) and determine the most suitable amount. Alternatively, use the normal dosage, that is, 0.5 mL of S9 mixture per dish.

Heat and melt two bottles of bottom culture medium. To one bottle, do not add histidine, and add 0.6 mL of 0.5 mmol D-biotin to every 100 mL of bottom culture medium; to the other bottle, add histidine, and add 1 mL of L-histidine and 0.6 mL of 0.5 mmol D- biotin to every 100 mL of bottom culture medium. Cool to about 50 °C and pour two plates of each bottom culture medium.

5.2.2.2.2 Vaccination

Take one medium plate with histidine and one without histidine; draw an inoculating loop of bacterial liquid in order of strain number and draw a straight line on the surface of the medium; incubate at 37 °C for 48 hours.

5.2.2.2.3 Result judgment

Each strain grew a bacterial film on the surface of the medium plate with histidine. On the medium plate without histidine, if there was no sterile film except for spontaneous reversion colonies, it indicates that the tested strain is indeed histidine deficient.

5.2.2.3 Identification of lipopolysaccharide barrier defects (rfa)

5.2.2.3.1 Vaccination

Heat and melt the nutrient broth agar medium. Take 0.1 mL of bacterial liquid and transfer it to the plate; quickly pour an appropriate amount of nutrient broth agar culture medium (cooled to about 50 °C) into the plate; mix well; lay it flat to solidify. Place a piece of sterile filter paper into the center of the solidified culture medium plate; use a pipette to drop 10 µL of 0.1% crystal violet solution on the filter paper piece; incubate at 37 °C for 24 hours. Make a plate for each strain.

5.2.2.3.2 Result determination

In positive cases, a transparent inhibition zone appears around the paper, indicating the presence of rfa mutation. This change allows certain macromolecules to enter the bacteria and inhibit their growth.

5.2.2.4 Identification of R factor (ampicillin resistance)

5.2.2.4.1 Vaccination

Heat and melt the nutrient broth agar culture medium; cool it to about 50 °C; pour an appropriate amount into the plate; lay it flat to solidify; use a pipette to suck 10 µL of

0.8% ampicillin; apply a strip along the midline on the surface of the solidified culture medium. After the ampicillin solution dries, use an inoculation loop to take the bacterial liquid of each strain and inoculate it crosswise with the ampicillin band; inoculate a strain without R factor as a control for ampicillin resistance; incubate at 37 °C for 24 hours. One plate can be used to identify several strains simultaneously.

5.2.2.4.2 Result determination

The strains still grow uninhibited around the ampicillin band, that is, they are resistant to ampicillin, proving that they all carry R factors.

5.2.2.5 Identification of tetracycline resistance

5.2.2.5.1 Vaccination

Use a pipette to draw 5 µL ~ 10 µL of 0.8% tetracycline solution and 0.8% ampicillin solution each; apply a strip along the midline on the surface of the nutrient broth agar medium plate. After the tetracycline and ampicillin solutions dry, use the inoculation loop to take the bacterial liquid of each strain and cross-streak the tetracycline and ampicillin bands to inoculate TA102 and a strain with R factor (as a control for tetracycline resistance); culture at 37 °C for 24 hours.

5.2.2.5.2 Result determination

The growth of the TA102 strain is not inhibited, and the control strain had a growth inhibition zone, indicating that the TA102 strain had an anti-tetracycline effect.

5.2.2.6 Identification of uvrB repair defective types

5.2.2.6.1 Vaccination

Use an inoculation loop to streak the surface of the nutrient broth agar medium plate to inoculate the desired strain. Cover half of the inoculated plate with ink paper; irradiate for 8 seconds at a distance of 33 cm from a 15 W ultraviolet sterilizing lamp; incubate at 37 °C for 24 hours.

5.2.2.6.2 Result determination

The three strains sensitive to ultraviolet light (TA97, TA98, and TA100) grew only in the half that is not irradiated, while the strain TA102 with the wild-type excision repair enzyme is still able to grow.

5.2.2.7 Identification of biotin deficiency (bio)

5.2.2.7.1 Preparation of bottom culture dish

Heat and melt two bottles of bottom culture medium. Add biotin to one bottle, and add

0.6 mL of 0.5 mmol D-biotin and 1 mL of L-histidine to every 100 mL of bottom culture medium; do not add biotin to the other bottle, and add 1 mL of L-histidine to every 100 mL of bottom culture medium. Cool to about 50 °C; pour two plates for each bottom culture medium.

5.2.2.7.2 Vaccination

The principles for determining the maximum dose of the test substance are the toxicity of the test substance to the test strain and the solubility of the test substance. Conducting a preliminary test will help to understand the toxicity of the test substance to the strain and the solubility of the test substance. The recommended maximum dose for soluble test substances without bacterial toxicity is 5 mg/dish or 5 µL/dish; for test substances with poor solubility, suspension can be used, but the degree of turbidity of the solution (the amount of precipitation) cannot affect the colony count. When the maximum dose cannot reach 5 mg/dish or 5 µL/dish due to solubility or toxicity limitations, the highest dose shall be the dose that causes precipitation or bacterial toxicity. When evaluating test substances containing potentially mutagenic impurities, the test dose can be higher than 5 mg/dish or 5 µL/dish. For test substances that require pre-treatment (such as liquid beverages, tea bags, oral liquids and samples with large excipient content, etc.), the dose design shall be based on the processed sample.

