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SIST EN ISO 10993-5:2009
01-september-2009
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SIST EN ISO 10993-5:2000
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Biological evaluation of medical devices - Part 5: Tests for in vitro cytotoxicity (ISO 10993
-5:2009)
Biologische Beurteilung von Medizinprodukten - Teil 5: Prüfungen auf in vitro-
Zytotoxizität (ISO 10993-5:2009)
Évaluation biologique des dispositifs médicaux - Partie 5: Essais concernant la
cytotoxicité in vitro (ISO 10993-5:2009)
Ta slovenski standard je istoveten z: EN ISO 10993-5:2009
ICS:
11.100.20 %LRORãNRRYUHGQRWHQMH Biological evaluation of
PHGLFLQVNLKSULSRPRþNRY medical devices
SIST EN ISO 10993-5:2009 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN ISO 10993-5:2009
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SIST EN ISO 10993-5:2009
EUROPEAN STANDARD
EN ISO 10993-5
NORME EUROPÉENNE
EUROPÄISCHE NORM
June 2009
ICS 11.100.20 Supersedes EN ISO 10993-5:1999
English Version
Biological evaluation of medical devices - Part 5: Tests for in
vitro cytotoxicity (ISO 10993-5:2009)
Évaluation biologique des dispositifs médicaux - Partie 5: Biologische Beurteilung von Medizinprodukten - Teil 5:
Essais concernant la cytotoxicité in vitro (ISO 10993- Prüfungen auf In-vitro-Zytotoxizität (ISO 10993-5:2009)
5:2009)
This European Standard was approved by CEN on 17 April 2009.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European
Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national
standards may be obtained on application to the CEN Management Centre or to any CEN member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by translation
under the responsibility of a CEN member into its own language and notified to the CEN Management Centre has the same status as the
official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland,
France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,
Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2009 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 10993-5:2009: E
worldwide for CEN national Members.
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SIST EN ISO 10993-5:2009
EN ISO 10993-5:2009 (E)
Contents Page
Foreword .3
Annex ZA (informative) Relationship between this International Standard and the Essential
Requirements of EU Directive 93/42/EEC .4
Annex ZB (informative) Relationship between this International Standard and the Essential
Requirements of EU Directive 90/385/EEC .5
2
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SIST EN ISO 10993-5:2009
EN ISO 10993-5:2009 (E)
Foreword
This document (EN ISO 10993-5:2009) has been prepared by Technical Committee ISO/TC 194 "Biological
evaluation of medical devices" in collaboration with Technical Committee CEN/TC 206 “Biological evaluation
of medical devices” the secretariat of which is held by NEN.
This European Standard shall be given the status of a national standard, either by publication of an identical
text or by endorsement, at the latest by December 2009, and conflicting national standards shall be withdrawn
at the latest by December 2009.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights.
This document supersedes EN ISO 10993-5:1999.
This document has been prepared under a mandate given to CEN by the European Commission and the
European Free Trade Association, and supports essential requirements of EC Directives.
For relationship with EC Directives, see informative Annex ZA and ZB, which are integral parts of this
document.
According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following
countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Cyprus, Czech
Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia,
Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain,
Sweden, Switzerland and the United Kingdom.
Endorsement notice
The text of ISO 10993-5:2009 has been approved by CEN as EN ISO 10993-5:2009 without any
modifications.
3
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SIST EN ISO 10993-5:2009
EN ISO 10993-5:2009 (E)
Annex ZA
(informative)
Relationship between this International Standard and the Essential
Requirements of EU Directive 93/42/EEC
This International Standard has been prepared under a mandate given to CEN by the European Commission
and the European Free Trade Association to provide a means of conforming to Essential Requirements of the
New Approach Directive 93/42/EEC on medical devices.
Once this standard is cited in the Official Journal of the European Communities under that Directive and has
been implemented as a national standard in at least one Member State, compliance with the clauses of this
standard given in Table ZA.1 confers, within the limits of the scope of this standard, a presumption of
conformity with the corresponding Essential Requirements of that Directive and associated EFTA regulations.
Table ZA.1— Correspondence between this International Standard and Directive 93/42/EEC
Clause(s)/sub-clause(s) of Essential Requirements (ERs) of Directive Qualifying
this International Standard 93/42/EEC remarks/Notes
4, 5, 6, 7, 8, 9, 10 Annex I:
7.1, 7.2, 7.5
WARNING: Other requirements and other EU Directives may be applicable to the product(s) falling within the
scope of this International standard.
4
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SIST EN ISO 10993-5:2009
EN ISO 10993-5:2009 (E)
Annex ZB
(informative)
Relationship between this International Standard and the Essential
Requirements of EU Directive 90/385/EEC
This International Standard has been prepared under a mandate given to CEN by the European Commission
and the European Free Trade Association to provide a means of conforming to Essential Requirements of the
New Approach Directive 90/385/EEC on active implantable medical devices.
