GB 15193.12-2014National Food Safety Standard -- HGPRT gene mutation test (English PDF)

2.1 HGPRT gene

Hypoxanthine guanine phosphoribosyl transferase gene in mammals. In humans, HGPRT gene located on the X chromosome length Arm coordinates Xq26.1; in mice also located in the X chromosome.

2.2 Mutation frequency

In some cell lines, a particular gene mutant cells (colonies) accounted cells (colonies) of the total percentage.

4.1 Cell

Common Chinese hamster lung cell line (V79) and Chinese hamster ovary cell line (CHO), others such as mouse lymphoma cells (L5178Y) and human lymphoblastoid cell lines (TK6) also. Cells should be carried out before use to check whether the mycoplasma contamination.

4.2 Broth

According to the test system should be used to select the appropriate cell type and medium. For V79 and CHO cells, common minimum essential medium Yang group (MEM, Eagle), Modified Eagle Medium (DMEM) with 10% fetal calf serum and an appropriate amount of antibiotics. For TK6 and L5178Y cells, commonly used RPMI1640 medium, 10% horse serum (culture flasks) or 20% horse serum (96 well plates) and an appropriate amount of antibiotics (penicillin, streptomycin).

4.3 Trypsin/EDTA solution

With calcium and magnesium in PBS, concentration of trypsin was 0.05%, the concentration of EDTA is 0.02% trypsin EDTA solution by Mixed 1. -20 °C storage.

4.4 Activation system

S9 mixture is generally used. S9 is prepared as follows.

Choose healthy male adult SD or Wistar rats weighing about 150g ~ 200g, weeks about five weeks to 6 weeks. 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, 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 solution of potassium chloride (0.15mol/L) continuous flushing liver several times to remove it can inhibit the activity of microsomal hemoglobin. Per gram of liver (wet weight) was added potassium chloride solution (0.1mol/L) 3mL, along with an ice bath and transferred to a beaker the liver minced with sterile scissors in glass homogenizer (less than 4000r/min, 1min ~ 2min) or tissue homogenizer (less than 20000r/min, 1min) is made in the liver homogenates. The above operation should pay attention to the local cold and sterile environment.

The liver homogenates prepared at low temperature (0 °C ~ 4 °C) high-speed centrifuge at 9000g centrifugation 10min, the supernatant was aspirated S9 fraction, Aliquot in sterile freezer tubes or ampoules, each ampoule about 2mL, frozen with liquid nitrogen or dry ice rear -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 carcinogens (mutagens) Qualified identify their biological activity after storage in cryogenic freeze or dry, a period not exceeding one year.

S9 is used at a concentration of 1% to 10% (final concentration).

4.5 Selection agent

6- thioguanine (6-TG), recommend using a final concentration of 5µg/mL ~ 15µg/mL, formulated with sodium bicarbonate solution (0.5%).

4.6 Pretreated broth (THMG/T HG)

To reduce the frequency of spontaneous mutation of cells, prior to testing, the first cell culture plus 24h in culture medium containing THMG, the spontaneous killing the mutant cells, then cells were seeded in THG (without methotrexate in THMG broth) in cultured cells 1d ~ 3d to recovery Normal growth cycle and morphology.

THMG final concentration of each substance contained in the following (in addition to the culture medium ingredients outside).

--- Thymidine, 5 × 10-6mol/L;

--- Hypoxanthine, 5 × 10-5mol/L;

--- Methotrexate, 4 × 10-7mol/L;

--- Glycine, 1 × 10-4mol/L.

5.1 Test substance

5.1.1 Formulated 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. Test substance shall be obtained before using now, otherwise you must confirm the storage does not affect its stability.

5.1.2 Vehicle selection

Solvent must be non-mutagenic not react chemically with the test substance, does not affect cell survival and S9 activity. The preferred solvent is distilled Water; for water-insoluble test substance optionally other solvents, preferred dimethyl sulfoxide (DMSO), but it should not be used at a concentration greater than 0.5%.

5.1.3 Control

Each one trial, in the presence of metabolic activation system and the absence shall be provided with positive and negative (vehicle) control group.

