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GB 31655-2021: National food safety standard - In vivo mammalian alkaline comet assay
GB 31655-2021
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National Food Safety Standard -
In Vivo Mammalian Alkaline Comet Assay
ISSUED ON: FEBRUARY 22, 2021
IMPLEMENTED ON: AUGUST 22, 2021
Issued by: National Health Commission of the People’s Republic of China;
State Administration for Market Regulation.
Table of Contents
1 Scope ... 3 
2 Terms and Definitions ... 3 
3 Test Purposes and Principles ... 4 
4 Instruments and Reagents ... 4 
5 Test Methods ... 5 
6 Test Procedures ... 7 
7 Data Processing and Result Evaluation ... 10 
8 Test Report ... 11 
9 Test Interpretation ... 12 
Appendix A Recommended Positive Control Substances and Corresponding
Target Tissues (for rodents) ... 14 
National Food Safety Standard -
In Vivo Mammalian Alkaline Comet Assay
1 Scope
This Standard specifies the basic test methods and technical requirements for in vivo
mammalian alkaline comet assay.
This Standard is applicable to the evaluation of the DNA damage effect of the test
substance on mammalian tissue cells.
2 Terms and Definitions
2.1 Comet
Comet refers to the tailing phenomenon of damaged DNA fragments under the action
of an electric field, which looks like a “comet” in the microscopic image. The comet
head is nuclear DNA; the comet tail is composed of damaged DNA fragments that
migrate out of the nucleus in an electric field.
2.2 Scorable Cell
Scorable cell refers to a cell with a clear outline of comet head and tail and is not
disturbed by neighboring cells.
2.3 “Hedgehog” Shaped Cell
“Hedgehog” shaped cell refers to a cell that consists of a small or fuzzy head and a
large and diffused tail in the microscopic image.
2.4 DNA Percentage of Tail
DNA percentage of tail refers to the ratio of DNA content of the comet tail to the total
DNA content (the sum of the head and tail). It reflects the relative degree of DNA
damage, which is expressed as a percentage.
2.5 Key Variables
Key variables refer to test parameters, whose minor changes may have a relatively
significant influence on the test results. Key variables include sampling time, lysis
conditions and electrophoresis time, etc. The key variables may be tissue specific.
4.2.4 Electrophoresis buffer: weigh-take EDTA disodium salt and sodium hydroxide
(analytically pure); use pure water to prepare a solution (final concentrations are: 0.001
mol/L EDTA disodium salt and 0.3 mol/L sodium hydroxide). Prepare it right before use.
Adjust pH to make pH ≥ 13. Before use, keep it refrigerated at below 10 °C for storage.
4.2.5 Neutralization buffer: weigh-take tris (hydroxymethyl) methyl aminomethane
(analytically pure); use pure water to prepare a solution; the final concentration is 0.4
mol/L. Adjust pH to 7.5. Before use, keep it refrigerated at below 10 °C for storage.
4.2.6 Shearing buffer: weigh-take EDTA disodium salt (analytically pure); use Hank’s
balanced salt solution (HBSS) (pH 6.7 ~ 7.8, excluding Ca2+, Mg2+ and phenol red) to
dissolve it and prepare a solution; the final concentration is: 0.02 mol/L. Adjust pH to
7.5. Right before use, add dimethyl sulfoxide; the final concentration is 10% (V/V).
Before use, keep it refrigerated at below 10 °C for storage.
4.2.7 Staining solution: DNA fluorescent dyes (for example, SYBR Gold, SYBR Green
I, Gelred, propidium iodide or ethidium bromide); in accordance with the product
requirements, prepare and use it.
5 Test Methods
5.1 Test Substance
5.1.1 Method of preparation: the test substance shall be dissolved or suspended in a
suitable solvent. The optimal solvent is water. For test substances that are insoluble in
water, vegetable oils (such as: olive oil and corn oil, etc.) may be used. For test
substances that are insoluble in water or oil, carboxymethyl cellulose and starch may
also be used to prepare suspensions or pastes. The test substance shall be prepared
right before use, unless there is data suggesting that its solution or suspension is stable
in storage.
5.1.2 Administration route: the test substance shall be administrated by gavage.
Generally speaking, the gavage volume does not exceed 10 mL/kg body weight for
rats and 20 mL/kg body weight for mice. If it is an aqueous solution, the maximum
gavage volume may reach 20 mL/kg body weight; if it is an oily liquid, the gavage
volume shall not exceed 4 mL/kg body weight; the gavage volume shall be consistent
in each group.
5.2 Positive Control
The positive control substance shall be able to induce DNA strand breaks in the target
tissue of the test substance. Ethyl methane sulfonate (EMS) may be selected as the
positive control substance. The mode, in which, the positive control substance is
handled is not necessarily the same as that of the test substance. The positive control
substances and their corresponding target tissues (for rodents) are shown in Appendix
A.
group shall follow the following sequence:
a) 10 g/kg body weight;
b) 100 times of possible intake by human beings;
c) Maximum gavage volume at one time.
In addition, there is also a negative (solvent) control group. The positive control
substance may be administrated by oral gavage of 200 mg/kg body weight of ethyl
methane sulfonate.
6 Test Procedures
6.1 Animal Observation
During the test, observe and record animal health at least once a day (preferably at
the same time). Meanwhile, the peak period of expected effects after the test
substance is administrated shall be taken into consideration.
6.2 Sampling Time
6.2.1 The sampling time is a key variable, which shall be after the DNA strand is
induced to break, and before the break is removed and repaired, or before cell death.
