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GB 15193.28-2020 (GB15193.28-2020)

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GB 15193.28-2020: PDF in English
GB 15193.28-2020
National Food Safety Standard -
Test of Mammalian Cell Micronucleus in Vitro
Issued by: National Health Commission of P. R. China;
State Administration for Market Regulation.
Table of Contents
1 Scope ... 3
2 Terms and Definitions ... 3
3 Purpose and Principle of Test ... 4
4 Instruments and Reagents ... 4
5 Test Methods ... 7
6 Data Processing and Result Judgment ... 12
7 Test Report ... 12
8 Interpretation of the Test ... 13
National Food Safety Standard -
Test of Mammalian Cell Micronucleus in Vitro
1 Scope
This Standard specifies the basic test methods and technical requirements for test of
mammalian cell micronucleus in vitro.
This Standard is applicable to evaluate the genotoxic effects of test substances.
2 Terms and Definitions
2.1 Micronucleus
When the chromosomes regularly enter the daughter cells to form the nucleus at the
late stage of mitosis, the entire chromatids or acentric fragments or rings of
chromosomes that remain in the cytoplasm. In the final stage, one or several regular
subnuclei are formed separately, which are contained in the cytoplasm of the cell.
2.2 Centromere
During cell division, the area where the chromosomes connect with the spindle fibers
allows the daughter chromosomes to move to the two poles of the daughter cells in an
orderly manner.
2.3 Aneuploidy
A variation type in which one or more complete chromosomes are missed or added to
the diploid chromosome complement.
2.4 Aneuploidy mutagen
A substance that acts on the mitosis or meiotic cycle of cells to cause abnormal cell
division and induce aneuploidy.
2.5 Chromosome break
A chromosome arm has a gap whose length is greater than the width of the
chromosome arm.
2.6 Chromosome clastogen
min, sterilize or filter. Coenzyme-II (oxidized) solution
Weigh Coenzyme-II under aseptic conditions and dissolve it with sterile distilled water
to prepare a 0.025 mol/L solution, which is prepared immediately before use. Glucose-6-phosphate sodium salt solution
Weigh glucose-6-phosphate sodium salt, dissolve it with distilled water to prepare a
0.05 mol/L solution, and filter and sterilize it. It shall be prepared immediately before
4.3.2 Induction and preparation of S9 component of rat liver
Choose healthy male adult SD or Wistar rats, with a body weight of 150g~200g, about
5 weeks old ~ 6 weeks old. Dissolve the polychlorinated biphenyl (Aroclor1254) in corn
oil at a concentration of 200g/L; and perform an intraperitoneal injection aseptically at
a weight of 500mg/kg. Then the animals are sacrificed 5d later; and the animals are
fasted for 12h before sacrifice.
It may also be prepared by the combined induction method of phenobarbital sodium
and β-naphthoflavone. Rats are given phenobarbital sodium and β-naphthoflavone by
oral gavage. The doses are both 80 mg/kg body weight for 3d. After fasting for 16h,
the animals are decapitated and sacrificed. Other operations are the same as
polychlorinated biphenyl induction.
After the animals are sacrificed, the liver is taken out. After weighing, the liver is
washed with fresh ice-cold 0.15 mol/L potassium chloride solution for several times in
order to remove hemoglobin, which can inhibit the activity of microsomal enzymes.
Add 3 mL of 0.1mol/L potassium chloride solution per gram of liver (wet weight); and
move it into an ice bath together with the beaker. Cut the liver with sterile scissors and
place it in a glass homogenizer (less than 4000 r/min, 1 min~2 min) or tissue
homogenizer (less than 20000 r/min, 1 min) to make liver homogenate. The above
operations should pay attention to aseptic and local cold environment.
Centrifuge the prepared liver homogenate in a low-temperature (0°C~4°C) high-speed
centrifuge at 9000g for 10 min. Aspirate the supernatant as the S9 component and
distribute it in sterile cryotubes, about 2mL per tube. It is best to use liquid nitrogen or
dry ice to quickly freeze and store at a low temperature of -80°C.
After the S9 component is prepared, the protein content (Lowry method) shall be
determined by sterility inspection; and the protein content per mL should not exceed
40mg; and the biological activity of the indirect mutagenic agent shall be qualified and
stored at -80°C low temperature or frozen and dried, and the shelf life is no more than
1 year.
c) Take 50mL of the first solution and add it to 50mL of the second solution; and mix
well, which is 1/15 mol/L phosphate buffer solution with pH 6.8.
5 Test Methods
5.1 Test substance
The solid test substance shall be dissolved in a suitable dissolvent and diluted to an
appropriate concentration. The liquid test substance may be used directly or diluted to
an appropriate concentration for use. The test substance shall be prepared aseptically
and immediately before use; otherwise, it must be confirmed that storage shall not
affect its stability.
