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GB 15193.6-2014: National Food Safety Standard -- Mammalian Bone Marrow Cell Chromosome Aberration Test
Status: Valid GB 15193.6: Evolution and historical versions
| Standard ID | Contents [version] | USD | STEP2 | [PDF] delivered in | Standard Title (Description) | Status | PDF |
| GB 15193.6-2014 | English | 189 |
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National Food Safety Standard -- Mammalian Bone Marrow Cell Chromosome Aberration Test
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GB 15193.6-2014
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| GB 15193.6-2003 | English | 199 |
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Mammalian bone marrow cell chromosome aberration test
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GB 15193.6-2003
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| GB 15193.6-1994 | English | 199 |
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Chromosome aberration test of marrow cell
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GB 15193.6-1994
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PDF similar to GB 15193.6-2014
Basic data | Standard ID | GB 15193.6-2014 (GB15193.6-2014) | | Description (Translated English) | National Food Safety Standard -- Mammalian Bone Marrow Cell Chromosome Aberration Test | | Sector / Industry | National Standard | | Classification of Chinese Standard | C53 | | Classification of International Standard | 07.100 | | Word Count Estimation | 8,827 | | Date of Issue | 1/28/2015 | | Date of Implementation | 5/1/2015 | | Older Standard (superseded by this standard) | GB 15193.6-2003 | | Regulation (derived from) | National Health and Family Planning Committee Announcement 2015 No. 2 | | Issuing agency(ies) | National Health and Family Planning Commission of the People's Republic of China | | Summary | This Standard specifies the mammalian chromosome in bone marrow cells of distortion test basic test methods and technical requirements. This Standard applies to the evaluation of the test substance mammalian bone thin full genotoxicity. | GB 15193.6-2014: National Food Safety Standard -- Mammalian Bone Marrow Cell Chromosome Aberration Test
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(National Food Safety Standard mammalian bone marrow chromosome aberration test)
National Standards of People's Republic of China
National Food Safety Standard
Mammalian bone marrow chromosome aberration test
Issued on.2015-01-28
2015-05-01 implementation
People's Republic of China
National Health and Family Planning Commission released
Foreword
This standard replaces GB 15193.6-2003 "mammalian bone marrow chromosome aberration test."
This standard compared with GB 15193.6-2003, the main changes are as follows.
--- Standard name was changed to "national food safety standard mammalian bone marrow chromosome aberration test";
--- Modify the scope;
--- Increasing the terms and definitions;
--- Modified test purposes and principles;
--- Revised test methods;
--- Modify the data processing.
National Food Safety Standard
Mammalian bone marrow chromosome aberration test
1 Scope
This standard specifies the mammalian bone marrow chromosome aberration test basic test methods and technical requirements.
This standard applies to the evaluation of the test substance to mammalian bone marrow cells genotoxicity.
2 Terms and definitions
2.1 structural chromosome aberrations
It can be directly observed by a microscope to take place in the cell mitotic metaphase chromosome structural changes. Such as chromosome deletions and
Fragments, chromosomes and swap within the exchange. It can be divided into structural aberrations chromosome aberrations (chromosome-typeaberration) and single staining
Body distortion (chromatid-typeaberration).
2.2 Chromosome aberrations
Chromosome structural damage, the performance of the two chromatids of the same sites are broken or appear to change the broken reorganization.
2.3 chromatid aberrations
Structural chromosome damage, manifested as chromatid breaks or chromatid recombination fracture injury.
2.4 The number of chromosome aberrations
Chromosome number changes, unlike normal karyotype.
2.5 endoreduplication
After the S phase of DNA replication, mitosis starts another S phase nuclei process was not performed. As a result, chromosome
There 4,8,16 times chromatids.
2.6 crack
The length of the chromosome or chromatid damage is less than the width of a chromatid, arranged in a minimum error chromatids.
2.7 mitotic index
The number of cells and the medium-term phase of the observed ratio of the total number of cells.
3 test purposes and principles
After administration of the test substance test animals, with metaphase blocker (eg colchicine or colchicine amine) process, inhibiting cell division when spinning
Forming a hammer in order to increase the proportion of mitotic cells in the medium-term, and then drawn, producer, staining, chromosome aberration analysis.
This test can detect the whole animal test substances can cause chromosomal aberrations in bone marrow cells, the test was to evaluate the possibility of mutagenic. If the
There is evidence that the test substance or its metabolites can not reach the bone marrow, are not applicable to this method.
4 instruments and reagents
4.1 Instrument
Commonly used laboratory equipment, constant temperature water bath (37 ℃ ± 5 ℃), centrifuges, biological microscope.
4.2 Reagents
4.2.1 colchicine (0.4mg/mL). placed in a brown bottle, refrigerator.
4.2.2 potassium chloride solution (0.075mol/L).
