GB 15193.8-2014National Food Safety Standard -- Chromosome aberration test of mouse spermatogonia or spermatocyte (English PDF)

2.1 Spermatogonia

Male mammal seminiferous epithelium can after several mitosis and meiosis proliferation of spermatocytes produce stem cells for the original male germ cells. The same number of chromosomes somatic cells have.

2.2 Spermatocytes

Eventually differentiate into mature sperm cells through meiosis spermatogonia, divided into primary spermatocytes and secondary spermatocytes.

Secondary spermatocytes chromosome number is halved to 1n.

2.3 Structural chromosome aberrations

In mitotic metaphase cells through a microscope it can be directly observed chromosomal structural changes. Structure can be divided into chromosomal aberrations Chromatid aberrations and distortion.

2.4 Chromosome aberrations

Structural chromosome damage, showing at the same site two chromatids are a fracture or break reconnection.

2.5 Chromatid aberrations

Structural chromosome damage, manifested as chromatid breaks or break reconnection.

2.6 The number of chromosome aberrations Chromosome number change, different from the normal diploid karyotype, including aneuploidy and aneuploidy.

4.1 Commonly used laboratory equipment

Anatomy of commonly used laboratory instruments, electronic scales, refrigerators, centrifuges and the like.

4.2 Reagents

4.2.1 0.1% colchicine Placed in a brown bottle, refrigerator.

4.2.2 1% sodium citrate Take 1g trisodium citrate (AR), add distilled water to 100mL.

4.2.3 60% glacial acetic acid Take 60mL glacial acetic acid (AR), add distilled water to 100mL.

4.2.4 Fixative

Methanol. acetic acid = 3, using now.

4.2.5 Phosphate buffer (pH 7.4)

Disodium hydrogen phosphate solution (1/15mol/L). disodium hydrogen phosphate (Na2HPO4, analytical grade) 9.47 gm was dissolved in 1000mL of distilled water.

Potassium dihydrogen phosphate solution (1/15mol/L). potassium dihydrogen phosphate (KH2PO4, AR) was dissolved in 9.07 g of distilled water 1000mL.

Disodium-hydrogen phosphate solution (1/15mol/L) 80mL solution of potassium dihydrogen phosphate (1/15mol/L) 20mL mixed, the pH adjusted to 7.4.

4.2.6 Giemsa dye

Weigh Giemsa dye 3.8g, was added 375mL of methanol (AR) grinding until completely dissolved before adding 125mL glycerin. Put 37 °C incubator insulation 48h shaken several times. After filtration with two weeks.

4.2.7 Giemsa Application Solution

Take 1 part of Giemsa dye was mixed with 9 parts of a phosphate buffer solution made using now.

5.1 Experimental Animals

5.1.1 Animal selection

Experimental animals should be selected in line with the relevant provisions of GB GB 14922.2 and 14922.1 of. Healthy adult male mice, 7 weeks to weeks 12 weeks, when the start of the test animal weight difference should not exceed ± 20% of average weight. Animals should be randomly assigned to each group comprising at least 5 it can be used to analyze the animals. If the test sample has several points of time is required for each sampling time points are only used for analysis of at least 5 animal.

5.1.2 Animal Preparation

Before the test, animals in the experimental animal room should be at least 3d ~ 5d acclimatization and quarantine observation.

5.1.3 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.

5.2 Test substance formulation

Test substances should be dissolved or suspended in a suitable vehicle in. The preferred solvent is water, water-insoluble test substance may be used vegetable oil (such as corn Oil, etc.), do not dissolve in water or oil test substance may be used as carboxymethyl cellulose, starch, etc. dubbed suspension or paste. The test substance should be now with the existing, Data show that, excluding its storage solution or suspension stabilizer.

5.3 Dose

5.3.1 the control group each test shall be provided with a corresponding negative (vehicle) and positive control, negative control group, except for not using the test sample, the other dealing with subject Consistent test was set.

Positive control group should be in spermatogonia and spermatocytes was observed to increase above background chromosomal aberrations. The positive control group exposed to passers-by Diameter may be different from the route of administration of the test substance, at a point in time can only sample. The positive control used cyclophosphamide (40mg/kg body Heavy, single intraperitoneal injection) or mitomycin C (1.5mg/kg body weight ~ 2mg/kg body weight, a single intraperitoneal injection).

5.3.2 Dose and grouping

The test substance should be located three dose groups, the highest dose group in principle poisoning and (or) individual animals died at a dose of animals, is generally preferable to acute oral toxicity LD50 of 50% of the high dose, a geometric ratio of 2 to 4 to set down in the low-dose, group should not Exhibit toxicity. Acute oral toxicity test LD50 can not come, the high-dose group in the following order.

a) 10g/kg body weight;

100 times b) who might intake;

c) a maximum oral dose design, and then under the low-dose group.

5.4 Test procedure and outcome measures

5.4.1 Experimental animals treated

5.4.1.1 Spermatogonia

By oral gavage administration of the test substance, the test substance is usually administered once. The maximum capacity of the sample solution to be administered once should not exceed 20mL/kg of body weight. If given a larger dose, but also twice within 1d administration of the test substance, the interval is preferably 4h ~ 6h.

