GB 15193.23-2014National Food Safety Standard -- In Vitro Mammalian Cells 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 Fragment, chromosome swaps. Structural chromosome aberrations can be divided into chromosomal aberrations (chromosome-typeaberration) and chromatid Aberrations (chromatid-typeaberration).

2.1.1 Chromosome aberrations

Chromosome structural damage, the performance of the two chromatids of the same sites all appear to change broken or broken restructuring.

2.1.2 Chromatid aberrations

Structural chromosome damage, manifested as chromatid breaks or chromatid breakage restructuring and other changes.

2.2 Mitotic index

The number of cells and the medium-term phase ratio of the total number of cells observed; reflect the degree of proliferation cell index.

2.3 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.

2.4 Crack

The length of the chromosome or chromatid damage is less than the width of a chromatid, arranged in a minimum error chromatids.

4.1 Instrument

Incubator, inverted microscope, ultra-clean table, centrifuges.

4.2 Broth

Common Eagle'sMEM broth (minimumessentialmedium, MEM), can also be used other suitable medium. Join Antibiotics (penicillin press 100IU/mL, streptomycin 100µg/mL), inactivated fetal bovine serum or calf serum added in a proportion of 10% Broth.

4.3 Metabolic activation system

4.3.1 S9 preparation cofactors

4.3.1.1 Magnesium potassium solution

1.9g of magnesium chloride and potassium chloride 6.15g add distilled water to 100mL.

Disodium hydrogen phosphate (Na2HPO4,28.4g/L) 440mL, sodium dihydrogen phosphate (NaH2PO4 · H2O, 27.6g/L) 60mL, pH adjusted 7.4,0.103MPa20min sterilization or to filter bacteria.

4.3.1.3 Coenzyme -II (oxidized) solution

He said under sterile conditions to take coenzyme -II, with sterile distilled water formulated to 0.025mol/L solution using now.

4.3.1.4 Glucose-6-phosphate sodium salt solution

Glucose-6-phosphate sodium salt was weighed, dissolved in distilled water formulated as 0.05mol/L, sterilized by filtration. Using now.

4.3.2 Induced rat liver S9 components and formulation

Choose healthy male adult SD or Wistar rats weighing 150g ~ 200g, about 5 weeks to 6 weeks of age. The PCBs (Aroclor1254) was dissolved in corn oil at a concentration of 200g/L, according to 500mg/kg body weight by intraperitoneal injection once aseptic, 5d after death Animals were sacrificed before fasting 12h.

It may also be used phenobarbital and beta- naphthalene flavones and induction methods for preparing orally administered to rats fed sodium phenobarbital and beta- naphthalene Flavonoids, doses were 80mg/kg body weight, continuous 3d, 16h after fasting animals were decapitated. Other operating with PCBs induction.

Animals were sacrificed after the liver was removed, weighed fresh ice-cold 0.15mol/L potassium chloride solution was continuously flushed several times the liver, can be suppressed in order to remove System microsomal enzyme activity of hemoglobin. Per gram of liver (wet weight) plus 0.1mol/L potassium chloride solution 3mL, along with an ice bath and transferred to a beaker with a sterile Scissors minced liver, in a glass homogenizer (less than 4000r/min, 1min ~ 2min) or tissue homogenizer (less than 20000r/min, 1min) Made in liver homogenates. The above operation should pay attention to the local cold and sterile environment.

Aliquot in sterile freezer tubes, each tube about 2mL, the best home after liquid nitrogen or dry ice frozen -80 °C cryopreservation.

After S9 fraction prepared by the sterility test, measuring protein content (Lowry method) per milliliter protein content of not more than 40mg is appropriate, and by Indirect mutagens qualified to identify its biological activity after storage at -80 °C low temperature or freeze dry, a period not exceeding one year.

Preparation 4.3.3 10% S9 mixture Usually by the S9 fraction and cofactors press 1.9 mixture consisting of 10% S9, sterile using now. 10% S9 mixture 10mL Prepared as follows.

Take the above phosphate buffer 6.0mL, magnesium potassium solution 0.4mL, glucose-6-phosphate salt solution 1.0mL, coenzyme -II solution 1.6mL, Liver S9 fraction 1.0mL, mix, set in an ice bath until ready to use.

