GB/T 14233.2-2005_English: PDF (GB/T14233.2-2005)
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Test methods for infusion, transfusion, injection equipment for medical use -- Part 2: Biological test methods
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Standard ID | GB/T 14233.2-2005 (GB/T14233.2-2005) | Description (Translated English) | Test methods for infusion, transfusion, injection equipment for medical use - Part 2: Biological test methods | Sector / Industry | National Standard (Recommended) | Classification of Chinese Standard | C31 | Classification of International Standard | 11.040.20 | Word Count Estimation | 31,353 | Date of Issue | 2005-11-17 | Date of Implementation | 2006-05-01 | Older Standard (superseded by this standard) | GB/T 14233.2-1993 | Quoted Standard | GB/T 16886.1; GB/T 16886.4; GB/T 16886.5; GB/T 16886.6; GB/T 16886.10; GB/T 16886.11; GB/T 16886.12 | Drafting Organization | Jinan, the State Food and Drug Administration Medical Device Quality Supervision and Inspection Center | Administrative Organization | State Food and Drug Administration | Regulation (derived from) | Announcement of Newly Approved National Standards No. 1, 2006 (No. 88 overall) | Proposing organization | State Food and Drug Administration | Issuing agency(ies) | General Administration of Quality Supervision, Inspection and Quarantine of the People Republic of China, China National Standardization Administration Committee | Summary | This standard specifies: Infusion, transfusion, injection equipment biological test methods. This standard applies to: medical infusion, transfusion, injection equipment. | Standard ID | GB/T 14233.2-1993 (GB/T14233.2-1993) | Description (Translated English) | Test methods for infusion, transfusion, injection equipment for medical use. Part 2: Biological test methods | Sector / Industry | National Standard (Recommended) | Classification of Chinese Standard | C31 | Classification of International Standard | 11.040.20 | Word Count Estimation | 21,210 | Date of Issue | 1993/3/16 | Date of Implementation | 1993/11/1 | Regulation (derived from) | Announcement of Newly Approved National Standards No. 1, 2006 (No. 88 overall) | Proposing organization | National Standard Committee on Medical Infusion Equipment | Issuing agency(ies) | State Bureau of Technical Supervision |
GB/T 14233.2-2005
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
ICS 11.040.20
C 31
Replacing GB/T 14233.2-1993
Test Methods for Infusion, Transfusion, Injection
Equipment for Medical Use –
Part 2. Biological Test Methods
ISSUED ON. NOVEMBER 17, 2005
IMPLEMENTED ON. MAY 1, 2006
Jointly Issued by. General Administration of Quality Supervision,
Inspection and Quarantine (AQSIQ);
Standardization Administration (SAC) of the People's
Republic of China.
Table of Contents
Foreword ... 3
Introduction ... 4
1 Scope ... 5
2 Normative References ... 5
3 Sterility Test ... 6
4 Bacterial Endotoxin Test ... 8
5 Pyrogen Test ... 11
6 Acute Systemic Toxicity Test... 13
7 Hemolytic Test ... 15
8 Cytotoxicity Test ... 17
9 Sensitization Test (Maximum Dose Method) ... 21
10 Intradermal Reaction Test ... 23
11 Implantation Test ... 24
Appendix A (Informative) Sub-acute (Sub-chronic) Systemic Toxicity Test
... 29
Appendix B (Informative) Interaction Test With Blood (Devices) ... 35
Appendix C (Informative) Preparation of Common Cell Culturing
Solution and Culture Solution ... 48
Foreword
GB 14233 "Test Methods for Infusion, Transfusion, Injection Equipment for Medical
Use" is divided into two parts.
- Part 1. Chemical Analysis Method;
- Part 2. Biological Test Methods.
This Part is Part 2 of GB/T 14233. This Part replaces GB/T 14233.2-1993 "Test
Methods for Infusion, Transfusion, Injection Equipment for Medical Use - Part 2.
Biological Test Methods". For the 3 tests - sterile, pyrogen and bacterial endotoxin,
this revision directly quotes the applicable chapters and sections of "Chinese
Pharmacopoeia (Part II)", so as to keep in line with the updated and revised edition of
Chinese Pharmacopoeia; cytotoxicity test methods were modified by reference to
GB/T 16886 "Biological Evaluation of Medical Devices"; GB/T 16886 "Biological
Evaluation of Medical Devices" was directly quoted by reference for sensitization,
intradermal reaction and implantation test; test methods for sub-acute (sub-chronic)
systemic toxicity and blood (equipment) interaction were added; product acceptance
judgment indexes were deleted. Appendix A, Appendix B and Appendix C in this Part
are informative.
This Part was proposed by China Food and Drug Administration.
This Part shall be under the jurisdiction of National Technical Committee on Medical
Syringes of Standardization Administration of China.
Drafting organizations of this Part. Jinan Center for Medical Equipment Quality
Supervision and Testing of State Food and Drug Administration, and Tianjin Medical
Biomaterial Monitoring Research Center.
