GB 15193.4-2014 National food safety standard — Bacterial reverse mutation test
1 Scope
The bacterial reverse mutation test includes salmonella typhimurium reverse mutation test and escherichia coli bacterial reverse mutation test. This standard specifies the basic technical requirements of salmonella typhimurium typhimurium reverse mutation test. Reference should be made to the relevant literature when selecting escherichia coli for bacterial reverse mutation test.
This standard is applicable to evaluate the mutagenicity of test substances.
2 Terms and definitions
2.1
bacterial reverse mutation test
in vitro test of gene mutation using a mutant strain of trophic deficiency type as indicator organism. The commonly used strains are salmonella typhimurium with histidine dystrophia and escherichia coli with tryptophan dystrophia
2.2
base substitution gene mutation
abnormal DNA base sequence caused by one base in the DNA polynucleotide chain is replaced by another base
2.3
frame shift gene mutation
when one or more (except 3 and the multiples of 3) bases are inserted or missing in the DNA base sequence, the codon composition after that site is all changed, and the polypeptide chain guiding synthesis is also all changed according to the rule of continuous reading of the triple code.
3 Test objective and principle
The gene mutation effect of test substance on microorganisms (bacteria) is detected to predict its genotoxicity and potential carcinogenic effect.
Bacterial reverse mutation tests use salmonella typhimurium and escherichia coli to detect point mutations involving the substitution, insertion, or deletion of one or more base pairs of DNA. See Annex A. The experimental strains of salmonella typhimurium and escherichia coli are histidine deficient mutant and tryptophan deficient mutant respectively, which could not grow on the medium without histidine or tryptophan, but could grow normally on the medium with histidine or tryptophan. In the presence of mutagens, they can revert to the prototrophic form, and they can also grow on a medium without histidine or tryptophan. Therefore, the number of colonies formed can be used to measure whether the test substance is a mutagen.
Some mutagens require metabolic activation in order for the bacteria to produce revert mutations, and the test substance is tested in both the presence and absence of metabolic activation systems.
4 Apparatus and reagents
4.1 Apparatus
Common laboratory equipment, low temperature and high speed centrifuge, low temperature refrigerator (-80°C) or liquid nitrogen tank, biosafety cabinet, constant temperature incubator, constant temperature water bath, sterilization equipment, homogenizer, etc.
4.2 Reagents
4.2.1 Nutrient broth medium
Beef extract: 2.5g
Tryptone: 5.0g
Sodium chloride: 2.5g
Dipotassium hydrogen phosphate (K2HPO4·3H2O): 1.3g
Add distilled water to 500mL, dissolve with heat, adjust the pH to 7.4, sterilize at 0.103MPa for 20min after subpacking, store at 4°C for later use. The shelf life is not more than half a year.
4.2.2 Nutrient broth agar medium
Agar powder: 1.5g
Add nutrient broth medium to 100mL, heat to melt, adjust the pH to 7.4, sterilize at 0.103MPa for 20min.
Foreword I
1 Scope
2 Terms and definitions
3 Test objective and principle
4 Apparatus and reagents
5 Strain identification and preservation
6 Experimental design and treatment of test substances
7 Test methods
8 Data processing and result evaluation
9 Report
10 Interpretation of test
Annex A The mutant genes, detection types, biological characteristics and spontaneous reverse mutation colonies of test strains
Annex B Standard diagnostic mutagen
Annex C Positive mutagen recommended by OECD and USEPA
