GB 15193.6-2014National Food Safety Standard -- Mammalian Bone Marrow Cell Chromosome Aberration Test (English PDF)

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.

4.1 Instrument

Commonly used laboratory equipment, constant temperature water bath (37 °C ± 5 °C), 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 °C 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.

5.1 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, 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 °C 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.

6.1 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 chi2 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.