5.1.3.1 Positive Control

When using a metabolic activation system, the positive control is to require metabolic activation must be, and can cause mutations in substance, you can use 3-methyl MCA (3-methylcholanthrene), N- NDMA (N-nitroso-dimethylamine), 7,12- dimethyl-benz [a] anthracene (7, 12-dimethylbenz [a] anthracene) and the like. In the absence of metabolic activation system, the positive control can be used ethyl methane sulfonate (ethyl methanesulphonate), ethylnitrosourea (ethylnitrosourea) and the like. It may also be used other suitable positive controls.

5.1.3.2 the negative control Negative control (including vehicle control) in addition to absence of the test substance, the other test substances should be treated the same. In addition, not having a laboratory history when data confirm vehicle used no mutagenic effect and no other harmful effects, but also blank control.

5.2 Dose

5.2.1 Select the highest concentration

The factors that determine the cytotoxic highest concentration, pH or osmotic pressure and changing the solubility test substance in the assay system.

5.2.2 Determine cytotoxicity

Integrity should be used and an indication of cell growth indicators, to determine the cytotoxicity of metabolic activation system in the presence and absence of two conditions , For example colony formation relative or relative survival rate. It shall determine cytotoxicity and solubility in the pre-test.

5.2.3 Density settings and select the highest concentration

It should be set at least four concentrations available for analysis. When cytotoxic concentration should include toxicity from the largest to the almost non-toxic, Typically the concentration factor in the interval between 2 to 10; as the highest concentration is based on cytotoxicity then the cell concentrations relative colony forming efficiency or the relative survival rates of 10% to be 20% (not less than 10%). For those low cytotoxic compounds the maximum concentration should be 5µL/mL, 5mg/mL or 0.01mol/L. For relatively insoluble substances, the highest concentration should meet or exceed the dendritic cells in culture the solubility limit of the state; the best and the end of the trial process begins in solubility were evaluated, such as the presence of S9, the internal system test Exposure during the solubility may vary; insolubility can be visually identified, but should not affect the precipitation was observed.

5.3 Test procedure and outcome measures

5.3.1 Adherent cell growth test procedures and outcome measures

5.3.1.1 Cell preparation

The 5 × 105 cells were seeded in 100mm diameter dish at 37 °C, 5% carbon dioxide incubator for 24h.

5.3.1.2 Contact with the test substance

Culture fluid was aspirated, PBS washed twice, adding a certain amount of serum-free medium, a certain concentration of the test substance and S9 mixture (without metabolic Activated by serum-free culture medium supplement), placed in an incubator in 3h ~ 6h, after the aspirated medium containing the test substance, the cells were washed with PBS Twice, change into the culture medium containing 10% serum, cultured 19h ~ 22h.

5.3.1.3 Expression

Contacting the test cells were cultured for 19h ~ 22h after digestion with trypsin -EDTA, until the cells fall off, containing 10% serum Medium to terminate digestion, and mix into a centrifuge tube to 800r/min ~ 1000r/min speed centrifugal 5min ~ 7min, the supernatant was discarded, Cell suspension, counted to 5 × 105 cells were seeded in 100mm diameter plates, 3d passaged, inoculated with 5 × 105 Ge still fine Cell culture 3d (optimal expression time 6d ~ 8d).

5.3.1.4 Cytotoxicity assay

The first count after digestion of said cells per dish inoculated with 200, five each dish, at 37 °C, 5% carbon dioxide cultured 7d, Fixed, Giemsa staining, counting the number of colonies per dish.

Select and set the rate of colony formation assay 5.3.1.5 mutants After expression, digestive cells, minutes, each group of five dishes, each dish inoculated with 200 cells, without 6-TG, 7d after fixed, Giemsa staining Color, the number of colonies per dish statistical calculation colony formation rate. Another mutation frequency was measured while doing, each set of five dishes, each dish was inoculated 2 × 105 cells, to be adherent cells were added to 6-TG (recommended final concentration of 5µg/mL ~ 10µg/mL), placed in incubator 8d ~ 10d after the solid Fixed, Giemsa staining, statistical colonies per dish, and calculate the frequency of mutation.