Chief drafting staffs of this Part. You Shaohua, Zhu Xuetao, Liu Xin, Huang Jingchun,
Wang Xin, Zhu Junmei, Wang Kelei, and Hao Shubin.
This Part was first-time issued in March, 1993.
Introduction
The biological test methods provided in this Part are based on the basic principles of
GB/T 16886.1 "Biological Evaluation of Medical Devices. Part 1. Evaluation and
Testing", and are established especially in accordance with the biological evaluation
demand of medical infusion, transfusion and injection equipment. This revision is
made on the basis of the corresponding test methodology principle and test
procedures in GB/T 16886 "Biological Evaluation of Medical Devices" and "Chinese
Pharmacopoeia (Part II)", and according to the characteristics of medical infusion,
transfusion and injection equipment. Therefore, this Part shares methodology identity
property with GB/T 16886 and the methods of "Chinese Pharmacopoeia"; it is
applicable to the method standard of biological performance test of medical infusion,
transfusion and injection equipment.
For the sterile, pyrogen and bacterial endotoxin tests, this revision directly quotes the
applicable chapters and sections of "Chinese Pharmacopoeia", without the year
number of pharmacopoeia noted, so as to be in line with the updated and revised
edition of "Chinese Pharmacopoeia". For the test items without detailed test
procedure given in GB/T 16886, they are refined, e.g. cytotoxicity, acute systemic
toxicity, sub-acute (sub-chronic) systemic toxicity and tests in interaction with blood
(equipment). For the stimulation, sensitization and implantation tests with test
procedures given in detail in GB/T 16886, this revision directly adopts the
corresponding sections of GB/T 16886.
This Part acts as the test method standard; this revision deletes the product
acceptance judgment indexes in the main body, but provides the current general
judgment indexes for reference in article note. The relevant product standards, when
quoting this Part, shall specify the suitable acceptance judgment indexes according to
the product application characteristics.
Test Methods for Infusion, Transfusion, Injection
Equipment for Medical Use –
Part 2. Biological Test Methods
1 Scope
This Part of GB/T 14233 specifies the biological test methods of medical infusion,
transfusion and injection equipment.
This Part is applicable to medical infusion, transfusion and injection equipment.
2 Normative References
The following normative documents contain provisions which, through reference of
this Part of GB/T 14233, constitute provisions of this standard. For dated references,
subsequent amendments (excluding corrigendum) or revisions of these publications
do not apply. However, all parties who enter into an agreement according to this
standard are encouraged to study whether the latest editions of these documents are
applicable. For undated references, the latest edition of the normative documents
referred to applies.
GB/T 16886.1 Biological Evaluation of Medical Devices Part 1. Evaluation and
Testing (GB/T 16886.1-2001, IDT ISO 10993-1.1997)
GB/T 16886.4 Biological Evaluation Of Medical Devices - Part 4. Selection of
Tests for Interactions with Blood (GB/T 16886.4-2003, ISO 10993-4.2000, IDT)
GB/T 16886.5 Biological Evaluation of Medical Devices - Part 5. Test for in Vitro
Cytotoxicity (GB/T 16886.5-2003, ISO10993-5.1999, IDT)
GB/T 16886.6 Biological Evaluation of Medical Devices - Part 6. Tests for Local
Effects after Implantation (GB/T 16886.6-1997, IDT ISO 10993-6.1996)
GB/T 16886.10 Biological Evaluation of Medical Devices - Part 10. Tests for
Irritation and Delayed-type Hypersensitivity (GB/T 16886.10-2005, ISO
10993-10.2002, IDT)
GB/T 16886.11 Biological Evaluation of Medical Devices - Part 11. Tests for
Systemic Toxicity (GB/T 16886.11-1997, IDT ISO 10993-11.1993)
GB/T 16886.12 Biological Evaluation of Medical Devices - Part 12. Sample
Preparation and Reference Materials (GB/T 16886.12-2005, ISO 10993-12.2002,
IDT)
Pharmacopoeia of People's Republic of China Part II
3 Sterility Test
3.1 Purpose
This test inoculates the medical equipment or its vat liquor into culture medium, so as
to test if the test substance is subject to bacteria and fungus contamination.
3.2 Reagent
Sodium chloride solution of which the mass concentration is 9 g/L, and other diluent
and flushing fluid that meet the requirements of Chinese Pharmacopoeia.
3.3 Main Equipment
Super-clean bench, optical microscope, constant temperature incubator, thermostatic
water bath, pressure steam sterilizer, and electric drying oven
3.4 Preparation Before the Test
3.4.1 Instrument sterilization
All the equipment that contact with the test solution shall be sterilized by reliable
method - Put in pressure steam sterilizer at 121°C for 30min or in electric drying oven
at 160°C for 2h.