The duration of some damages that cause DNA strand breaks detected by the alkaline
comet assay is probably extremely short. If such transient DNA damage is suspected,
then, measures shall be taken to reduce the leak detection of DNA damage and ensure
that tissue sampling is carried out as soon as possible, which is probably earlier than
the sampling time provided below.
6.2.2 The best sampling time depends on the test substance itself or the mode, in
which, the test substance is administrated. When conditions permit, the sampling time
shall be determined by the toxicokinetic data [for example, reaching the peak plasma
or tissue concentration (Cmax) and time (Tmax), or the steady state of multiple
administrations of the test substance]. In the absence of the toxicokinetic data, the
method of administrating the test substance (including the positive control substance)
to the animals twice (with an interval of 21 h) is often adopted. 3 h after the last
administration of the test substance, conduct one-time sampling. When the alkaline
comet assay is integrated with the repeated feeding test (for example, the 28-day oral
toxicity test), the same applies to the one-time sampling 3 h after the last administration
of the test substance.
The test may also adopt the following two sampling modes:
a) The test substance is administrated to the animals once; 2 h ~ 6 h and 16 h ~
26 h after the administration of the test substance, conduct sampling twice.
The preparation of glass slides shall be completed within 1 h after the preparation of
the single cell / nucleus suspension.
When preparing the glass slides, the amount of single cell / nucleus suspension added
to the low melting-point agarose (usually, 0.5% w/V ~ 1.0% w/V) shall not reduce the
concentration of the low melting-point agarose to 0.45% w/V. The optimal cell density
(cells are not overlapped, and with a concentration convenient for image analysis) shall
be determined in junction with the image analysis system of comet scoring.
6.6 Lysis
The lysis conditions are a key variable. In the same test, the lysis conditions of all slides
shall be kept as consistent as possible.
Immerse the slides in the pre-cooled cell lysis solution (the lysis solution shall cover
the slides); at 2 °C ~ 10 °C, perform lysis in the dark for at least 1 h (or overnight). After
lysis, rinse the slides to remove the residual detergent and salt. For rinsing, pure water,
neutralization buffer or phosphate buffer may be used, or electrophoresis buffer may
also be used.
6.7 Unwinding and Electrophoresis
Randomly place the slides on a horizontal electrophoresis tank containing enough
electrophoresis buffer. The electrophoresis buffer shall cover the slides (the coverage
depth of the electrophoresis buffer shall also be consistent each time). The slides shall
be placed for at least 20 min to unwind the DNA, then, set the electrophoresis
conditions. The electric potential is usually 0.7 V/cm; the electrophoresis time is not
less than 20 min. The electrophoresis time is a key variable, whose dynamic range
needs to be optimized. In each test, the voltage shall be kept constant, and the other
parameters shall change within a specific narrow range. During the whole
electrophoresis process, the depth of the electrophoresis buffer shall be ensured; the
current at the beginning and the end of electrophoresis shall be recorded. Unwinding
and electrophoresis are performed at 2 C ~ 10 °C in the dark.
The position where the slides are placed in the electrophoresis tank shall be balanced,
so as to reduce the influence of edge effects and minimize the difference between
batches. In each electrophoresis, samples for the negative control and positive control
in different dose groups shall have the same number of slides.
6.8 Neutralization and Dehydration
After the electrophoresis is completed, take out the slides. Use the pre-cooled
neutralization buffer to rinse them for at least 5 min. Then, naturally dry them in the air
or completely dry them at 37 °C. Under the circumstance when slide reading needs to
be extended, the slides may be immersed in absolute ethanol, which is pre-frozen to -
20 °C to stabilize for 5 min, dried and stored at room temperature, or refrigerated.
8.4 Test summary.
8.5 Test substance: name, batch No., dose form, properties (including sensory
properties, package integrity and identification), quantity, pre-treatment methods,
solvents and relevant information of positive control substance.
8.6 Laboratory animals: species, strains, grade, quantity, weight, gender, source
(supplier name and laboratory animal production license No.), animal quarantine,
adaptation, feeding environment (temperature, relative humidity, laboratory animal
facility license No.) and feed source (supplier name and laboratory animal feed
production license No.).
8.7 Test methods: test grouping, number of animals in each group, basis for dose
selection, route and duration of test substance administration, tissue and time point of
sampling, specimen preparation method, observation indicators, number of cells
observed and analyzed per animal, comet scoring and measurement methods,
statistical methods and judgment criteria.
8.8 Test results: record the health condition and physical sign observation of each
animal, and the number of cells observed and analyzed per animal; report the DNA
percentage of the tail and the frequency of the occurrence of “hedgehog” shaped cells
of each group of animals, the dose-response relationship, the statistical data of
provided data and the results of histopathological examination and whether there is
pathological damage in a tabular manner.
8.9 Test conclusion: positive result suggests that the test substance has the effect of
causing DNA damage in mammalian target tissue cells under the test conditions;
negative result suggests that the test substance does not cause DNA damage in
mammalian target tissue cells under the test conditions.
9 Test Interpretation
This test is not applicable to the detection of DNA strand breaks in mature germ cells.
This test is not applicable to the detection of DNA-DNA and DNA-protein cross-linking
reactions, and other base modifications, for example, base oxidation.
This test is not applicable to the detection of aneuploidy mutagens.
This test can be combined with other toxicological studies, for example, repeated dose
toxicity studies, and can also be combined with the endpoints of other genotoxicity
tests, for example, in vivo mammalian erythrocyte micronucleus test.
In this test, the influence on the test results after the key variables are changed must
be considered before changing the key variables, and there must be support of positive
control and negative control. The key variables shall not be arbitrarily changed, and if