Procedures and observed indicators 5.3.2 Suspension cell growth

5.3.2.1 Preparation of cells and contacting the test substance

Take good cell growth, adjusting a density of 5 × 105/mL, 1% by volume of a series of concentrations of the test substance and S9 mix (without Metabolic activation by serum-free culture medium supplement), 37 °C shaking process 3h ~ 6h, to 800r/min ~ 1000r/min speed centrifugation 4min ~ 6min, the supernatant with PBS or serum-free medium cells were washed twice, resuspended in 10% horse serum RPMI cells 1640, and adjust the cell density of 2 × 105/mL.

5.3.2.2 PE0 (0 days plated efficiency) Determination

Take appropriate cell suspension for dilution to 108 cells/mL, inoculated 96 (per well 0.2mL, an average of 1.6 cells /Well), inoculated with each dose of 1 to 2 plates, at 37 °C, 5% carbon dioxide and saturated humidity cultured 9d ~ 11d, counting each plate has Growth of colonies set number of holes.

5.3.2.3 Expression

5.3.2.1 The resulting cell suspension was taken, as 6d expression culture, the cell density counted daily and maintained at a density 1 × 106/mL or less.

5.3.2.4 PE6 (the sixth day of plating efficiency) Determination

After the end of the expression of the culture, take appropriate cell suspension, according to "5.3.2.2" method of measuring PE6.

5.3.2.5 Mutation frequency (MF) Determination

After the expression of culture, take the appropriate cell suspension, adjusting the cell density of 1 × 105/mL, was added 6-TG (recommended final concentration of 5µg/mL ~ 15µg/mL), mixed, seeded 96 well plates (0.2mL added per well, i.e., 2 × 104 cells/well), inoculated with 2 ~ each dose 4 plates, at 37 °C, 5% carbon dioxide and saturated humidity cultured 11d ~ 14d, count mutation colonies growing number of holes.

6.1 Data Processing

6.1.1 The growth of adherent cells HGPRT Test Data Processing

6.1.1.1 Cytotoxicity

With respect to the vehicle control group set off the formation rate indicates cytotoxicity. Namely vehicle control colonies was 100% (1.00) is formed, seeking the relative value of each test substance group.

Relative colony forming efficiency calculation see formula (1).

A = B/C × 100% (1)

Where.

A --- relative colony forming rate,%;

B --- test substance group colony formation rate,%;

Vehicle control group C --- colony formation rate,%.

6.1.1.2 Colony formation and mutation frequency

Colony formation rate calculation see formula (2).

D = E/F × 100% (2)

Where.

D --- colony formation rate,%;

E --- actual viable cell colonies;

F --- inoculated cells.

See the mutation frequency calculation formula (3).

Where.

G --- mutation frequency;

H --- mutant colonies;

I --- inoculated cells;

D --- colony formation rate.

6.1.2 Suspension grown cells HGPRT Test Data Processing

6.1.2.1 Plating efficiency (PE0, PE6)

Plating efficiency calculation see formula (4).

ln (EW/TW)

1.6 × 100%

Where.

EW --- no colony growth of the number of holes;

TW --- Total number of holes;

1.6 --- inoculated cells per well.

6.1.2.2 Relative survival (RS)

Relative survival calculation see formula (5).

PE0 (test substance group) PE0 (vehicle control group) × 100% (5)

6.1.2.3 Mutation frequency (MF)

Mutation frequency calculation see formula (6).

MF (× 10-6) = -

ln (EW/TW)/N

PE6

Where.

EW --- no colony growth of the number of holes;

TW --- Total number of holes;

N --- per seeded cell number that is 2 × 104;

PE6 --- sixth day of plating efficiency.

6.2 Evaluation Results

6.2.1 Determination positive results

6.2.1.1 mutation frequency test substance group in any one or more doses of negative (vehicle) control group three times or three times, can be determined Positive.

6.2.1.2 mutation frequency test group was increased, and negative (vehicle) control group was statistically significant and dose - response trend, the it may be assessed as positive.

6.2.1.3 the test was set to cause a statistically significant increase in any condition and has a dose reproducibility can be judged as positive.

6.2.2 Determination negative results

Positive results do not meet these criteria, it is judged as negative.