GB 15193.20-2014National Food Safety Standard -- TK gene mutation test (English PDF)

2.1 TK gene

Mammalian thymidine kinase gene. TK human genes located in the distal end of the long arm of chromosome 17; mice are located in the on the 11th chromosome.

2.2 Mutation frequency

In some cell lines, a particular gene mutant cells (colonies) occupy cells (colonies) of the total proportion (unit usually 10-6).

4.1 Instrument

Commonly used laboratory equipment, a deep freezer (-80 °C) or liquid nitrogen tank, biological safety cabinet, incubator, inverted microscope, centrifuge.

4.2 Medium

4.2.1 Complete medium

RPMI1640 medium, 10% horse serum (culture flasks) or 20% horse serum (96 well plates) and the amount of antibiotic (Green Final concentration of neomycin, streptomycin were 100IU/mL and 100µg/mL).

4.2.2 THMG and THG selection medium

THMG medium. 3µg/mL thymidine (thymidine, T) 5µg/mL hypoxanthine (hypoxanthine, H) 0.1µg/mL methotrexate (methotrexate, M) 7.5µg/mL glycine (glycine, G).

THG medium. 3µg/mL thymidine (T) 5µg/mL hypoxanthine (H) 7.5µg/mL glycine (G).

Above a concentration of each reagent at a final concentration in the medium. Practical tests, often according to the method of Table 1 and THG dubbed THMG 100-fold concentration.

Configuration Table 1 T, H, M, G reagent Reagent relative molecular mass mass/mg and the volume of solvent concentration/(mg/mL) T 242.2 30 10mLH2O 3 H 136.1 5 1mL1mol/LHCl 5 M 454.5 5 50mLH2O 0.1 G 75.07 75 10mLH2O 7.5 the above-described four kinds of reagents formulated in a 1000-fold concentration, this concentration then were taken of T, H, M, G 5mL each solution (20 mL total), or T, H, G solution each 5mL (a total of 15mL), were diluted with distilled water to 50mL, dubbed 100X THMG or THG stock solution.

Finally filtered through a membrane filter sterilization, packaging, stored at -20 °C. 1% of the proportion of complete medium when applying.

4.2.3 CHAT selection medium and CHT

CHAT media. 1 × 10-5mol/L deoxycytidine (cytosinedeoxyriboside, C) 2 × 10-4 mol/L hypoxanthine (Hypoxnathine, H) 1 × 10-7mol/L aminopterin (aminopterin, A) 1.75 × 10-5mol/L thymidine (thymidine, T).

CHT medium. 1 × 10-5mol/L deoxycytidine (C) 2 × 10-4mol/L hypoxanthine (H) 1.75 × 10-5mol/L Chest Glycosides (T).

Above a concentration of each reagent at a final concentration in the medium. Practical tests, often according to the method of Table 2 and CHT dubbed the CHAT 100-fold concentration.

Table Configuration 2 C, H, A, T reagent

Reagent relative molecular mass mass/mg and the volume of solvent concentration/(mol/L) C 227.2 113.6 50mLH2O 1 × 10-2 H 136.1 1361.0 50mL1mol/LHCl 2 × 10-1 A 440.4 2.2 50mLH2O 1 × 10-4 T 242.2 423.85 10mLH2O 1.75 × 10-2 the four kinds of reagents formulated in a 1000-fold concentration, and then were taken of the concentration C, H, A, T 5mL each solution (20 mL total), or C, H, T Solution of each 5mL (a total of 15mL), were diluted with distilled water to 50mL, dubbed 100X CHAT or CHT stock solution. Finally filter Membrane filtration sterilization, packaging, stored at -20 °C. 1% of the proportion of complete medium when applying.

4.3 Metabolic activation system

4.3.1 S9 cofactors

4.3.1.1 Magnesium potassium solution

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

4.3.1.2 0.2mol/L phosphate buffer (pH 7.4) 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

Under sterile conditions weigh 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 Preparation of rat liver S9 fraction

Choose healthy male adult SD or Wistar rats weighing about 150g ~ 200g, about 5 weeks to 6 weeks of age. Using phenobarbital Flavonoids and beta- naphthalene induced by the method of preparation by oral gavage to rats phenobarbital and beta- naphthalene flavonoids, doses were 80mg/kg Weight, continuous 3d, 16h after fasting decapitated animals were sacrificed before fasting 12h.