4.2.3 fixative. 3 to 1 mix of methanol and acetic acid, temporary use when the existing service.
4.2.4 Giemsa (Giemsa) reserve bath. Take a small amount of methanol and Giemsa dye 3.8g finely ground in a mortar was gradually added methanol
To 375mL, until completely dissolved, then add 125mL glycerin and mix well. Incubator at 37 ℃ incubated 48h. During vibration insulation
Shake several times, prompting sufficiently soluble dyes. Remove the filter, store at room temperature, two weeks after use.
4.2.5 Application Giemsa dye. Take 1 part by Giemsa dye stock solution with 9 parts phosphate buffer (1/15mol/L) mixture. Temporary use
When formulated.
4.2.6 phosphate buffer (pH6.8).
4.2.6.1 disodium hydrogen phosphate solution (1/15mol/L). disodium hydrogen phosphate (Na2HPO4) 9.47g was dissolved in 1000mL of deionized water.
4.2.6.2 potassium dihydrogen phosphate solution (1/15mol/L). potassium dihydrogen phosphate (KH2PO4) 9.07g was dissolved in 1000mL of deionized water.
4.2.6.3 take disodium hydrogen phosphate solution (1/15mol/L) 50mL and potassium dihydrogen phosphate solution (1/15mol/L) 50mL mixed.
4.2.7 positive control. conventional cyclophosphamide, mitomycin C and the like.
5 Test methods
5.1 test substance
The test substance should be used in the original sample, if it can not use the original sample, the test should be treated in accordance with the principles of the test substance for proper disposal.
5.2 Experimental Animals
5.2.1 animal selection
Common healthy young adult rats or mice, such as using the mouse to select 7 weeks to 12 weeks, when the start of the test animal body weight difference
Isobutyl should not exceed 20% of the average weight. Animals should be randomly divided into groups of male and female animals each at least five. If the trial has several
Sampling time points, the requirements of each sex in each group for each sampling time point for the analysis has only 5 animals.
5.2.2 Animal Preparation
Before the test, animals in the experimental animal room should be at least 3d ~ 5d acclimatization and quarantine observation.
5.2.3 Animal Feeding
Animal breeding conditions should be consistent with GB 14925, drinking water should be consistent with GB 5749, feed should comply with the relevant provisions of GB 14924.
During the test animals free access to water and food, the number of animals per cage should meet the minimum space required for experimental animals in order not to affect the free movement of animals and
Animals were observed for signs appropriate.
5.3 dose
Pre-test should be carried out to select the highest dose. If the test substance is toxic, should be set three doses, the highest dose group the principles of animal
Now serious poisoning and (or) individual animals a dose of death, is generally preferable to 1/2LD50, low-dose group should not exhibit toxicity, respectively
Take 1/4LD50 and 1/8LD50 as medium and low dose. For substances with specific biological activity at a low or non-toxic doses (such as hormones
And mitogen source) can inhibit cell mitosis index (50%) as an indicator to determine the highest dose, set down in a geometric ratio 2
Medium and low dose group. Acute toxicity test was administered the highest dose tested (maximum concentration and maximum capacity gavage) no animal death for the sake of not
The LD50, and when the test substance is genotoxic, you do not have to set up three doses of structurally related substances according to the information can not be inferred. Follow along
Order only set a dose.
a) 10g/kg body weight;
100 times b) who might intake;
c) a maximum oral dose, continuous exposure 14d. Separate vehicle control group and positive control group, if there is no literature or history
Use of information confirming the vehicle does not have harmful effects or mutagenic effects, but also blank control group. Positive control wire available
Mitomycin C (1.5mg/kg body weight ~ 2.0mg/kg body weight) or cyclophosphamide (40mg/kg body weight) intraperitoneal injection or orally
give.
5.4 Test procedure
5.4.1 The administration of the test substance way
Oral administration of the test substance, the test solution should not exceed the amount of time intragastric 20mL/kg body weight. Using one or more exposure exposed side
formula. Exposure time should be twice to collect specimens, each animal that is divided into two subgroups, subgroups 1 12h ~ 18h after exposure the animals were killed collection
The first specimen, subgroup 2 in subgroup 1 Animals were sacrificed 24h after collecting a second sample. If multiple exposure mode, the administration of the test
Was 2 to 4 times, each time interval of 24h, after the last exposure 12h ~ 18h collect a specimen. Before the animals were sacrificed 3h ~ 5h, press 4mg/kg
Body weight by intraperitoneal injection of colchicine.
5.4.2 coverage
Animals were sacrificed by cervical dislocation, quickly removed the femur, picked off the muscle, wiping blood, cut both ends of the epiphysis, with a syringe with a needle
5mL normal saline, insert the bone marrow cavity, bone marrow washed into 10mL centrifuge tube, followed by pipetting clumps uniformly marrow, the cells were suspended
Liquid to 1000r/min centrifugal 10min, supernatant was discarded.
