GB 15193.5-2014 PDF in English
GB 15193.5-2014 (GB15193.5-2014) PDF English
| Standard ID | Contents [version] | USD | STEP2 | [PDF] delivered in | Name of Chinese Standard | Status |
| GB 15193.5-2014 | English | 95 |
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National Food Safety Standard -- Mammalian Erythrocyte Micronucleus Test
| Valid |
| GB 15193.5-2003 | English | 199 |
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Bone marrow cell micronucleus test
| Obsolete |
| GB 15193.5-1994 | English | 199 |
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Micronucleus test of bone marrow cell
| Obsolete |
Standards related to (historical): GB 15193.5-2014
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GB 15193.5-2014: PDF in English GB 15193.5-2014
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National food safety standard – Mammalian erythrocyte
micronucleus 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 ... 5
4 Apparatus and reagents ... 5
5 Test methods ... 6
6 Data processing and result evaluation ... 8
7 Test report ... 9
8 Interpretation of the test ... 9
National food safety standard - Mammalian erythrocyte
micronucleus test
1 Scope
This Standard specifies the basic test methods and technical requirements for
mammalian erythrocyte micronucleus test.
This Standard is applicable to the evaluation of the genotoxicity of test substances.
2 Terms and definitions
2.1 Micronucleus
During the cell mitosis anaphase, when the chromosomes regularly enter the daughter
cells to form the cell nucleus, the entire chromatids or acentric fragments or rings of
chromosomes that remain in the cytoplasm. In the telophase, one or several regular
subnuclei are formed separately and are contained in the cytoplasm of the cell.
2.2 Centromere
During the phase of cell division, the region where chromosomes connect to spindle
fibers allows the orderly movement of daughter chromosomes to the poles of daughter
cells.
2.3 Normochromatic erythrocytes
Mature erythrocytes that lack ribosomes and can be distinguished from immature
polychromatic erythrocytes by selective ribosome dyes.
2.4 Polychromatic erythrocytes
Immature erythrocytes are in the intermediate stage of development and still contain
ribosomes, so they can be distinguished from mature normochromatic erythrocytes with
selective ribosome dyes.
2.5 Total erythrocytes
The sum of normochromatic erythrocytes and polychromatic erythrocytes.
5 Test methods
5.1 Test substances
5.1.1 Preparation of test substances: The test substances shall be dissolved or suspended
in a suitable solvent, where water is the preferred solvent; for water-insoluble test
substances, vegetable oils (such as olive oil, corn oil, etc.) can be used; for water-
insoluble or oil-insoluble test substances, carboxymethyl cellulose, starch, etc. can also
be used to prepare suspensions or pastes. Test substances shall be prepared before use,
unless there is data indicating that their solutions or suspensions are storage stable.
5.1.2 Administration route: Oral gavage shall be used. Positive control substances can
also be injected intraperitoneally. The gavage volume shall generally not exceed 10
mL/kg body weight. If it is an aqueous solution, the maximum gavage volume can 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 of each group shall be the same.
5.2 Experimental animals
5.2.1 Animal species and strain selection: When using bone marrow, it is recommended
to use mice or rats. When using peripheral blood, it is recommended to use mice. If it
has been proven that the spleen of a certain strain of animal does not clear
polychromatic erythrocytes with micronuclei, or is sufficiently sensitive to detect
chemicals that cause structural or numerical aberrations of chromosomes, such animals
can be used. Usually, mice aged 7 ~ 12 weeks and weighing 25 g ~ 35 g or rats weighing
200 g ~ 300 g are used. At the beginning of the test, the difference in animal weight
shall not exceed ±20% of the average weight of each gender. Use at least 5 animals of
each gender in each group.
5.2.2 Animal preparation: Before the test, the animals shall undergo environmental
acclimatization and quarantine observation for at least 3 ~ 5 days in the experimental
animal room.
5.2.3 Animal feeding: Experimental animal feeding conditions, drinking water, and feed
shall comply with national standards and relevant regulations (GB 14925, GB 5749,
GB 14924.1, GB 14924.2, GB 14924.3). The animals in each test group shall be housed
in separate cages according to gender, with 5 animals in each cage. During the
experiment, the experimental animals shall be fed basic feed and have free access to
water.