S9 mixture concentration is generally 1% to 10%, the actual concentration determined by individual laboratories, but its activity needs identified, We must be able to significantly activate the positive control, and cells without significant toxicity.

4.4 Colchicine solution

PBS solution with an appropriate concentration of formulation stock solution, filter sterilized, refrigerated in the dark condition can be stored for at least 6 months.

5.59g KCl add distilled water to 1000mL.

4.6 Fixative

Methanol. acetic acid as 3.1, prepared before use. According to the experimental conditions, the concentration of glacial acetic acid can be adjusted to improve the dispersion of chromosomes Degrees, but not too large, leading to cell rupture.

Take Giemsa dye 3.8g, set in a mortar, add a small amount of methanol grinding. Gradually add methanol to 375mL, until completely dissolved, plus 125mL glycerin, into 37 °C incubator insulation 48h. During the incubation shaking several times to fully dissolve. Remove the filter, two weeks after use, as as Giemsa stock solution. When in use, take 1 part Giemsa stock solution was mixed with 9 parts 1/15mol/L phosphate buffer (pH6.8), its application was dubbed, now with the current.

Phosphate buffer (1/15mol/L, pH6.8) formulated as follows.

a) First solution. Take disodium hydrogen phosphate (Na2HPO4) 9.47g was dissolved in 1000mL of deionized water, dubbed 1/15mol/L solution;

b) Second solution. take potassium dihydrogen phosphate (KH2PO4) 9.07g was dissolved in 1000mL of deionized water, dubbed 1/15mol/L solution;

c) taking a first liquid applied to the second liquid 50mL 50mL and mix well, pH6.8 is the 1/15mol/L phosphate buffer.

5.1 Test substance

Solid test substances should be dissolved or suspended in a suitable solvent and diluted to the appropriate concentration. Liquid test substances can be used directly or diluted to Appropriate concentration. Sterile test substance should now use the existing, it shall be confirmed that the storage does not affect its stability.

5.2 Cell line

Choice of Chinese hamster lung (CHL) cell line or ovary (CHO) cell lines, human or other mammalian peripheral blood lymphocytes (Lymphocyte). Check before the test karyotype and chromosome number of cells, the cells detect the presence or absence of mycoplasma contamination. We recommend using Chinese hamster Lung (CHL) cell lines.

5.3 Dose

5.3.1 Dose setting

Test substances should be taken at least three doses tested. Of cytotoxic test substance, the dose range should include the maximum from almost no toxicity to Cytotoxicity (cell viability in the range of 20% to 100% in); concentration typically is not greater than the spacing factor of 2 to 10 Sang.

5.3.2 Selection highest dose

When the cells were harvested, the highest dose should be able to significantly reduce the cell count or mitotic index (greater than 50%, such as toxicity is too large, should be properly Increase the number of cells inoculated); at the same time should consider the impact on the solubility of the test substance, pH and osmolarity; for non-cytotoxic, or cell Compounds having low toxicity, the highest dose should reach 5µL/mL, 5mg/mL or 10mmol/L.

For low solubility substances, still no toxicity when the maximum dissolved concentration, the highest dose should be in the final broth solubility limit A concentration value or more. In some cases, the concentration should be more than a visible precipitation, differential solubility naked eye, but no precipitate We can observe the effects.

5.3.3 Cytotoxicity determination

Cytotoxicity assay can be used indicating cellular integrity and growth indicators such as relative colony forming efficiency or relative cell growth rate.

Cytotoxicity should be determined in the S9 system, the presence or absence of.

5.3.4 Positive control

The nature and structure of the test substance may be the choice of the appropriate positive control in accordance with, should be aware of the cleavage agent, can cause can be detected and can be heavy Complex positive results. When there is no exogenous metabolic activation system, the positive control can be used are methyl methanesulfonate (methyl methanesulphonate, MMS), ethyl methanesulfonate (ethylmethanesulphonate, EMS), mitomycin C (mytomycinC), Ethylnitrosourea (ethylnitrosourea, ENU), nitroquinoline oxide -N- (4-nitroquinoline-N-oxide) and the like. When there is a foreign when endogenous activation system, the positive control can have benzo [Rao] pyrene [benzo (Rao) pyrene, BaP], cyclophosphamide (Cyclophosphamide) and the like.

