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.
GB/T 16886 consists of the following parts under the general title Biological Evaluation of Medical Devices:
——Part 1: Evaluation and Testing within a Risk Management Process;
——Part 2: Animal Welfare Requirements;
——Part 3: Test for Genotoxicity, Carcinogenicity and Reproductive Toxicity;
——Part 4: Selection of Tests for Interactions with Blood;
——Part 5: Test for In Vitro Cytotoxicity;
——Part 6: Tests for Local Effects after Implantation;
——Part 7: Ethylene Oxide Sterilization Residuals;
——Part 9: Framework for Identification and Quantification of Potential Degradation Products;
——Part 10: Tests for Irritation and Skin Sensitization;
——Part 11: Tests for Systemic Toxicity;
——Part 12: Sample Preparation and Reference Materials;
——Part 13: Identification and Quantification of Degradation Products from Polymerical Medical Devices;
——Part 14: Identification and Quantification of Degradation Products from Ceramics;
——Part 15: Identification and Quantification of Degradation Products from Metals and Alloys;
——Part 16: Toxicokinetic Study Design for Degradation Products and Leachables;
——Part 17: Establishment of Allowable Limits for Leachable Substances;
——Part 18: Chemical Characterization of Materials;
——Part 19: Physico-Chemical Morphological and Topographical Characterization of Materials;
——Part 20: Principles and Methods for Immunotoxicology Testing of Medical Devices.
This is Part 5 of GB/T 16886.
This standard is developed in accordance with the rules given in GB/T 1.1-2009.
This part replaces GB/T 16886.5-2003 Biological Evaluation of Medical Devices - Part 5: Test for in Vitro Cytotoxicity, compared with which, the following technical changes have been made:
——Relevant contents such as “preparation of liquid extracts of material”, “test procedure” and “assessment of results” are modified; indexes for qualitative and quantitative assessment of cytotoxicity are provided (see 4.2, Chapters 8 and 10; 4.2, Chapters 8 and 10 in 2003 edition) are given;
——Neutral Red Uptake (NRU) Cytotoxicity Test (see Annex A) is added;
——Colony Formation Cytotoxicity Test (see Annex B) is added;
——MTT Cytotoxicity Test (see Annex C) is added;
——XTT Cytotoxicity Test (see Annex D) is added.
This part is identical to ISO 10993-5: 2009 Biological Evaluation of Medical Devices - Part 5: Tests for in Vitro Cytotoxicity by means of translation.
Chinese counterparts of the international documents given as normative references in this part are:
GB/T 16886.1-2011 Biological Evaluation of Medical Devices - Part 1: Evaluation and Testing within a Risk Management Process (ISO 10993-1:2009, IDT)
GB/T 16886.12-2017 Biological Evaluation of Medical Devices - Part 12: Sample Preparation and Reference Materials (ISO 10993-12: 2012, IDT)
This part was proposed by China Food and Drug Administration.
This part is under the jurisdiction of the National Technical Committee on Biological Evaluation on Medical Device of Standardization Administration of China (SAC/TC 248).
The previous editions of the standard replaced by this part are as follows:
——GB/T 16886.5-1997;
——GB/T 16886.5-2003.
Introduction
Due to the general applicability of in vitro cytotoxicity tests and their widespread use in evaluating a large range of devices and materials, it is the purpose of this part of GB/T 16886, rather than to specify a single test, to define a scheme for testing which requires decisions to be made in a series of steps. This should lead to the selection of the most appropriate test.
Three categories of test are listed: extract test, direct contact test, indirect contact test.
The choice of one or more of these categories depends upon the nature of the sample to be evaluated, the potential site of use and the nature of the use.
This choice then determines the details of the preparation of the samples to be tested, the preparation of the cultured cells, and the way in which the cells are exposed to the samples or their extracts.
At the end of the exposure time, the evaluation of the presence and extent of the cytotoxic effect is undertaken. It is the intention of this part of GB/T 16886 to leave open the choice of type of evaluation. Such a strategy makes available a battery of tests, which reflects the approach of many groups that advocate in vitro biological tests.
The numerous methods used endpoints measured in cytotoxicity determination can be grouped into the following categories of evaluation:
——assessments of cell damage by morphological means;
——measurements of cell damage;
——measurements of cell growth;
——measurements of specific aspects of cellular metabolism.