3.4.2 Disinfection chamber requirements
3.4.2.1 Partial disinfection chamber operating table or super-clean bench shall meet
the requirements of unidirectional airflow area at cleanliness class 100. After
completion of disinfection treatment on the disinfection chamber, the colony number in
the air shall be inspected, with the following methods. Take a petri dish with the
diameter of about 90 mm; for the aseptic operation, inject about 20mL of melted
nutrient agar medium; cultivate at 30°C~35°C for 48h. After it is confirmed as sterile,
take 3 petri dishes; open the upper cover at the mean position of disinfection chamber
operating table or super-clean bench; after exposing for 30min, cover it and place at
30°C~35°C to cultivate for 48h; take out for inspection. The colony number growing on
the 3 petri dishes shall not exceed 1.
3.4.2.2...
......
GB/T 14233.2-1993
NATIONAL STANDARD OF THE
PEOPLE’S 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 symmetrical intradermal injection in the hair removal area of
each guinea pig. The interval between two points is 1~2cm. The injection
locations are shown below:
10.4.2.1 Use healthy New Zealand rabbits, weighing 2.5~3kg. Each lot of test
items uses 6 rabbits.
10.4.2.2 In 24h before test, cut 10cm×15cm area of rabbit hair on both sides of
the rabbit's spine. It shall avoid skin damage.
10.4.3 Processing and treatment of test item and control sample
10.4.3.1 The control sample is recommended to use pre-qualified medical
grade heat-cured methyl vinyl silicone rubber.
10.4.3.2 Test items and control samples are processed into cylindrical test bars
with a diameter of 1mm and a length of 10mm. The surface shall be smooth.
The edges shall be flat.
10.4.3.3 Use soap water, clean water and distilled water to clean the test strip.
After the filter paper dries, immerse it into 75% ethanol solution for 20min.
Before implantation, use sterile saline for sand washing 3 times.
10.5 Test methods
10.5.1 Anesthesia and disinfection
Anesthesia uses intravenous injection to inject 25~30mL/kg pentobarbital
sodium or 15~20mg/kg thiopental sodium. According to the requirements for
conventional surgical procedures, use carbonic anhydride and 75% ethanol to
thoroughly disinfect the surgical area.
10.5.2 Surgical procedures
Operate according to the requirements for conventional surgical procedures.
Select 4 implantation points at equal distances of approximately 2.5mm on each
side of the rabbit's spine. The interval between two points is 2cm. Implant test
item to one side, control sample to the other side. Use a scalpel to cut the skin
of the implantation site and separate the subcutaneous tissue and fascia. The
surgery method uses hemostats to separate muscles and pass the test strip
into the muscle with a depth of 1~2cm. The injection method uses a puncture
needle to penetrate into the muscle at a 30° angle parallel to the spine. Take a
probe bar into the needle, push the test strip into the muscle, and withdraw the
needle and probe bar. Use surgical sutures to suture myofascial and skin
incisions. If there is excessive bleeding during implantation, a spare sample
shall be re-implanted in another location.
10.5.3 Result observation
10.5.3.1 Clinical observation
In 1, 3, 5d after implantation, observe skin reaction at implantation site whether
B4 Eagle culture solution
B4.1 Prepare into several stock solutions first. Mix them before use.
B4.1.1 Stock solution 1
Prepare Eagle saline free of glucose and NaHCO3. Sterilize at a high pressure.
Store at 4°C.
B4.1.2 Stock solution 2
Heat 100mL of stock solution 1 to 80°C. Dissolve the following amino acids:
1.26g of arginine, 0.38g of histidine, 0.72g of lysine, 0.52g of leucine, 0.52g of
isoleucine, 0.15g of methionine, 0.32g of phenylalanine, 0.48g of threonine,
0.10g of tryptophan, 0.48g of valine.
B4.1.3 Stock solution 3
Use 100mL of 0.1mol/L HCl solution to dissolve 0.36g of tyrosine and 0.24g of
cystine.
B4.1.4 Stock solution 4
Use 100mL of stock solution 1 to dissolve the following B vitamins: 0.1g of
choline chloride, 0.1g of nicotinamide, 0.1g of calcium metapolyacid, 0.1g of
pyridoxal, 0.01g of riboflavin, 0.1g of thiamine and 0.2g of inositol.
B4.1.5 Stock solution 5
Add several drops of 0.5mol/L NaOH in 100mL of stock solution 1. Then use it
to dissolve 0.1g of folate.
B4.1.6 Stock solution 6
Use 100mL of stock solution 1 to dissolve 10g of glucose.
B4.1.7 Stock solution 7
Use 100mL of distilled water to dissolve 2g of NaHCO3.
B4.1.8 Stock solution 8
Use 100mL of stock solution 1 to dissolve 2.92g of glutamine.
B4.2 Respectively use glass bacteria filter to filtrate and sterilize stock solutions
2~7. Store at 4°C. After stock solution 8 is filtrated and sterilized, sub-package
it into 10mL/bottle. Store in a low-temperature refrigerator.
B4.3 When using, respectively take 10mL of stock solution 2, 3, 5, 6, 8 to mix
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