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, each tube 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 mutagens qualified to identify its biological activity after storage at -80 °C low temperature or freeze dry, a period not exceeding one year.

4.3.3 10% S9 mixture Usually by the S9 fraction and cofactors press 1.9 mixture consisting of 10% S9, sterile, now with the existing or filter sterilized. 10% S9 10mL mixture prepared as follows.

6.0mL phosphate buffer Magnesium potassium solution 0.4mL Glucose-6-phosphate sodium 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, it should be able to significantly activate the positive control, and cells without significant toxicity.

4.4 Phosphate buffer (phosphatebuefferdsaline, PBS)

The 8.0gNaCl, 0.20gKCl, 2.74gNa2HPO4 · 7H2O, 0.20gKH2PO4 dissolved in distilled water and dilute to 1000mL, pH7.2 ~ 7.4.

4.5 preparation of trifluorothymidine (trifluorothymidine, TFT) of Take TFT30mg, was dissolved in PBS was added to 10mL, dubbed the stock solution 3mg/mL of. The application by 1 per mille volume Bijiarupei Support group.

5.2 Test substance

5.2.1 Formulated test substance

Test substance prior to use now with the existing, it shall be verified under specific storage conditions do not affect its stability.

5.2.2 Test substance dose setting

Shall be at least 4 for analysis of the concentration of 3 ~. For the cytotoxicity of the test substance, the test should be based on the results of pre-cytotoxic, in the RS or RSG 20% to 80% are within a three to four-dose (concentration) level, and should consider the test substance solubility, pH and osmolarity affected. The method is. take good cell growth, adjusting a density of 5 × 105/mL, 1% by volume by adding different Concentration of the test substance, 37 °C shaking process 3h (L5178Y cells) or 4h (TK6 cells), cells were washed by centrifugation, for 2d (L5178Y Cell) or 3d (TK6 cells) expression of culture, counting each day and calculate the relative density of the cell suspension growth (RSG). Or a fine after taking the above treatment Cell suspension, for dilution to 108 cells/mL, inoculated 96 (per well 0.2mL, an average of 1.6 cells/well), each dose Species Cultivation of a ~ two plates, 37 °C, 5% carbon dioxide, saturated humidity 12d, counting each plate has a number of holes colony growth is calculated Relative survival (RS).

For extremely low cytotoxicity of the test substance, the concentration should be set up to 5mg/mL, 5µL/mL or 0.01mol/L. For relatively insoluble Substance solution, which should be set to achieve the highest concentration does not affect the cell culture may be added to the maximum concentration.

5.2.3 Control settings

Under normal circumstances, each one trial, in the presence of metabolic activation system and the absence shall be provided with positive and negative (vehicle) on Control group.

When using a metabolic activation system, the positive control was requested metabolic activation should be used, and can cause mutation colonies typical substances that can make with 3-methyl-MCA (3-methylcholanthrene), cyclophosphamide (cyclophosphamide, CP) and the like. In the absence of metabolic activation system, Positive controls may be used methyl methanesulfonate (methylmethanesulfonate, MMS), mitomycin C (mitomycinC, MMC), ethyl methane sulfonate (ethylmethanesulfonate, EMS) and the like. It may also be used other suitable positive controls.

Solvent should be non-mutagenic not react chemically with the test substance, does not affect cell survival and S9 activity. The preferred solvent of distilled water, such as Non-aqueous solvent (optionally dimethylsulfoxide, acetone, ethanol, etc.), need additional vehicle control.

5.3 Processing

Take good cell growth, adjusting a density of 5 × 105/mL, at 1% by volume of added test substance (required metabolic activation, while adding the final concentration of 1% to 10% of a mixture of S9), 37 [deg.] C shaking treatment 3h (L5178Y cells) or 4h (TK6 cells) to Speed centrifugal 800r/min ~ 1000r/min for 4min ~ 6min, the supernatant with PBS or serum-free medium cells were washed 2 times, cells were resuspended in RPMI1640 medium containing 10% horse serum solution, and adjust the cell density of 2 × 105/mL.

5.4 PE0 (0d plate plating efficiency) Determination

Take appropriate cell suspension for dilution to 108 cells/mL, inoculated 96 (per well 0.2mL, an average of 1.6 cells /Kong), one species per dose ~ 2 plates, cultured 12d at 37 °C, 5% carbon dioxide and saturated humidity, counting each plate there are colonies Growth of the number of holes.