There are several means of producing results in each of these four categories. The investigator should be aware of the test categories and into which category a particular technique fits, in order that comparisons be able to be made with other results on similar devices or materials both at the intra- and interlaboratory level. Examples of quantitative test protocols are given in annexes. Guidance for the interpretation of the results is given in this part of GB/T 16886.
Biological Evaluation of Medical Devices - Part 5: Tests for in Vitro Cytotoxicity
1 Scope
This part of GB/T 16886 describes test methods to assess the in vitro cytotoxicity of medical devices.
These methods specify the incubation of cultured cells in contact with a device and/or extracts of a device either directly or through diffusion.
These methods are designed to determine the biological response of mammalian cells in vitro using appropriate biological parameters.
2 Normative References
The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies.
ISO 10993-1 Biological Evaluation of Medical Devices - Part 1: Evaluation and Testing within a Risk Management Process
ISO 10993-12 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 ISO 10993-1 and the following apply.
3.1
culture vessels
vessels appropriate for cell culture including glass petri dishes, plastic culture flasks or plastic multiwells and microtitre plates
Note: these can be used interchangeably in these methods provided that they meet the requirements of tissue culture grade and are suitable for use with mammalian cells.
3.2
positive control material
material which, when tested in accordance with this part, provides a reproducible cytotoxic response
Note: The purpose of the positive control is to demonstrate an appropriate test system response. For example, an organotin-stabilized polyurethane has been used as positive control for solid materials and extracts. Dilutions of phenol, for example, have been used as a positive control for extracts. In addition to a material, pure chemicals can also be used to demonstrate the performance of the test system.
3.3
blank
extraction vehicle not containing the test sample, retained in a vessel identical to that which holds the test sample and subjected to conditions identical to those to which the test sample is subjected during its extraction
Note: The purpose of the blank is to evaluate the possible confounding effects due to the extraction vessel, vehicle and extraction process.
3.4
negative control material
material which, when tested in accordance with this part, does not produce a cytotoxic response
Note: The purpose of the negative control is to demonstrate background response of the cells. For example, high-density polyethylene for synthetic polymers, and aluminum oxide ceramic rods for dental material have been used as negative controls.
3.5
test sample
material, device, device portion, component, extract or portion thereof that is subjected to biological or chemical testing or evaluation
3.6
subconfluency
approximately 80% confluency, i.e. the end of the logarithmic phase of growth
4 Sample and Control Preparation
4.1 General
The test shall be performed on:
a) an extract of the test sample,
b) the test sample itself.
Sample preparation shall be in accordance with ISO 10993-12.
Negative and positive controls shall be included in each assay.
4.2 Preparation of liquid extracts of material
4.2.1 Principles of extraction
Extracting conditions should attempt to simulate or exaggerate the clinical use conditions so as to determine the potential toxicological hazard without causing significant changes in the test sample, such as fusion, melting or any alteration of the chemical structure, unless this is expected during clinical application. Due to the nature of certain materials (e.g. biodegradable materials), alteration of the chemical structure can occur during the extraction procedure.
Note: the concentration of any endogenous or extraneous substances in the extract, and hence the amount exposed to the test cells, depends on the interfacial area, the extraction volume, pH, chemical solubility, diffusion rate, osmolality, agitation, temperature, time and other factors.
For devices that involve mixing two or more components in the patient to arrive at the final device (for example bone cement), the final device should not be washed prior to extraction. Washing the test sample can reduce or remove residuals present on the device. If the test sample is to be used in a sterile environment, a sterilized test sample should be used to extract chemical constituents.
Foreword i Introduction iv 1 Scope 2 Normative References 3 Terms and Definitions 4 Sample and Control Preparation 5 Cell Lines 6 Culture Medium 7 Preparation of Cell Stock Culture 8 Test Procedures 9 Test Report 10 Assessment of Results Annex A (Informative) Neutral Red Uptake (NRU) Cytotoxicity Test Annex B (Informative) Colony Formation Cytotoxicity Test Annex C (Informative) MTT Cytotoxicity Test Annex D (Informative) XTT Cytotoxicity Test 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.
