Codeofchina.com is in charge of this English translation. In case of any doubt about the English translation, the Chinese original shall be considered authoritative.
This document is developed in accordance with the rules given in GB/T 1.1-2020 Directives for standardization — Part 1: Rules for the structure and drafting of standardizing documents.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. The issuing body of this document shall not be held responsible for identifying any or all such patent rights.
This document was proposed by the National Medical Products Administration of People's Republic of China.
This document is under the jurisdiction of the National Technical Committee on Biological Evaluation on Medical Device of Standardization Administration of China (SAC/TC 248).
Introduction
The normal keratinocytes obtained from healthy volunteers may be cultured at the gas-liquid interface on the film or filter paper for several days to form a three-dimensional skin model, including the basal layer, stratum spinosum, granular layer and functional stratum corneum, that is, the reconstructed human epidermis model. This model was originally developed to detect outer skin irritation of pure chemical objects. In recent years, such models have also been used to detect stimulating substance in medical devices.
In vitro skin irritation test for medical devices
1 Scope
This document specifies a method for test for medical devices using a reconstructed human epidermis (RhE) model.
This document is applicable to in vitro skin irritation test using RhE model to evaluate the potential skin irritation of medical devices.
2 Normative references
The following documents, in whole or in part, are normatively referenced in this document and are indispensable for its application. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies.
GB/T 16886.10 Biological evaluation of medical devices — Part 10: Tests for irritation and skin sensitization
GB/T 16886.12 Biological evaluation of medical devices — Part 12: Sample preparation and reference materials
3 Terms and definitions
For the purposes of this document, the terms and definitions given in GB/T 16886.10 and GB/T 16886.12 apply.
4 Test principle
The polar and non-polar extracts of medical devices/materials or the devices/materials themselves may directly contact the upper surface of the RhE model; wash and remove the test samples on the epidermis after incubation for a certain time, and then detect the cell activity of the RhE model by methyl thiazolyl tetrazolium (MTT) test; compare with the negative control to obtain the tissue activity, and predict the irritation of the test samples according to the tissue activity.
5 Material
5.1 RhE model
The commercial RhE model is used for the test. See Annex A for instructions of RhE model.
Epidermal cells shall be taken from healthy volunteers whose antigens are negative for human immunodeficiency virus (HIV) 1 and 2 antibodies, HCV-Ab, hepatitis B. Users of this document should establish corresponding safety and health regulations to ensure biosafety.
5.2 Instruments and apparatus
Instruments and apparatus are as follows:
a) bechtop;
b) biosafety cabinet;
c) cell incubator;
d) balance;
e) microplate reader;
f) horizontal shaking table;
g) vortex oscillator;
h) pipettor (200 μL, 1 mL), piston displacement pipettor (100 μL), continuous liquid separator;
i) timer.
5.3 Reagent consumables
Reagent consumables are as follows:
a) skin model culture medium (provided by the skin model manufacturer and used together with the skin model);
b) Dulbecco's phosphate-buffered saline (DPBS, 1 ×);
c) sodium chloride injection (mass concentration: 0.9%);
d) sesame oil (high purity or medicinal grade);
e) MTT;
f) sodium dodecyl sulfate (SDS, mass concentration: 20%);
g) isopropanol (analytically pure);
h) 6-well culture plate, 24-well culture plate and 96-well culture plate (flat bottom).
i) aseptic centrifuge tube;
j) flushing liquid collection bottle;
k) angled blunt nose tweezers;
l) large funnel;
m) sealing film;
n) tin foil;
o) sterile cotton swab.
6 Test procedure
6.1 Preparation of extract and control solution
6.1.1 Preparation of extract
The extracts of medical devices and/or materials shall be prepared according to GB/T 16886.12, and attention shall be paid to aseptic operation.
0.9% sodium chloride injection should be used as polar extraction vehicle to prepare polar extraction solution, and sesame oil should be used as non-polar extraction vehicle to prepare non-polar extraction solution. If other extraction vehicle is used, it shall be proved that the extraction vehicle will not affect the test system.
The extraction time and temperature should be demonstrated based on GB/T 16886.12.
When testing medical devices and/or materials that are not suitable for extraction, their suitability should be demonstrated.
6.1.2 Preparation of control solution
6.1.2.1 Negative control
Sterile DPBS (1 ×).
6.1.2.2 Vehicle control
The vehicle control shall be placed in the extraction container, and the same extraction procedure as the medical device and/or material shall be carried out.
6.1.2.3 Positive control
1% SDS solution. Take 0.5 mL of 20% SDS aqueous solution and 9.5 mL of corresponding extraction vehicle, and mix well with vortex oscillator.
Note 1: The positive control prepared freshly on the day of test is used, and it is recommended to use 20% commercial SDS solution to prepare positive control. SDS positive control may be prepared by sterile DPBS when the test samples are not suitable for extraction.
Note 2: If the test sample reacts with MTT reagent, or the tissues or cells are stained; and there is still a certain amount of residue after washing, the test results may be disturbed.
In this case, an appropriate control may be set to eliminate interference.
Foreword i
Introduction ii
1 Scope
2 Normative references
3 Terms and definitions
4 Test principle
5 Material
6 Test procedure
7 Data calculation steps
8 Acceptance criteria for test
9 Result judgment
10 Test report
Annex A (Informative) Description of the RhE model
Bibliography
Codeofchina.com is in charge of this English translation. In case of any doubt about the English translation, the Chinese original shall be considered authoritative.