5.5 Expression

Take 5.3 resulting cell suspension for 2d (L5178Y cells) or 3d (TK6 cells) expression of culture, the cell density counted every day and keep Density of 106/mL or less, calculated relative survival (RSG).

5.6 PE2 (L5178Y cells) or PE3 (TK6 cells) measured

After the end of the expression of the culture, take appropriate cell suspension was measured PE2/PE3 by 5.4 methods.

5.7 Mutation frequency (MF) Determination

5.7.1 L5178Y cells

After L5178Y cells cultured 2d, take appropriate cell suspension, adjusting the cell density of 1 × 104/mL, was added TFT (final concentration 3µg/mL), mix inoculated 96 (per well 0.2mL, an average of 2000 cells/well), each dose for 2 to 4 plates, Cultured 12d at 37 °C, 5% carbon dioxide and saturated humidity conditions, mutations count the number of colonies growing hole. Mutant colonies by large colonies (LargeColony, LC. diameter >=1/4 aperture, low density) and small colonies (smalcolony, SC. diameter 6 Data processing and evaluation of results

6.1 Data Processing

6.1.1 Plating efficiency (PE0, PE2/PE3)

Plating efficiency (%) is calculated, see equation (1).

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 Relative survival (RS)

Relative survival rate (%) is calculated, see formula (2).

PE treatment

PE negative/vehicle control × 100% (2) Note. When using a non-aqueous solvent vehicle, compared with the vehicle control.

6.1.3 Relative suspension growth (RSG)

Calculate the relative suspension growth (%) see formula (3).

RSG =

Expression of cell proliferation in multiples treated during Cell proliferation in multiples during the negative/vehicle control group was × 100% (3) Note. When using a non-aqueous solvent vehicle, compared with the vehicle control.

6.1.4 Relative total growth (relativetotalgrowth, RTG)

Calculating the relative total growth (%) see formula (4).

RTG = RSG × RSn × 100% (4) where.

RSn --- Day 2 (L5178Y cells) or three days (TK6 cells) relative survival.

6.1.5 Mutation frequency (MF)

Mutation frequency calculation see formula (5).

MF (× 10-6) = -

ln (EW/TW)/N

PE2/3

Where.

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

TW --- Total number of holes;

N --- per seeded cells (L5178Y cells 2000, TK6 cells 30,000);

PE2/3 --- Day 2 (L5178Y cells) or three days (TK6 cell) of plating efficiency.

In addition, L5178Y cells, large colonies can be calculated separately mutation frequency (L-MF), small colonies mutation frequency (S-MF) and total mutation Frequency (T-MF). For TK6 cells can calculate normal colony mutation frequency (N-MF), slowly growing colony mutation frequency (S-MF) and total mutation frequency (T-MF).

6.1.6 small colony mutants percentage (smalcolonymutation, SCM) or slow the growth of mutant colonies percentage (slowly-growth colonymutation, SCM) Small percentage of mutant colonies or slow the growth of mutant colonies percentage calculation see formula (6).

SCM =

S-MF

T-MF × 100%

6.2 Evaluation Results

6.2.1 Test conditions established

L5178Y cells used in the test spontaneous mutation frequency should be between 50 × 10-6 ~ 200 × 10-6; spontaneous mutation frequency TK6 cells Rate should be between 1.5 × 10-6 ~ 5.5 × 10-6, while spontaneous mutation frequency should be within the range of laboratory history. Negative/vehicle control the PE0 between 60% ~ 140%, PE2/PE3 value of between 70% to 130%. Positive control and negative T-MF/vehicle control There are significant differences, or negative/vehicle control more than 3 times.

6.2.2 The test substance is determined positive and negative results

6.2.2.1 The results of positive determination. More than one dose of the test substance (concentration) of T-MF group was significantly higher than the negative/vehicle control or female More sexual/vehicle control three times, and a dose - response trend, it is judged as positive. However, if only in relative survival rate of less than 20% of the high dose by the amount of positives cases, the result is judged as "suspicious." Determination 6.2.2.2 negative results. In the case of increase relative survival rate of no less than 20% mutation frequency, it can be judged to be negative.