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GB/T 14233.2-1993 English PDF (GBT14233.2-1993)

GB/T 14233.2-1993 English PDF (GBT14233.2-1993)

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GB/T 14233.2-1993: Test methods for infusion, transfusion, injection equipment for medical use. Part 2: Biological test methods

This Standard specifies biological performance test methods for finished products and materials of infusion, transfusion, injection equipment for syringes. This Standard is applicable to biological performance tests of infusion, transfusion, injection and supporting equipment that are made of medical polymer materials. Other medical polymer products can also refer to this Standard for use.
GB/T 14233.2-1993
NATIONAL STANDARD OF THE
PEOPLE REPUBLIC OF CHINA
UDC 615.473
C 31
GB/T 14233.2-93
Infusion, transfusion, injection equipment for medical
use - Part 2: Biological test methods
APPROVED ON: MARCH 16, 1993
IMPLEMENTED ON: NOVEMBER 01, 1993
Issued by: National Technical Supervision Bureau
Replaced
Table of Contents
1 Subject content and application scope ... 3
Chapter One -- Finished product test ... 3
2 Sterility test ... 3
3 Rabbit pyrogen test ... 6
4 Bacterial endotoxin test ... 10
5 Acute systemic toxicity test ... 13
6 Hemolysis test ... 15
Chapter Two -- Material test ... 17
7 Cytotoxicity test ... 17
8 Intradermal stimulation test ... 20
9 Skin sensitization test ... 22
10 Short-term muscle implantation test ... 25
Annex A Preparation of mediums ... 29
Annex B Preparations of cell culture solution, digestive solution, balanced salt solution (Supplementary) ... 31
Additional information: ... 33
Infusion, transfusion, injection equipment for medical
use - Part 2: Biological test methods
1 Subject content and application scope
This Standard specifies biological performance test methods for finished products and materials of infusion, transfusion, injection equipment for syringes. This Standard is applicable to biological performance tests of infusion, transfusion, injection and supporting equipment that are made of medical polymer materials. Other medical polymer products can also refer to this Standard for use.
Chapter One -- Finished product test
2 Sterility test
2.1 Definition
Sterility test is a method to check whether the test article is sterile. 2.2 Main equipment
Ultra-clean bench, optical microscope, constant-temperature incubator,
pressure steam sterilizer, electric heating oven.
2.3 Reagents
Peptone, beef extract, yeast extract, agar, glucose, potassium dihydrogen phosphate, sodium chloride, sodium hydroxide, magnesium sulfate, trypsin, L- cystine, sodium thioglycolate, 0.9% sodium chloride injection liquid.
2.4 Medium
2.4.1 Preparation of medium
2.4.1.1 First, peptone, beef extract, yeast extract, agar used for medium shall prepare a small amount of medium to observe if they are cloudy after
sterilization and if the growth of bacteria is good. Try again when changing factory designation.
must not exceed 3, no more than 4 colonies in a single dish.
Check the sterility of the sterile room during the sterility test in the same method as above. At the beginning of the test, open the dish cover and expose it to the air. At the end of the test, cover it and incubate according to the method mentioned above, according to the above requirements.
2.6 Preparation of control bacterial solution
Staphylococcus aureus bacterial solution: Take common agar beveled fresh culture of staphylococcus aureus (26 003) to inoculate a platinum ear into the aerobic and anaerobic medium. After incubating at 30~35??C for 16~18h, use sterile 0.9% sodium chloride injection to dilute into 1:106 to obtain.
2.7 Test methods
2.7.1 Number of test items
The same lot at least has 3 units of test items.
2.7.2 Extraction medium
0.9% sterile sodium chloride injection.
2.7.3 Preparation and inoculation of test solution
2.7.3.1 Preparation and inoculation of test solution shall be performed according to aseptic method.
2.7.3.2 Tube appliances: According to 1mL of extraction medium per 10cm2 of the internal surface area of the tube, the flow is 10mL/min.
2.7.3.3 Containers: If the container is already filled with liquid, the liquid in the container can be directly extracted as test solution. If it is not filled with liquid, it shall add 1mL of extraction medium per 10cm2 of surface area in the container. Shake several times.
2.7.3.4 Small accessories or solid appliances: Small accessories or solid appliances can be dropped directly into the medium. For large solid appliances, add 1mL of extraction medium per 10cm2 of surface area. Shake several times. 2.7.3.5 Test solution shall be used within 2h after preparation.
2.7.3.6 Each lot of test solutions are respectively inoculated into 5 tubes of aerobic and anaerobic medium. One tube is used to inoculate 1mL of
staphylococcus aureus solution for positive control. Two tubes are used to inoculate mold medium. The inoculation volume of each tube of test solution and the amount of medium to be sub-packed are according to Table 1.