Once this standard is cited in the Official Journal of the European Communities under that Directive and has
been implemented as a national standard in at least one Member State, compliance with the clauses of this
standard given in Table ZB.1 confers, within the limits of the scope of this standard, a presumption of
conformity with the corresponding Essential Requirements of that Directive and associated EFTA regulations.
Table ZB.1 — Correspondence between this International Standard and Directive 90/385/EEC
Clause(s)/sub-clause(s) of Essential Requirements (ERs) of Qualifying
this International Standard Directive 90/385/EEC remarks/Notes
4, 5, 6, 7, 8, 9, 10 Annex I:
9
WARNING — Other requirements and other EU Directives may be applicable to the product(s) falling within
the scope of this standard.
5
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SIST EN ISO 10993-5:2009
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SIST EN ISO 10993-5:2009
INTERNATIONAL ISO
STANDARD 10993-5
Third edition
2009-06-01
Biological evaluation of medical
devices —
Part 5:
Tests for in vitro cytotoxicity
Évaluation biologique des dispositifs médicaux —
Partie 5: Essais concernant la cytotoxicité in vitro
Reference number
ISO 10993-5:2009(E)
©
ISO 2009
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SIST EN ISO 10993-5:2009
ISO 10993-5:2009(E)
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ii © ISO 2009 – All rights reserved
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SIST EN ISO 10993-5:2009
ISO 10993-5:2009(E)
Contents Page
Foreword. iv
Introduction . vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions. 1
4 Sample and control preparation. 2
4.1 General. 2
4.2 Preparation of liquid extracts of material. 3
4.3 Preparation of material for direct-contact tests .4
4.4 Preparation of controls . 5
5 Cell lines . 5
6 Culture medium. 5
7 Preparation of cell stock culture. 6
8 Test procedures . 6
8.1 Number of replicates . 6
8.2 Test on extracts . 6
8.3 Test by direct contact. 7
8.4 Test by indirect contact. 7
8.5 Determination of cytotoxicity . 9
9 Test report . 10
10 Assessment of results. 11
Annex A (informative) Neutral red uptake (NRU) cytotoxicity test. 12
Annex B (informative) Colony formation cytotoxicity test. 19
Annex C (informative) MTT cytotoxicity test . 24
Annex D (informative) XTT cytotoxicity test. 29
Bibliography . 34
© ISO 2009 – All rights reserved iii
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SIST EN ISO 10993-5:2009
ISO 10993-5:2009(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies
(ISO member bodies). The work of preparing International Standards is normally carried out through ISO
technical committees. Each member body interested in a subject for which a technical committee has been
established has the right to be represented on that committee. International organizations, governmental and
non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the
International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. ISO shall not be held responsible for identifying any or all such patent rights.
ISO 10993-5 was prepared by Technical Committee ISO/TC 194, Biological evaluation of medical devices.
This third edition cancels and replaces the second edition (ISO 10993-5:1999) which has been technically
revised.
ISO 10993 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: Tests for genotoxicity, carcinogenicity and reproductive toxicity
⎯ Part 4: Selection of tests for interactions with blood
⎯ Part 5: Tests 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 polymeric medical devices
⎯ Part 14: Identification and quantification of degradation products from ceramics
⎯ Part 15: Identification and quantification of degradation products from metals and alloys
iv © ISO 2009 – All rights reserved
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SIST EN ISO 10993-5:2009
ISO 10993-5:2009(E)
⎯ 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 [Technical
Specification]
⎯ Part 20: Principles and methods for immunotoxicology testing of medical devices [Technical Specification]
© ISO 2009 – All rights reserved v
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SIST EN ISO 10993-5:2009
ISO 10993-5:2009(E)
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 ISO 10993, 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 ISO 10993 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 and 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 ISO 10993.
vi © ISO 2009 – All rights reserved
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SIST EN ISO 10993-5:2009
INTERNATIONAL STANDARD ISO 10993-5:2009(E)
Biological evaluation of medical devices —
Part 5:
Tests for in vitro cytotoxicity
1 Scope
This part of ISO 10993 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 system
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 of ISO 10993, provides a reproducible cytotoxic
response
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SIST EN ISO 10993-5:2009
ISO 10993-5:2009(E)
NOTE The purpose of the positive control is to demonstrate an appropriate test system response. For example, an
1)
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 of ISO 10993, does not produce a cytotoxic response
NOTE The purpose of the negative control is to demonstrate background response of the cells. For example,
2)
high-density polyethylene for synthetic polymers, and aluminium 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
and/or
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.
