EN ISO 10993-3:2009

SLOVENSKI STANDARD
01-september-2009
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SIST EN ISO 10993-3:2004
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Biological evaluation of medical devices - Part 3: Tests for genotoxicity, carcinogenicity
and reproductive toxicity (ISO 10993-3:2003)
Biologische Beurteilung von Medizinprodukten - Teil 3: Prüfungen auf Gentoxizität,
Karzinogenität und Reproduktionstoxizität (ISO 10993-3:2003)
Évaluation biologique des dispositifs médicaux - Partie 3: Essais concernant la
génotoxicité, la cancérogénicité et la toxicité sur la reproduction (ISO 10993-3:2003)
Ta slovenski standard je istoveten z: EN ISO 10993-3:2009
ICS:
11.100.20 %LRORãNRRYUHGQRWHQMH Biological evaluation of
PHGLFLQVNLKSULSRPRþNRY medical devices
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EUROPEAN STANDARD
EN ISO 10993-3
NORME EUROPÉENNE
EUROPÄISCHE NORM
May 2009
ICS 11.100.20 Supersedes EN ISO 10993-3:2003
English Version
Biological evaluation of medical devices - Part 3: Tests for
genotoxicity, carcinogenicity and reproductive toxicity (ISO
10993-3:2003)
Évaluation biologique des dispositifs médicaux - Partie 3: Biologische Beurteilung von Medizinprodukten - Teil 3:
Essais concernant la génotoxicité, la cancérogénicité et la Prüfungen auf Gentoxizität, Karzinogenität und
toxicité sur la reproduction (ISO 10993-3:2003) Reproduktionstoxizität (ISO 10993-3:2003)
This European Standard was approved by CEN on 28 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 10993-3:2009: E
worldwide for CEN national Members.

Contents Page
Foreword .3
Annex ZA (Informative) Relationship between this European Standard and the Essential
Requirements of EU Directive 93/42/EEC .4

Foreword
The text of ISO 10993-3:2003 has been prepared by Technical Committee ISO/TC 194 “Biological evaluation
of medical devices” of the International Organization for Standardization (ISO) and has been taken over as EN
ISO 10993-3:2009 by 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 November 2009, and conflicting national standards shall be withdrawn
at the latest by March 2010.
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 10993-3:2003.
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 Directive.
For relationship with EC Directive, see informative Annex ZA, which is an integral part 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 10993-3:2003 has been approved by CEN as a EN ISO 10993-3:2009 without any
modification.
Annex ZA
(Informative)
Relationship between this European Standard and the Essential
Requirements of EU Directive 93/42/EEC
This European 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 European Standard and EU Directives

Clause(s)/sub-clause(s) of this Essential Requirements (ERs) of Qualifying
EN Directive 93/42/EEC remarks/Notes
4, 5, 6, 7
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 standard.
INTERNATIONAL ISO
STANDARD 10993-3
Second edition
2003-10-15
Biological evaluation of medical
devices —
Part 3:
Tests for genotoxicity, carcinogenicity
and reproductive toxicity
Évaluation biologique des dispositifs médicaux —
Partie 3: Essais concernant la génotoxicité, la cancérogénicité et la
toxicité sur la reproduction
Reference number
ISO 10993-3:2003(E)
©
ISO 2003
ISO 10993-3:2003(E)
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ii © ISO 2003 — All rights reserved

ISO 10993-3:2003(E)
Contents Page
Foreword. iv
Introduction . vi
1 Scope. 1
2 Normative references. 1
3 Terms and definitions. 2
4 Genotoxicity tests. 3
4.1 General. 3
4.2 Test strategy. 3
4.3 Sample preparation. 4
4.4 Test methods. 4
4.4.1 In vitro genotoxicity tests. 4
4.4.2 In vivo genotoxicity tests . 4
5 Carcinogenicity tests. 5
5.1 General. 5
5.2 Test strategy. 5
5.3 Sample preparation. 5
5.4 Test methods. 5
6 Reproductive and developmental toxicity tests . 6
6.1 General. 6
6.2 Test strategy. 6
6.3 Sample preparation. 6
6.4 Test methods. 7
7 Test report. 7
Annex A (informative) Cell transformation test system . 8
Annex B (informative) Rationale of test systems. 9
Annex C (informative) Role of implantation carcinogenicity studies. 11
Bibliography . 13