3.3 Reagents
0.9% sodium chloride injection.
3.4 Rabbit for trial
3.4.1 General requirements
The rabbit for trial shall be healthy and free of injuries. It shall have smooth hair, a normal anus. Its weight shall be 1.7~3.0kg. It can be male or female. If it is a female rabbit, it shall not be pregnant. In 7d before temperature measurement, it shall be raised on the same feed. During this period, it shall not lose weight. There shall be no abnormalities in spirit, appetite, excretion, etc.
3.4.2 Selection of rabbit for trial
3.4.2.1 For a new rabbit that hasn?€?t used for pyrogen inspection, it shall pre- measure the temperature of the rabbit in 7d before the test to select. The conditions for selecting the test are the same as for the test item. But there is no injection. Measure the body temperature once every 1h, 4 times in total. When the temperatures measured are within 38.0~39.6??C and the difference between the highest temperature and the low temperature does not exceed 0.4??C, it shall comply with the provisions and can be used for test.
3.4.2.2 For a rabbit that has been used for pyrogen inspection, if the test item complies with the provisions, it shall rest for at least 48h before it is used for the second inspection. If the rabbit has a temperature rise of 0.6??C, it shall rest for more than two weeks. Then measure its temperature as for a new rabbit. It can be used for the test after its temperature meets the requirements. If the test item fails to comply with the provisions and when the average rabbit
temperature in the group reaches 0.8??C or greater, all the rabbits in the group are no longer used. Rabbits that two temperature rises or drops outside the qualified range are also no longer used.
3.4.2.3 If the interval between two uses is more than 3 weeks, the temperature shall be measured as for a new rabbit.
3.4.2.4 Each rabbit shall not be used more than 10 times.
3.5 Preparation before test
3.5.1 Appliance sterilization and depyrogen
Place all appliances that are in contact with the test solution in the electric drying oven to dry at 180??C for 2h or at 250??C for 30min.
3.5.2 Verification of anal thermometer
The interval time is 30~60min. The difference between two temperatures shall not exceed 0.2??C. Take the average of two body temperatures as the normal temperature of the rabbit. The body temperature of the rabbit used on the day shall be in the range of 38.0~39.6??C. For test rabbits in the same lot, the difference in normal body temperature between rabbits shall not exceed 1??C. 3.6.4.3 Temperature measurement method: Put the rabbit in a holder to prevent it from commotion. Start the first temperature measurement in 30min. Slowly insert an anal thermometer or temperature probe into the rabbit's anus, with a depth of 6cm. The temperature measurement time for each rabbit shall at least be 2min. If the rabbit is turbulent during temperature measurement, wait for 10min before measuring temperature again.
3.6.5 Injection of test solution and temperature measurement after
injection
3.6.5.1 Within 15min after the normal body temperature measured of rabbits meet the requirements, from the rabbit ear vein, slowly inject the test solution that has been warmed up to 38??C. The injection dose is 10mL/kg.
3.6.5.2 Measure the body temperature every 1h after injection, 3 times in total. Subtract the normal temperature from the highest of 3 temperatures, which shall be the degree of temperature increase of this rabbit.
3.6.6 Temperature drop of rabbits
When the temperature drop is less than or equal to 0.4??C, it is the range of normal temperature fluctuations of rabbits, calculated as ?€?0?€?. It shall re-test when the temperature drop is greater than or equal to 0.6??C. When the
temperature drop is greater than 0.4??C and less than 0.6??C, if only one of 3 rabbits is in this range, it shall be calculated as ?€?0?€?. If 2 or more of 3 rabbits are in this range, it shall re-test.
3.6.7 Precautions
3.6.7.1 Keep quiet inside and outside of the pyrogen test room. Avoid strong direct sunlight or lights and other stimuli.
3.6.7.2 During the entire test process, avoid rabbit commotion and keep the rabbit body temperature stable.
3.6.7.3 In 1~2d before the test, the test rabbits shall be in the same temperature environment. The temperature difference between the test room and the
breeding room shall not be greater than 5??C. The test room temperature is 17~28??C. During the entire test, the room temperature shall not change more than 5??C, and the relative humidity shall be kept stable.
4.3.1 National standard product of bacterial endotoxin: used to arbitrate the sensitivity of tachypleus amebocyte lysate and for positive control in test. 4.3.2 Working standard product of bacterial endotoxin: used to calibrate the sensitivity of tachypleus amebocyte lysate and for positive control in test. 4.3.3 Tachypleus amebocyte lysate: sensitivity is 0.25EU/mL, specification is 0.5mL.
4.3.4 Pyrogen-free water: endotoxin content is less than 0.05EU/mL.
4.4 Preparation before test
4.4.1 Appliance de-pyrogen
All appliances in contact with test solution shall be subject to de-pyrogen. Glass appliances are placed in the electric drying oven at 180??C to dry 2h or at 250??C to dry 30min. Plastic appliances are placed in 30% hydrogen peroxide to soak 4h. Then use pyrogen-free water to rinse and dry at 60??C for use.