Should be given high-dose group at 24 hours after the test substance and the animals were sacrificed 48 h sampling at the end of times, animals in the low dose group were last After administration of the test animals were sacrificed 24h sampling.

5.4.1.2 Spermatocytes

Oral administration of the test substance, once a day for 5d. The maximum capacity of the sample solution to be administered once should not exceed 20mL/kg body weight. The first 12 days of each group were in the first administration of the test substance - after the animals were sacrificed on day 14 sampling.

5.4.2 The use of colchicine 3h ~ 5h before the animals were sacrificed by intraperitoneal injection of colchicine 4mg/kg body weight ~ 6mg/kg body weight (injection volume. 10mL/kg body weight ~ 20mL/kg of body weight). Colchicine should now use the existing day.

5.4.3 Sample preparation

5.4.3.1 Drawn

Mice were sacrificed by cervical dislocation, open the abdominal cavity, remove the testes, to the net fat, wash hair and bloodstained in hypotonic, insert containing an appropriate amount of 1% trisodium citrate or 0.4% potassium chloride solution in a small dish.

5.4.3.2 Hypotonic

5.4.3.2.1 Spermatogonia

Ophthalmology tear film tweezers, gently separate the seminiferous tubules, adding 1% sodium citrate solution 10mL, with a dropper percussion seminiferous tubules, Still 2min, so sinking seminiferous tubules, sperm will contain many carefully aspirate supernatant. Leaving the seminiferous tubules to re-use 10mL1% Trisodium citrate treatment 10min.

5.4.3.2.2 Spermatocytes

Ophthalmology tear film tweezers, gently separate the seminiferous tubules, adding 1% sodium citrate solution 10mL, with a dropper percussion seminiferous tubules, Stand at room temperature 20min.

5.4.3.3 Fixed

The supernatant was carefully exhaustion, plus fixative 10mL fixed. The first no more than 15min, drained after fixative, then add a new fixative Fixed 20min or more. As in the refrigerator (0 °C ~ 4 °C) better fixed overnight.

5.4.3.4 Centrifugal

Exhaustion fixative, plus 60% acetic acid 1mL ~ 2mL, until most of the seminiferous tubules after softening, times the amount of fixative was added immediately, playing Uniform, transferred to a centrifuge tube to 1000r/min centrifugal 10min.

5.4.3.5 Drop sheet

Most of the supernatant was discarded, leaving about 0.5mL ~ 1.0mL, made full playing uniform cell suspension, the cell suspension evenly drops Ice slides. Each sample was prepared 2 ~ 3. Hot air drying or drying slightly.

5.4.3.6 Dyeing

Application Solution with 1.10Giemsa staining 10min (based on room temperature dyeing at different times), washed with distilled water, dry.

5.5 Reading sheet

5.5.1 Number

All slides, including positive and negative controls, before the examination have to be numbered.

5.5.2 Microscopy

At low magnification in order to find a clear background, well dispersed, moderate contraction of chromosome metaphase, and then carried out in the oil microscope analysis.

5.5.3 Chromosome Analysis

Note. Each animal in mind at least 100 the number of metaphase cells, each dose group was observed at least 500 metaphase. When the cells were observed in the number of aberrations when the amount is more, it can reduce the number of cells observed. Since the fixed method often leads to loss of chromosomes, so the count should contain spermatogonia chromosome number Metaphase cells 2n ± 2, the count should contain spermatocytes chromosome number 1n ± metaphase cells 1.

5.5.3.1 Spermatogonia

5.5.3.1.1 Determine the mitotic index

Each animal was observed at least 1000 cells to determine spermatogonial mitotic index. High-dose group spermatogonia mitosis means Number should be not less than 50% of the control group.

5.5.3.1.2 The number of chromosomal alterations Normal spermatogonial metaphases common to polyploidy, polyploid therefore clarify the meaning should be careful.

5.5.3.1.3 Structural chromosome aberrations

Structural chromosome aberrations, including broken fragments, small body, no kinetochore ring, ring chromosome, the centromere of chromosome bi- or single Body swaps.

5.5.3.2 Spermatocytes

In addition to visible cracks, fragments, small body, but also to analyze reciprocal translocation, XY and autosomal univalents.

6.1 Data Processing

For each animal the number of chromosome aberrations record number of cells and each cell containing chromosomal aberrations, and each group is given a list of different types Structural chromosome aberrations and the number of frequencies. Fragments test group and negative control group, translocation, cell distortion rate, autosomal univalents sex Univalent chromosome, respectively, according to the binomial distribution for statistical processing, chromosome fracture, univalents should be recorded and reported separately, generally not included abnormal Variability.

6.2 Evaluation Results

Chromosomal aberration test dose rate or cell distortion rate compared with the negative control group, the difference was statistically significant, and there is a significant dose -Response relationship results as positive. It appears in a chromosomal aberration test group dose rate or cell distortion rate difference was statistically significant, but no dose - response relationship, you need to repeat the test, reproducible results were defined as positive.