The positive control without S9 common mitomycin C, which commonly used at a concentration of 0.2µg/mL ~ 0.8µg/mL. A pH of 6 to 9 water Solution at 0 °C ~ 5 °C dark preservation can be stored for one week. S9 plus cyclophosphamide positive control common, their usual concentration 8µg/mL ~ 15µg/mL. Their solution instability, should now with the current.

5.3.5 Negative control

Solvent should be non-mutagenic not react chemically with the test substance, does not affect cell survival and S9 activity. The preferred vehicle control is not Serum-containing medium and water, may also be used dimethyl sulfoxide (DMSO), but the concentration should not exceed 0.5%.

5.3.6 Blank

If there is no literature or historical data confirm that vehicle should be used no mutagenic effect blank control.

5.4 Test procedure

5.4.1 Cell culture and exposure

Need to test with and without added S9 (S9 final concentration is usually 1% to 10%, subject to the results of toxicity tests cells) into the condition Row. Day before the test, a certain number of cells were seeded in culture dishes (bottles) in [to harvest cells, culture dishes (bottle) is not covered as a cell Standard, generally grow to about 85% better; as with CHL cells can be inoculated with 1 × 106 pcs], put in CO2 incubator. When absorption test to Petri dishes (bottles) of the culture solution, a series of concentrations of the test substance, S9 mix (without S9 mixture, the required culture medium supplement) to and a certain amount of serum-free culture medium, set incubator process 2h ~ 6h. After the process is finished, to absorb the culture medium containing the test substance, with PBS Solution cells were washed 3 times, and culture medium containing 10% FBS, returned to the incubator, the cells were harvested at 24h. At 2h ~ 4h before harvest, Added to the cells in metaphase blockers (such as colchicine, a final concentration of 0.1µg/mL ~ 1µg/mL).

When the test substance is a single chemical substance, if added above with and without S9 mixture conditions are obtained negative results, you need to Plus a test of a long process, that in the absence of S9 mixture conditions, the contact time of the test substance with the test system was extended to 24h.

When it is difficult to draw definitive conclusions, replace the test conditions, such as changes in the metabolic activation conditions, the test substance with the test system, the contact time repeat test.

5.4.2 The cells were harvested with producer

5.4.2.1 Digest

With 0.25% trypsin digestion solution cell, until the cell off, containing 10% fetal bovine calf serum broth or termination of trypsin the role of protease, and mix into a centrifuge tube to 800r/min ~ 1000r/min speed centrifugal 5min, the supernatant was discarded.

5.4.2.2 Hypotonic

Added 0.075mol/L potassium chloride solution 2mL, with a dropper and gently mix the cells were placed in 37 °C incubator in hypotonic treatment 30min ~ 40min.

5.4.2.3 Fixed

Added 2mL fixative, fixed more than 5min after mixing to 800r/min ~ 1000r/min speed centrifugal 5min, the supernatant was discarded liquid. Repeat, the supernatant was discarded.

5.4.2.4 Drop sheet

Adding a few drops of fresh fixative, and mix. Suspensions dropwise sheet and dried naturally. Before using the slides soaked with ice water.

5.4.2.5 Dyeing

5% to 10% Giemsa, 15min ~ 20min.

5.4.3 Reading sheet

In oil immersion read the piece, each dose group should be analyzed at least 100 well-dispersed metaphase chromosomes and each of the cells was observed Number 2 in the form ± 2 range. For atypical cells also record coordinates of the location and the type of distortion of the microscopic field.

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

Data according to a list of different doses, the cells were observed indicators include the number of aberrations cells, chromosome aberration, each dose group and the control group in different categories Type the number of chromosomal aberrations and distortion rate. Fissure should be recorded separately and reported but generally not included in the total distortion rate. Each group of chromosome aberrations Rates were statistically treated with chi2 test.

6.2 Evaluation Results

The following two situations can determine the test substance in this test system, a positive result.

a) the test substance causes an increase in the number of structural chromosome aberrations were statistically significant and dose-related;

b) increase the number of structural chromosome aberrations test substances at any doses, caused statistically significant, and there may be heavy Refolding.