GB 15193.4-2014 PDF English
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National food safety standard -- Bacterial reversion mutation test
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Salmonella typhimurium/mammal’s microsomal enzyme test (Ames test)
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GB 15193.4-2014: PDF in English GB 15193.4-2014
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National food safety standard - Bacterial reversion mutation
test
ISSUED ON: DECEMBER 24, 2014
IMPLEMENTED ON: MAY 01, 2015
Issued by: National Health and Family Planning Commission of the People’s
Republic of China
Table of Contents
Foreword ... 3
1 Scope ... 4
2 Terms and definitions ... 4
3 Test purpose and principle ... 4
4 Apparatus and reagents ... 5
5 Bacterial strains and their identification and preservation ... 10
6 Design of experiments and treatment of test substances ... 13
7 Test methods ... 15
8 Data processing and result evaluation ... 16
9 Report ... 17
10 Interpretation of the test ... 18
Appendix A Mutant genes, test types, biological characteristics and spontaneous
reversion of test strains ... 19
Appendix B Standard diagnostic mutagens ... 21
Appendix C Positive mutagens recommended by OECD and USEPA ... 23
National food safety standard -
Bacterial reversion mutation test
1 Scope
Bacterial reverse mutation test includes Salmonella typhimurium reversion mutation
test and Escherichia coli reversion mutation test. This Standard specifies the basic
technical requirements for Salmonella typhimurium reversion mutation test. When
selecting Escherichia coli for bacterial reversion mutation test, relevant literature shall
be referred to.
This Standard is applicable to the evaluation of the mutagenic effect of test substances.
2 Terms and definitions
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.
3 Test purpose and principle
Detect the genetic mutation effect of the test substance on microorganisms (bacteria)
and predict its genotoxicity and potential carcinogenic effects.
The bacterial reversion mutation test uses Salmonella typhimurium and Escherichia coli
to detect point mutations, involving the replacement, insertion or deletion of one or
several base pairs of DNA, as shown in Appendix A. The test strains of Salmonella
typhimurium and Escherichia coli are histidine-deficient mutants and tryptophan-
80 ℃, prepare plates at a rate of 25 mL per plate (relative to a 90 mm plate). After
condensation and solidification, place the plates upside down in a 37 ℃ incubator for
24 h for use.
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 ℃ 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 ℃ ~ 4 ℃) 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 ℃; 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 Test methods
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 ℃ 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 ℃ 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 ℃ 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 Report
9.1 Test name, test unit name and contact information, and report number.
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.
......
Source: Above contents are excerpted from the PDF -- translated/reviewed by: www.chinesestandard.net / Wayne Zheng et al.
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