4.4.2 Determination of tachypleus amebocyte lysate sensitivity
4.4.2.1 Before the test, it shall verify the sensitivity of the tachypleus amebocyte lysate used, which shall comply with the provisions.
4.4.2.2 Sensitivity determination: According to the labeled sensitivity range, use pyrogen-free water to dilute the working standard product of bacterial endotoxin in an equal ratio of 1???2. Select 4 serial dilutions that can have positive and negative results. Take several tachypleus amebocyte lysate of same lot. According to the labeled amount, respectively add pyrogen-free water to dissolve into the tachypleus amebocyte lysate solution. Take several
10mm??75mm test tubes. Respectively add 0.1mL of tachypleus amebocyte
lysate solution. Add 0.1mL of endotoxin dilution. Parallelly operate 4 tubes for each solution. Gently shake the test tubes to mix the contents. Seal the tubes. Place in the 37??1??C constant-temperature water bath to maintain 60??2min to observe the results. The 4 tubes with the highest concentration shall be positive. The 4 tubes with the lowest concentration shall be negative. Calculate the standard difference according to formula (1):
where,
s - Standard difference;
record as (-).
4.5.5.2 When the two tubes for test items are (-), it shall determine that the product complies with the provisions of this method and shall not conduct the pyrogen test.
4.5.5.3 When the two tubes for test items are (+) or one of them is (+), use pyrogen-free water to dilute the test solution in an equal ratio of 1???2 and re- test according to the above method. Operate 4 tubes for test items. If four tubes are all (-), it shall determine that the product complies with the provisions of this method and shall not conduct the pyrogen test.
4.5.5.4 When one of 4 tubes during the re-test is (+), it shall determine that the product fails to comply with the provisions of this method and it shall conduct the pyrogen test. Determine according to the test results.
4.5.5.5 The bacterial endotoxin limit of test item shall not exceed 0.5EU/mL. 4.5.5.6 Both two tubes for positive control shall be (+). Both two tubes for negative control shall be (-). Otherwise, the test shall be invalid.
5 Acute systemic toxicity test
5.1 Definition
This method injects a certain dose of test solution into white mice from veins. Observe the mice for toxic reaction and death within the specified time, so as to determine whether the test item complies with the provisions.
5.2 Main equipment and reagents
Pressure steam sterilizer, animal balance, 0.9% sodium chloride injection. 5.3 Preparation before test
5.3.1 Appliance sterilization
Place all appliances in contact with test solution in the pressure steam sterilizer at 115??C for 30min.
5.3.2 Preparation of test animal
5.3.2.1 The mice used for the test shall be healthy, free from injuries. The hair shall be smooth and eyes shall be brightly red. They must be raised under the same breeding conditions, of same source, of same breed. The female mice shall not be pregnant. The weight is 17~23g. The mice that have been tested by this test must not be reused.
the degree of hemolysis in vitro.
6.2 Main equipment
Constant-temperature water bath, spectrophotometer, centrifuge.
6.3 Reagents
2% potassium oxalate solution, 0.9% sodium chloride injection.
6.4 Test methods
6.4.1 Number of test items
The same lot shall at least have 2 units of test items.
6.4.2 Extraction medium
0.9% sodium chloride injection.
6.4.3 Preparation of test item
Weigh 15g of test item. For tube appliances, cut into 0.5cm segments. For other type appliances, cut into 0.5cm ?? 2cm strips or blocks.
6.4.4 Preparation of freshly diluted anticoagulated rabbit blood
6.4.4.1 Collect 20mL of blood from a healthy rabbit?€?s heart. Add 1mLof 2% potassium oxalate to prepare into fresh anticoagulated rabbit blood.
6.4.4.2 Take 8mL of fresh anticoagulated rabbit blood. Add 10mL of 0.9% sodium chloride injection to dilute.
6.4.5 Test steps
6.4.5.1 Prepare 3 test tubes for test items. Add 5g of test item and 10mL of 0.9% sodium chloride injection to each tube. Prepare 3 test tubes for negative control group. Add 10mL of 0.9% sodium chloride injection to each tube. Prepare 3 test tubes for positive control group. Add 10mL of distilled water to each tube. 6.4.5.2 Put all test tubes into constant-temperature water bath at 37??C to insulate for 30min. Add 0.2mL of diluted rabbit blood into each test tube. Gently mix well. Place in the 37??C water batch to keep insulation for 60min.