Each test substance is divided into 4 dose groups under the maximum allowable dose, including with and without S9. Set the dose interval according to the principle of equal group intervals, and it is recommended to use times the group interval. Three plates shall be prepared for each dose.

Generally, the minimum dose of the test substance is not less than 0.2 µg/dish.

The test material shall be sterile and, if necessary, sterilized or disinfected by appropriate methods.

6.3 Control group setting

The experiment shall be set up a positive control group, a solvent control group and an untreated control group, including conditions with and without S9.

The positive control shall be selected according to the strain used, and the appropriate dosage shall be selected to ensure the effectiveness of each test. Refer to Appendix B,

Appendix C or other materials.

The treatment method of the vehicle control group is the same as that of the treatment group except that no test substance is added.

When the positive mutagen is dissolved in DMSO and the test substance is not dissolved in DMSO, a DMSO solvent control shall be performed at the same time.

6.4 Test substances containing histidine

For test substances that are known and confirmed to contain histidine and may affect the test results, pretreatment (such as filtration through XAD-II resin column) may be required.

7.1 General

Commonly used test methods include plate incorporation method, pre-culture plate incorporation method and spot test method.

7.2 Plate incorporation method

7.2.1 Inoculate the strain culture from the master plate or frozen storage into nutrient broth medium and culture at 37 °C with shaking (100 times/min) for 10 hours or static culture for 16 hours to ensure that the number of viable bacteria is not less than 1×109

/mL ~ 2×109 /mL.

7.2.2 For the bottom culture medium plates, make three plates for each dose with or without S9.

7.2.3 Thaw the top culture medium and dispense into sterile capped small test tubes (the number of test tubes is the same as the number of plates), 2 mL per tube, and keep warm in a 45 °C water bath.

7.2.4 Add 0.1 mL of fresh enrichment liquid of the test strain to the insulated top culture medium (test tube) and mix well; add 0.05 mL ~ 0.2 mL of test substance to the test group (add 0.1 mL generally, and add 0.5 mL of 10% S9 mixture when activation is required); mix again; quickly pour it onto the plate with the bottom culture medium;

rotate the plate to make the top culture medium evenly distributed on the bottom culture medium; lay it flat for solidification; incubate at 37 °C for 48 hours to observe the results;

if necessary, extend the time to 72 h to observe the results.

7.2.5 Add the same volume of standard mutagen to the positive control group; add only the same volume of solvent to the solvent control group; only add bacterial liquid to the culture medium in the untreated control group; other methods are the same as those of the test group.

7.3 Pre-culture plate incorporation method

Pre-culture can achieve better results for some test substances. Therefore, whether to perform pre-culture can be determined according to the situation. Before adding the top culture medium, perform the following pre-culture steps:

a) Incubate the test substance (add 0.5 mL of 10% S9 mixture if activation is required) and bacterial solution at 37 °C for 20 min, or at 30 °C for 30 min;

b) Add 2 mL of top agar;

Other details are the same as 7.2.

8.4 Verification

Obviously, positive results do not need to be verified; suspicious results shall be verified using other methods; negative results need to be verified (i.e., repeated once), and the test conditions shall be changed, such as the dose interval (changed to 5 times the interval).

8.5 Result evaluation of control group

A positive result indicates that the test substance induces point mutations in the genome of the test strain. A negative result indicates that the test substance does not induce gene mutations in the test strain under the test conditions.

9.2 Name and contact information of the test entrusting unit, and sample acceptance

date.

9.3 Test start and end dates, test item leader, test unit technical leader, and issue date.

9.4 Test summary.

9.5 Test substances: name, identification information, CAS number (if known), purity, physical and chemical properties and stability of the test substance related to this test, etc.

9.6 Solvent: The basis for selecting the solvent, the solubility and stability of the test substance in the solvent.

9.7 Strain: source, name, concentration (number of bacteria/dish) and strain

characteristics (including time and results of strain identification).

9.8 Test conditions: dosage, metabolic activation system, standard mutagen, operating

procedures, etc.

9.9 Test results: toxicity of the test substance to the strain, growth of background bacterial moss, whether there is precipitation on the plate, the number of reversion colonies on each plate, the mean and standard deviation of the number of reversion colonies on each plate for each dose and each strain with and without S9, whether there is a dose-response relationship, statistical results, the mean and standard deviation of the simultaneous solvent control and positive control, and the historical range of the solvent control and positive control.

9.10 Conclusion: Whether the test substance has mutagenic effect under the test conditions.

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