This document is developed in accordance with the rules given in GB/T 1.1-2020 Directives for standardization — Part 1: Rules for the structure and drafting of standardizing documents.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. The issuing body of this document shall not be held responsible for identifying any or all such patent rights.
This document was proposed by the National Medical Products Administration of People's Republic of China.
This document is under the jurisdiction of the National Technical Committee on Biological Evaluation on Medical Device of Standardization Administration of China (SAC/TC 248).
Introduction
The normal keratinocytes obtained from healthy volunteers may be cultured at the gas-liquid interface on the film or filter paper for several days to form a three-dimensional skin model, including the basal layer, stratum spinosum, granular layer and functional stratum corneum, that is, the reconstructed human epidermis model. This model was originally developed to detect outer skin irritation of pure chemical objects. In recent years, such models have also been used to detect stimulating substance in medical devices.
In vitro skin irritation test for medical devices
1 Scope
This document specifies a method for test for medical devices using a reconstructed human epidermis (RhE) model.
This document is applicable to in vitro skin irritation test using RhE model to evaluate the potential skin irritation of medical devices.
2 Normative references
The following documents, in whole or in part, are normatively referenced in this document and are indispensable for its application. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies.
GB/T 16886.10 Biological evaluation of medical devices — Part 10: Tests for irritation and skin sensitization
GB/T 16886.12 Biological evaluation of medical devices — Part 12: Sample preparation and reference materials
3 Terms and definitions
For the purposes of this document, the terms and definitions given in GB/T 16886.10 and GB/T 16886.12 apply.
4 Test principle
The polar and non-polar extracts of medical devices/materials or the devices/materials themselves may directly contact the upper surface of the RhE model; wash and remove the test samples on the epidermis after incubation for a certain time, and then detect the cell activity of the RhE model by methyl thiazolyl tetrazolium (MTT) test; compare with the negative control to obtain the tissue activity, and predict the irritation of the test samples according to the tissue activity.
5 Material
5.1 RhE model
The commercial RhE model is used for the test. See Annex A for instructions of RhE model.
Epidermal cells shall be taken from healthy volunteers whose antigens are negative for human immunodeficiency virus (HIV) 1 and 2 antibodies, HCV-Ab, hepatitis B. Users of this document should establish corresponding safety and health regulations to ensure biosafety.
5.2 Instruments and apparatus
Instruments and apparatus are as follows:
a) bechtop;
b) biosafety cabinet;
c) cell incubator;
d) balance;
e) microplate reader;
f) horizontal shaking table;
g) vortex oscillator;
h) pipettor (200 μL, 1 mL), piston displacement pipettor (100 μL), continuous liquid separator;
i) timer.
5.3 Reagent consumables
Reagent consumables are as follows:
a) skin model culture medium (provided by the skin model manufacturer and used together with the skin model);
b) Dulbecco's phosphate-buffered saline (DPBS, 1 ×);
c) sodium chloride injection (mass concentration: 0.9%);
d) sesame oil (high purity or medicinal grade);
e) MTT;
f) sodium dodecyl sulfate (SDS, mass concentration: 20%);
g) isopropanol (analytically pure);
h) 6-well culture plate, 24-well culture plate and 96-well culture plate (flat bottom).
i) aseptic centrifuge tube;
j) flushing liquid collection bottle;
k) angled blunt nose tweezers;
l) large funnel;
m) sealing film;
n) tin foil;
o) sterile cotton swab.
6 Test procedure
6.1 Preparation of extract and control solution
6.1.1 Preparation of extract
The extracts of medical devices and/or materials shall be prepared according to GB/T 16886.12, and attention shall be paid to aseptic operation.
0.9% sodium chloride injection should be used as polar extraction vehicle to prepare polar extraction solution, and sesame oil should be used as non-polar extraction vehicle to prepare non-polar extraction solution. If other extraction vehicle is used, it shall be proved that the extraction vehicle will not affect the test system.
The extraction time and temperature should be demonstrated based on GB/T 16886.12.
When testing medical devices and/or materials that are not suitable for extraction, their suitability should be demonstrated.
6.1.2 Preparation of control solution
6.1.2.1 Negative control
Sterile DPBS (1 ×).
6.1.2.2 Vehicle control
The vehicle control shall be placed in the extraction container, and the same extraction procedure as the medical device and/or material shall be carried out.
6.1.2.3 Positive control
1% SDS solution. Take 0.5 mL of 20% SDS aqueous solution and 9.5 mL of corresponding extraction vehicle, and mix well with vortex oscillator.
Note 1: The positive control prepared freshly on the day of test is used, and it is recommended to use 20% commercial SDS solution to prepare positive control. SDS positive control may be prepared by sterile DPBS when the test samples are not suitable for extraction.
Note 2: If the test sample reacts with MTT reagent, or the tissues or cells are stained; and there is still a certain amount of residue after washing, the test results may be disturbed.
In this case, an appropriate control may be set to eliminate interference.
Contents of YY/T 1808-2021
Foreword i
Introduction ii
1 Scope
2 Normative references
3 Terms and definitions
4 Test principle
5 Material
6 Test procedure
7 Data calculation steps
8 Acceptance criteria for test
9 Result judgment
10 Test report
Annex A (Informative) Description of the RhE model
Bibliography