GB/T 16886 consists of the following parts under the general title Biological Evaluation of Medical Devices:
——Part 1: Evaluation and Testing within a Risk Management Process;
——Part 2: Animal Welfare Requirements;
——Part 3: Test for Genotoxicity, Carcinogenicity and Reproductive Toxicity;
——Part 4: Selection of Tests for Interactions with Blood;
——Part 5: Test for In Vitro Cytotoxicity;
——Part 6: Tests for Local Effects after Implantation;
——Part 7: Ethylene Oxide Sterilization Residuals;
——Part 9: Framework for Identification and Quantification of Potential Degradation Products;
——Part 10: Tests for Irritation and Skin Sensitization;
——Part 11: Tests for Systemic Toxicity;
——Part 12: Sample Preparation and Reference Materials;
——Part 13: Identification and Quantification of Degradation Products from Polymerical Medical Devices;
——Part 14: Identification and Quantification of Degradation Products from Ceramics;
——Part 15: Identification and Quantification of Degradation Products from Metals and Alloys;
——Part 16: Toxicokinetic Study Design for Degradation Products and Leachables;
——Part 17: Establishment of Allowable Limits for Leachable Substances;
——Part 18: Chemical Characterization of Materials;
——Part 19: Physico-Chemical Morphological and Topographical Characterization of Materials;
——Part 20: Principles and Methods for Immunotoxicology Testing of Medical Devices.
This is Part 5 of GB/T 16886.
This standard is developed in accordance with the rules given in GB/T 1.1-2009.
This part replaces GB/T 16886.5-2003 Biological Evaluation of Medical Devices - Part 5: Test for in Vitro Cytotoxicity, compared with which, the following technical changes have been made:
——Relevant contents such as “preparation of liquid extracts of material”, “test procedure” and “assessment of results” are modified; indexes for qualitative and quantitative assessment of cytotoxicity are provided (see 4.2, Chapters 8 and 10; 4.2, Chapters 8 and 10 in 2003 edition) are given;
——Neutral Red Uptake (NRU) Cytotoxicity Test (see Annex A) is added;
——Colony Formation Cytotoxicity Test (see Annex B) is added;
——MTT Cytotoxicity Test (see Annex C) is added;
——XTT Cytotoxicity Test (see Annex D) is added.
This part is identical to ISO 10993-5: 2009 Biological Evaluation of Medical Devices - Part 5: Tests for in Vitro Cytotoxicity by means of translation.
Chinese counterparts of the international documents given as normative references in this part are:
GB/T 16886.1-2011 Biological Evaluation of Medical Devices - Part 1: Evaluation and Testing within a Risk Management Process (ISO 10993-1:2009, IDT)
GB/T 16886.12-2017 Biological Evaluation of Medical Devices - Part 12: Sample Preparation and Reference Materials (ISO 10993-12: 2012, IDT)
This part was proposed by China Food and Drug Administration.
This part is under the jurisdiction of the National Technical Committee on Biological Evaluation on Medical Device of Standardization Administration of China (SAC/TC 248).
The previous editions of the standard replaced by this part are as follows:
——GB/T 16886.5-1997;
——GB/T 16886.5-2003.
Introduction
Due to the general applicability of in vitro cytotoxicity tests and their widespread use in evaluating a large range of devices and materials, it is the purpose of this part of GB/T 16886, rather than to specify a single test, to define a scheme for testing which requires decisions to be made in a series of steps. This should lead to the selection of the most appropriate test.
Three categories of test are listed: extract test, direct contact test, indirect contact test.
The choice of one or more of these categories depends upon the nature of the sample to be evaluated, the potential site of use and the nature of the use.
This choice then determines the details of the preparation of the samples to be tested, the preparation of the cultured cells, and the way in which the cells are exposed to the samples or their extracts.
At the end of the exposure time, the evaluation of the presence and extent of the cytotoxic effect is undertaken. It is the intention of this part of GB/T 16886 to leave open the choice of type of evaluation. Such a strategy makes available a battery of tests, which reflects the approach of many groups that advocate in vitro biological tests.
The numerous methods used endpoints measured in cytotoxicity determination can be grouped into the following categories of evaluation:
——assessments of cell damage by morphological means;
——measurements of cell damage;
——measurements of cell growth;
——measurements of specific aspects of cellular metabolism.