5.3 Dosage
The test substances shall be set up in three dose groups. In principle, the maximum dose
group is the dose at which animals show severe poisoning and/or individual animals
die, which can generally be 1/2 LD50. The low dose groups shall not show toxicity, and
1/4 LD50 and 1/8 LD50 shall be taken as the medium and low doses, respectively. When
the animal does not die at the maximum dose (maximum use concentration and
maximum gavage capacity) of the test substances in the acute toxicity test and the LD50
cannot be calculated, the high dose groups shall be in the following order:
a) 10 g/kg body weight;
b) 100 times the possible human intake;
c) Design the maximum gavage dose, and then set up medium and low dose groups.
Set up a solvent control group. The positive control can be given at
cyclophosphamide 40 mg/kg body weight orally or intraperitoneally (oral is
preferred).
5.4 Test procedures and observation indicators
5.4.1 Administration of test substances
Oral gavage. According to the cell cycle and the characteristics of the action of different
substances, a preliminary test can be done first to determine the sampling time. The 30-
hour test substance administration method is commonly used. That is, the test substance
is administered twice with an interval of 24 hours, and the bone marrow sample is
collected 6 hours after the second administration of the test substance. The experiment
can also have the following two sampling methods:
a) Administer the test substance to the animal once. Collect bone marrow samples
at least twice at appropriate intervals, starting no earlier than 24 hours after
administration and ending no later than 48 hours after administration. The
reason for sampling earlier than 24 hours after administration shall be stated.
Collect peripheral blood samples at least twice at appropriate intervals, starting
no earlier than 36 hours after administration and ending no later than 72 hours
after administration. If a positive result is found at one sampling time, no further
sampling is required.
b) Administer once a day for 2 or more times (24 h intervals). Bone marrow can be
collected once between 18 h and 24 h after the last administration, and peripheral
blood can be sampled once between 36 h and 48 h after the last administration.
If the rate of micronucleated cells in peripheral blood normochromatic
erythrocytes is used as the experimental observation endpoint, the animal shall
be administered for more than 4 weeks.
5.4.2 Specimen preparation
5.4.2.1 Bone marrow sample: After killing, take the sternum or femur; use hemostatic
forceps to squeeze out the bone marrow fluid and mix it with the calf serum at one end
of the slide; make a conventional smear. Alternatively, use calf serum to rinse the
femoral bone marrow cavity to make a cell suspension smear; after the smear is
naturally dried, put it in methanol to fix it for 5 min ~ 10 min. Store it after fixation on
but there is no dose-response relationship, the test shall be repeated. Repeatable results
can be considered positive.
7 Test report
7.1 Test name, test unit name and contact information, and report number.
7.2 Name and contact information of the test entrusting unit, and sample acceptance
date.
7.3 Test start and end dates, test item leader, test unit technical leader, and issue date.
7.4 Test summary.
7.5 Test substances: name, batch number, dosage form, status (including sensory,
properties, packaging integrity, and labeling), quantity, pretreatment method, and
relevant information of the positive control.
7.6 Experimental animals: species, strain, grade, quantity, age, weight, sex, source
(supplier name, experimental animal production license number), animal quarantine,
adaptation, breeding environment (temperature, relative humidity, experimental animal
facility use license number), feed source (supplier name, experimental animal feed
production license number).
7.7 Test method: test grouping, number of animals in each group, dose selection basis,
test substance administration route and period, sampling time point, specimen
preparation method, number of cells observed per animal, statistical methods and
judgment standards.
7.8 Test results: Record the number of polychromatic erythrocytes and the number of
micronucleated cells observed in each animal, and report the number of polychromatic
erythrocytes in each group of animals of different genders, the rate of micronucleated
cells and the proportion of polychromatic erythrocytes in total red blood cells, the dose-
response relationship, the historical data and range of negative controls in tabular form;
state the statistical method for the results.
7.9 Test conclusion: Based on the test results, make a conclusion on whether the test
substance can cause an increase in the rate of micronucleated cells in mammalian
polychromatic erythrocytes.
8 Interpretation of the test
A positive result indicates that the test sample can cause an increase in the rate of
micronucleated cells in mammalian polychromatic erythrocytes under the test
conditions. A negative result indicates that the test sample does not cause an increase in
the rate of micronucleated cells in mammalian polychromatic erythrocytes under the
......
Source: Above contents are excerpted from the PDF -- translated/reviewed by: www.chinesestandard.net / Wayne Zheng et al.
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