1) The ZDEC and ZDBC polyurethanes are available from the Food and Drug Safety Center, Hatano Research Institute,
Ochiai 729-5, Hadanoshi, Kanagawa 257, Japan.
2) High-density polyethylene can be obtained from the U.S. Pharmacopeia (Rockville, MD, USA) and from the Food and
Drug Safety Center, Hatano Research Institute (Ochiai 729-5, Hadanoshi, Kanagawa 257, Japan).
The information given in 1) and 2) is for the convenience of the user of this part of ISO 10993 and does not constitute an
endorsement by ISO of these products. Equivalent products may be used if they can be shown to lead to the same results.
2 © ISO 2009 – All rights reserved
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SIST EN ISO 10993-5:2009
ISO 10993-5:2009(E)
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,
osmolarity, 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.
4.2.2 Extraction vehicle
The choice of the extraction vehicle(s) taking into account the chemical characteristics of the test sample shall
be justified and documented. For mammalian cell assays one or more of the following vehicles shall be used:
a) culture medium with serum;
b) physiological saline solution;
c) other suitable vehicle.
The choice of vehicle should reflect the aim of the extraction. Consideration shall be given to the use of both a
polar and a non-polar vehicle. Culture medium with serum is the preferred extraction vehicle. The use of
culture medium with serum is preferred for extraction because of its ability to support cellular growth as well as
extract both polar and non-polar substances. In addition to culture medium with serum, use of medium without
serum should be considered in order to specifically extract polar substances (e.g. ionic compounds). Other
suitable vehicles include purified water and dimethyl sulfoxide (DMSO). DMSO is cytotoxic in selected assay
systems at greater than 0,5 % (volume fraction). The cellular exposure concentration of extractables in DMSO
will be lower due to the greater dilution as compared to extraction in culture medium with serum.
NOTE 1 Different types of serum (e.g. foetal, bovine/calf serum, newborn calf serum) might be used and the choice of
the serum is dependent on the cell type.
NOTE 2 It is important to recognise that serum/proteins are known to bind, to some extent, extractables.
4.2.3 Extraction conditions
4.2.3.1 The extraction shall be performed in sterile, chemically inert, closed containers by using aseptic
techniques, in accordance with ISO 10993-12.
4.2.3.2 With the exception of circumstances given below, the extraction shall be conducted under one of
the following conditions and shall be applied according to the device characteristics and specific conditions for
use:
a) (24 ± 2) h at (37 ± 1) °C;
b) (72 ± 2) h at (50 ± 2) °C;
c) (24 ± 2) h at (70 ± 2) °C;
d) (1 ± 0,2) h at (121 ± 2) °C.
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SIST EN ISO 10993-5:2009
ISO 10993-5:2009(E)
Extraction conditions described above, which have been used to provide a measure of the hazard potential for
risk estimation of the device or material, are based on historical precedent. Other conditions, e.g. prolonged or
shortened extraction times at 37 °C, which simulate the extraction that occurs during clinical use or provide an
adequate measure of the hazard potential, may be used, but shall be justified and documented. For medical
devices that are in short-term contact (no greater than 4 h cumulative contact duration) with intact skin or
mucosa and that are not implanted, this may include extraction times of less than 24 h but no less than 4 h, as
given in a) to c).
Cell culture medium with serum should only be used in accordance with a) because extraction temperatures
greater than (37 ± 1) °C can adversely impact chemistry and/or stability of the serum and other constituents in
the culture medium.
For polymeric test samples, the extraction temperature should not exceed the glass transition temperature as
the higher temperature can change the extractant composition.
4.2.3.3 If the extract is filtered, centrifuged or processed by other methods prior to being applied to the
cells, these details shall be recorded in the final report along with a rationale for the additional steps (see
Clause 9). Any pH adjustment of the extract shall be reported. Manipulation of the extract, such as by pH
adjustment, should be avoided because it could influence the result.
4.3 Preparation of material for direct-contact tests
4.3.1 Form of test samples
Materials that have various shapes, sizes or physical states (i.e. liquid, gels, solids, etc.) may be tested
without modification in the cytotoxicity assays.
The preferred test sample of a solid material should have at least one flat surface. If not, adjustments shall be
made to achieve flat surfaces.
4.3.2 Sterility of test samples
4.3.2.1 Sterility of the test sample shall be taken into account.
4.3.2.2 Test samples from sterilized devices shall be handled aseptically throughout the test procedure.
4.3.2.3 Test samples from devices that are normally supplied non-sterile but are sterilized before use
shall be sterilized by the method recommended by the manufacturer and handled aseptically throughout the
test procedure.
The effect of sterilization methods or agents on the device should be considered in defining the preparation of
the test sample prior to
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