There are several means of producing results in each of these four categories. The investigator should be aware of the test categories and into which category a particular technique fits, in order that comparisons be able to be made with other results on similar devices or materials both at the intra- and interlaboratory level. Examples of quantitative test protocols are given in annexes. Guidance for the interpretation of the results is given in this part of GB/T 16886.
Biological Evaluation of Medical Devices - Part 5: Tests for in Vitro Cytotoxicity
1 Scope
This part of GB/T 16886 describes test methods to assess the in vitro cytotoxicity of medical devices.
These methods specify the incubation of cultured cells in contact with a device and/or extracts of a device either directly or through diffusion.
These methods are designed to determine the biological response of mammalian cells in vitro using appropriate biological parameters.
2 Normative References
The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies.
ISO 10993-1 Biological Evaluation of Medical Devices - Part 1: Evaluation and Testing within a Risk Management Process
ISO 10993-12 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 ISO 10993-1 and the following apply.
3.1
culture vessels
vessels appropriate for cell culture including glass petri dishes, plastic culture flasks or plastic multiwells and microtitre plates
Note: these can be used interchangeably in these methods provided that they meet the requirements of tissue culture grade and are suitable for use with mammalian cells.
3.2
positive control material
material which, when tested in accordance with this part, provides a reproducible cytotoxic response
Note: The purpose of the positive control is to demonstrate an appropriate test system response. For example, an organotin-stabilized polyurethane has been used as positive control for solid materials and extracts. Dilutions of phenol, for example, have been used as a positive control for extracts. In addition to a material, pure chemicals can also be used to demonstrate the performance of the test system.
3.3
blank
extraction vehicle not containing the test sample, retained in a vessel identical to that which holds the test sample and subjected to conditions identical to those to which the test sample is subjected during its extraction
Note: The purpose of the blank is to evaluate the possible confounding effects due to the extraction vessel, vehicle and extraction process.
3.4
negative control material
material which, when tested in accordance with this part, does not produce a cytotoxic response
Note: The purpose of the negative control is to demonstrate background response of the cells. For example, high-density polyethylene for synthetic polymers, and aluminum oxide ceramic rods for dental material have been used as negative controls.
3.5
test sample
material, device, device portion, component, extract or portion thereof that is subjected to biological or chemical testing or evaluation
3.6
subconfluency
approximately 80% confluency, i.e. the end of the logarithmic phase of growth
4 Sample and Control Preparation
4.1 General
The test shall be performed on:
a) an extract of the test sample,
b) the test sample itself.
Sample preparation shall be in accordance with ISO 10993-12.
Negative and positive controls shall be included in each assay.
4.2 Preparation of liquid extracts of material
4.2.1 Principles of extraction
Extracting conditions should attempt to simulate or exaggerate the clinical use conditions so as to determine the potential toxicological hazard without causing significant changes in the test sample, such as fusion, melting or any alteration of the chemical structure, unless this is expected during clinical application. Due to the nature of certain materials (e.g. biodegradable materials), alteration of the chemical structure can occur during the extraction procedure.
Note: the concentration of any endogenous or extraneous substances in the extract, and hence the amount exposed to the test cells, depends on the interfacial area, the extraction volume, pH, chemical solubility, diffusion rate, osmolality, agitation, temperature, time and other factors.
For devices that involve mixing two or more components in the patient to arrive at the final device (for example bone cement), the final device should not be washed prior to extraction. Washing the test sample can reduce or remove residuals present on the device. If the test sample is to be used in a sterile environment, a sterilized test sample should be used to extract chemical constituents.
Contents of GB/T 16886.5-2017
Foreword i
Introduction iv
1 Scope
2 Normative References
3 Terms and Definitions
4 Sample and Control Preparation
5 Cell Lines
6 Culture Medium
7 Preparation of Cell Stock Culture
8 Test Procedures
9 Test Report
10 Assessment of Results
Annex A (Informative) Neutral Red Uptake (NRU) Cytotoxicity Test
Annex B (Informative) Colony Formation Cytotoxicity Test
Annex C (Informative) MTT Cytotoxicity Test
Annex D (Informative) XTT Cytotoxicity Test
Bibliography