6.4.5.3 Pour out the liquid in the tube and centrifuge for 5min (2,500rpm). 6.4.5.4 Pipette supernatant into a cuvette. Use a spectrophotometer to
determine the absorbance at the wavelength of 545nm.
sulfate, disodium ethylenediamine tetraacetate (EDTA), trypsin, Earle solution, Hanks solution, D-Hanks solution, Eagie culture solution, cell culture solution, cell digestive solution.
7.4 The preparation of several balanced salt solutions (BSS), cell culture solution, and digestive solution shall meet the requirements of Annex B. 7.5 Cell strain
It is recommended to use L-929 cells (mouse fibroblasts). The test uses L-929 to passage 48~72h vigorous cells.
7.6 Appliance sterilization
Place all appliances in contact with test solution in the pressure steam sterilizer at 121??C to sterilize for 30min.
7.7 Test methods
7.7.1 Preparation of test solution
7.7.1.1 Use soapy water to clean and remove oil stains on the test item sheet. Then use distilled water to rinse. After it is dried by filter paper, cut into 0.5cm ?? 2cm strips. Use pressure steam or ultraviolet radiation to sterilize.
7.7.1.2 Put the test item into a sterile culture flask. Add 10mL of cell culture solution per 1cm2 of the surface area of test item. Seal the flask and place at 37??C for 24h.
7.7.2 Cell culture
7.7.2.1 Cell culture shall be performed according to aseptic method.
7.7.2.2 Preparation of cell suspension: Use digestive fluid to digest L-929 cells that have grown vigorously for 48~72h for 5~10min. Use Hanks solution to wash 2~3 times. Add cell culture solution. Use a straw to blow the cells off the flask wall. After well mixing, take 0.9mL plus 0.1mLof 0.4% trypan blue solution. Mix them. In 4min, use a hemocytometer to count under a microscope. Calculate cell concentration (in cell number/mL of stock solution) according to formula (4): According to the measured cell concentration, add an appropriate amount of cell culture solution to the flask to prepare 40,000/mL cell suspension for use. 7.7.2.3 Take 33 culture flasks. Respectively add 4mL of cell culture solution and Total number of cells in 5 large cells in
the center and corners
8.5.6 Result evaluation
8.5.6.1 When the skin reaction on the test side does not exceed the skin reaction on the control side, it means that the test solution has no irritating effect on the skin.
8.5.6.2 When there are two or more skin reactions in the test side that significantly exceed the control side reaction, it means that the test solution has irritating effect on the skin.
8.5.6.3 When there is only one obvious or severe reaction on the test side, it shall select another 3 rabbits to re-test. When there is no significant difference in the response between the test side and the control side, it shall pass this test. 9 Skin sensitization test
9.1 Definition
This test contacts a certain amount of test solution with the skin of guinea pig, so as to test the possibility whether the test item has the potential to cause contact skin allergies.
9.2 Main equipment and appliances
Pressure steam sterilizer, electric razor, hypodermic syringe, glass mortar. 9.3 Reagents
75% ethanol, 0.9% sodium chloride injection, 5% formaldehyde solution, 10% sodium lauryl sulfate, Freund's complete adjuvant.
9.4 Preparation before test
9.4.1 Appliance sterilization
Put all appliances in contact with test solution in the pressure steam sterilizer to sterilize at 121??C for 30min.
9.4.2 Preparation of laboratory animal
9.4.2.1 White guinea pig, weighing 300~500g, male or female. Each group shall at least have 10.
9.4.2.2 In 24h before test, shave 4cm??6cm hair from shoulder and back of guinea pig.
9.4.3 Preparation of Freund's complete adjuvant (CFA)
9.4.3.1 The volume ratio of anhydrous lanolin to liquid paraffin is 4:6 (in winter, the use ratio is 3:5). After dissolving anhydrous lanolin by heating, take 40mL and put into the mortar. After slightly cooling, add liquid paraffin while grinding until 60mL of liquid paraffin is added. Place in the pressure steam sterilizer to sterilize at 115??C for 30min to prepare Freund's incomplete adjuvant (IFA). Store at 4??C for use.
9.4.3.2 In IFA, add dead or attenuated mycobacteria (such as BCG or
tuberculosis) according to 4~5mg/mL to obtain Freund's complete adjuvant. 9.5 Test methods
9.5.1 Extraction medium
0.9% sodium chloride injection.
9.5.2 Negative control solution
0.9% sodium chloride injection.
9.5.3 Positive control solution
5% formaldehyde solution.
9.5.4 Preparation of test solution
9.5.4.1 The preparation of extract solution of test item and the preparation of negative control solution are same as 8.5.3.
9.5.4.2 Mix the extract solution of test item or negative, positive control solution and CFA with an equal volume. Stir for several minutes until completely emulsified.
9.5.5 Induction
9.5.5.1 Use 75% ethanol to clean the back-hair removal area of guinea pig. Conduct 6-point symme...

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