5.2 Cell
Chinese hamster lung cell lines (V79, CHL) or ovarian cell lines (CHO), mouse
lymphoma cell lines (L5178Y), human peripheral blood lymphocyte lines (such as TK6)
and primary cultured cells may be selected. It is recommended to use the CHL or
L5178Y cell line. Cells shall be checked for stability of the chromosome number and
mycoplasma contamination before use.
5.3 Selection of test scheme
The test is divided into two schemes: using and not using cytoB. Scheme-1: After the
cells are treated with the test substance, use cytoB before mitosis; and then observe
and analyze the micronucleus rate of the cells (binuclear cells) that have completed
one mitosis. When using human lymphocytes, it is recommended to use Scheme-1;
because different sources of cells, their cycle are different; and not all cells respond to
Phytohemagglutinin (PHA). Scheme-2: Do not use cytoB; and then observe and
analyze the micronucleus rate of the cells after the cells are treated with the test
substance. If there is evidence that proves the test substance interferes with the activity
of cytoB, or cytoB may affect the growth of cells (such as mouse lymphoma cell lines),
then it is recommended to adopt the Scheme-2.
5.4 Dose
5.4.1 Dose setting
At least 3 detection doses shall be set. When the test substance is not cytotoxic, set
at least 2 doses from the highest dose downward. Generally, the interval coefficient
may be 2~3. When there is cytotoxicity, the dose range shall cover from 55%±5%
cytotoxicity to almost no cytotoxicity.
5.4.2 Selection of the highest dose
If there is no literature or historical data to prove that the used dissolvent is not
mutagenic, a blank control shall be set.
5.5 Test procedures
5.5.1 Preparation of cell
Inoculate a certain number of cells in a culture dish (bottle). When harvesting the cells,
if the cells in the culture dish (bottle) is not overgrown, it shall be regarded as the
standard. Adherent cells are generally preferred to grow to about 85%.
5.5.2 Treatment of test substance Application scheme-1: Aspirate the culture solution; wash the cells with
phosphate buffer solution. Add serum-free culture solution and a certain concentration
of the test substance (if metabolic activation is required, add S9 mix at the same time);
and place it in the incubator for 3h~6h. After the end, aspirate the culture solution
containing the test substance; wash the cells with PBS. Add fresh culture solution
containing 10% serum and cytoB; continue to culture for 1.5~2.0 normal cell cycles
and then collect the cells.
For lymphocytes, the most effective method is to start the test substance treatment
44h~48h after mitogen (e.g.: PHA) stimulation, at which time the cells begin to enter
the division cycle.
If the test result of the short-term treatment for 3h~6h is negative or unclear, a long-
term treatment test without S9 is required. The cells are treated with cytoB and the test
substance for 1.5~2.0 normal cell cycles; and the cells are collected after the treatment
is completed.
If it is known or suspected that the test substance (e.g.: nucleosides) may affect the
cell cycle (especially P53 active cells), then the cell harvest time shall be extended by
1.5~2.0 normal cell cycles. Application scheme-2: it is the same as, except that cytoB is not added.
5.5.3 Cell harvesting and slicing
Cells shall be harvested and sliced separately for each culture. If the cell mixed liquor
is well dispersed, hypotonic treatment is not required. Digestion
Adherent cells are digested with 0.25% trypsin solution. After the cells fall off, add a
culture solution containing 10% fetal bovine or calf serum to stop the trypsin effect; mix
well; and put it in a centrifuge tube to centrifugate at 800 r/min~ 1000 r/min for 5min;
and discard the supernatant. Suspended cells do not need to be digested and
6 Data Processing and Result Judgment
6.1 Data processing
The data is listed by different doses; and the indicators include cytotoxicity, the number
of observed cells, the number of micronucleus cells and micronucleus cell rate. The
micronucleus cell rate of each dose group of the test substance, the blank control
group, the negative control group (dissolvent control group), and the positive control
group are processed by appropriate statistical methods (such as X2 test).
6.2 Judgment of results
The following two situations may determine that the test substance is a positive result
in this test system:
a) The increase in the rate of micronucleus cells caused by the test substance is
statistically significant and is related to the dose;
b) The increase in the rate of micronucleus cells caused by the test substance under
any dosage condition is statistically significant and repeatable.
7 Test Report
7.1 Test name, test organization name, contact information, and report number.
7.2 The name and contact information of the test entrusting organization, and the date
of sample acceptance.
7.3 The start and end date of the test, the person in charge of the test project, the
technical person in charge of the test organization, and the date of issuance.
7.4 Test summary.
7.5 Test substance: name, identification information, CAS number (if known), purity,
physical and chemical properties and stability of the test substance related to this test.
7.6 Solvent.
7.7 Cell line: the source and name of the cell line.
7.8 Test conditions: dose, metabolic activation system, standard mutagens, operation
procedures, etc.
(Above excerpt was released on 2021-03-20, modified on 2021-06-07, translated/reviewed by: Wayne Zheng et al.)
Source: https://www.chinesestandard.net/PDF.aspx/GB15193.28-2020