5.4.3 hypotonic
After centrifugation, the precipitate was added 7mL0.075mol/L potassium chloride solution with a dropper gently pipetting the cells evenly into 37 ℃ water bath
The hypotonic 10min ~ 20min.
5.4.4 Pre-fixed
Fixative is added 1mL ~ 2mL immediately (methanol. acetic acid = 3) to 1000r/min centrifugal 10min, supernatant was discarded.
5.4.5 Fixed
Fixative was added 7mL, mix, after a fixed 15min, to 1000r/min centrifugal 10min, the supernatant was discarded, and then using the same method Consolidation
Was given 1 to 2 times, the supernatant was discarded.
5.4.6 drip sheet
Adding a few drops of fresh fixative, and mix well with a dropper. The cell suspension was dropped on a uniform ice slides, blow diffusion cell suspension level
Spread on slides. Each specimen made 2 ~ 3 slides, air dried naturally.
5.4.7 Dyeing
Stained with Giemsa dye 15min, rinsed with deionized water, air dried naturally.
5.4.8 reading sheet
At low magnification to check the quality of production, the producers should focus more on the whole chromosome, and each chromosome dispersion, do not overlap, the length of shrink fit
, The two monomers separately, clearly shows the position of the centromere, chromosome reddish purple. Oil mirror for cell metaphase chromosome analysis, each
Animals analysis 100 metaphase cells per dose group of not less than 1000 metaphase cells. Sheet should be read in the record for each observation
Chromosome number of cells, the cells should also be recorded for distortion coordinate location and the type of distortion of the microscopic field. Due to low permeability and other mechanical action
Damage will result in the middle of the chromosome is lost, so the number of metaphase chromosome observation should be controlled 2n ± 2.
5.5 OUTCOME MEASURES
5.5.1 chromosome number change
5.5.1.1 Aneuploidy. hypodiploidy or hyperdiploidy.
5.5.1.2 polyploid. chromosome doubling.
5.5.1.3 endoreduplication. phenomenon of times within a special form of the nuclear envelope.
5.5.2 change chromosome structure
5.5.2.1 Disruption. chromosome damage length greater than the width.
5.5.2.2 tiny body. fragments representing small and rounded.
5.5.2.3 has kinetochore ring. with centromere portion at both ends to form a cyclic structure and accompanied by one pair fragment without centromere.
5.5.2.4 No kinetochore ring. a ring structure.
5.5.2.5 monomer exchange. an image is formed three-spoke body, spoke four or more body shape.
5.5.2.6 tiny body double. a pair of chromatin body.
5.5.2.7 crack. the length of the damage is less than the width of the chromatids.
5.5.2.8 non-specific type of change. such as pulverization, the centromere of slender, adhesion and the like.
6 Data processing and evaluation of results
6.1 Data Processing
Each animal was observed as a unit, each animal were calculated by sex chromosome structural aberrations cell percentage. If male and female
No significant gender differences between animals can be combined to calculate the results. Available χ2 test methods for statistical analysis. Fissure should be recorded separately and
Report, but generally not included in the total distortion rate.
6.2 Evaluation Results
Evaluation results should be analyzed from the biological significance and statistically significant in two ways. Dose rate and chromosome aberration negative control group
Compared with a statistically significant dose - response relationship or a dose group showed significantly increased chromosomal aberrations in cells and statistically
Significance and confirmed by repeat test can confirm a positive result. If statistically significant, but not dose - response relationship when you should be
Repeat the test. The results can be repeated may determine positive.
7 Test report
7.1 Name of test, the test unit name and contact details, report number.
7.2 Test Requester name and contact information, sample acceptance date.
7.3 Test start and end dates, test project manager, technical director of the unit test or authorized signatory, the date of issue.
7.4 test summary.
7.5 test substance. name, batch number, dosage form, character (including sensory, character, integrity packaging, labeling), the number of pre-treatment methods, Solvents.
7.6 Experimental animals. species, strain, level, quantity, weight, sex, origin (supplier name, animal production license number), animal inspection
Phytophthora, adaptation, breeding environment (temperature, relative humidity, using experimental animal facility license number), feed sources (supplier name, laboratory animals
Animal feed production license number).
7.7 Test method. grouping, the number of animals in each group, according to dose selection, route of administration and duration of the test substance, observed indicators, statistical methods.
7.8 Test Results. text description and itemized summary form, including the number of cell observation and analysis, and the type and number of chromosomal aberrations
Distortion, given the results of statistical processing of the data.
7.9 Test Conclusions. give a clear conclusion.
Explanation 8 trial
A positive result indicates the test substance has the effect of causing bone marrow cells of the test animals chromosomal aberrations.
Negative results indicate that under the experimental conditions of the test substance does not cause chromosomal aberrations in bone marrow cells of the test animals.
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