ISO 10993-3:2003(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 10993-3 was prepared by Technical Committee ISO/TC 194, Biological evaluation of medical devices.
This second edition cancels and replaces the first edition (ISO 10993-3:1992), 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
 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 8: Selection and qualification of reference materials for biological tests
 Part 9: Framework for the identification and quantification of potential degradation products
 Part 10: Tests for irritation and delayed-type hypersensitivity
 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
iv © ISO 2003 — All rights reserved

ISO 10993-3:2003(E)
 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
Future parts will deal with other relevant aspects of biological testing.
ISO 10993-3:2003(E)
Introduction
The basis for biological evaluation of medical devices is often empirical and driven by the relevant concerns
for human safety. The risk of serious and irreversible effects, such as cancer or second-generation
abnormalities, is of particular public concern. It is inherent in the provision of safe medical devices that such
risks be minimized to the greatest extent feasible. The assessment of mutagenic, carcinogenic and
reproductive hazards is an essential component of the control of these risks. Not all test methods for the
assessment of genotoxicity, carcinogenicity or reproductive toxicity are equally well developed, nor is their
validity well established for the testing of medical devices.
Significant issues in test sample size and preparation, scientific understanding of disease processes and test
validation can be cited as limitations of available methods. For example, the biological significance of solid
state carcinogenesis is poorly understood. It is expected that ongoing scientific and medical advances will
alter our understanding of and approaches to these important toxicity test methods. At the time this part of
ISO 10993 was prepared, the test methods proposed were those most acceptable. Scientifically sound
alternatives to the proposed testing may be acceptable insofar as they address relevant matters of safety
assessment.
In the selection of tests needed to evaluate a particular medical device, there is no substitute for a careful
assessment of expected human uses and potential interactions of the medical device with various biological
systems. These considerations will be particularly important in such areas as reproductive and developmental
toxicology.
This part of ISO 10993 presents test methods for the detection of specific biological hazards, and strategies
for the selection of tests, where appropriate, that will assist in hazard identification. Testing is not always
necessary or helpful in hazard identification but, where it is appropriate, it is important that maximum test
sensitivity be achieved. Most tests included in this part of ISO 10993 refer to Guidelines for Testing of
Chemicals, prepared by the Organization for Economic Cooperation and Development (OECD).
The interpretation of findings and their implications for human health effects are beyond the scope of this part
of ISO 10993. Because of the multitude of possible outcomes and the importance of factors such as extent of
exposure, species differences and mechanical or physical considerations, risk assessment has to be
performed on a case-by-case basis.

vi © ISO 2003 — All rights reserved

INTERNATIONAL STANDARD ISO 10993-3:2003(E)

Biological evaluation of medical devices —
Part 3:
Tests for genotoxicity, carcinogenicity and reproductive toxicity
1 Scope
This part of ISO 10993 specifies strategies for hazard identification and tests on medical devices for the
following biological aspects:
 genotoxicity,
 carcinogenicity, and
 reproductive and developmental toxicity.
This part of ISO 10993 is applicable for evaluation of a medical device whose potential for genotoxicity,
carcinogenicity or reproductive toxicity has been identified.
NOTE Guidance on selection of tests is provided in ISO 10993-1.
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 10993-1:1997, Biological evaluation of medical devices — Part 1: Evaluation and testing
ISO 10993-2:1992, Biological evaluation of medical devices — Part 2: Animal welfare requirements
ISO 10993-6:1994, Biological evaluation of medical devices — Part 6: Tests for local effects after implantation
ISO 10993-12:2002, Biological evaluation of medical devices — Part 12: Sample preparation and reference
materials
ISO 10993-18, Biological evaluation of medical devices — Part 18: Chemical characterization of materials.
1)
OECD 414 , Prenatal Development Toxicity Study
OECD 415, One-Generation Reproduction Toxicity Study
OECD 416, Two-Generation Reproduction Toxicity

1) Organization for Economic Cooperation and Development.
ISO 10993-3:2003(E)
OECD 421, Reproduction/Developmental Toxicity Screening Test
OECD 451, Carcinogenicity Studies
OECD 453, Combined Chronic Toxicity/Carcinogenicity Studies
OECD 471, Bacterial Reverse Mutation Test
OECD 473, In vitro Mammalian Chromosome Aberration Test
OECD 476, In vitro Mammalian Cell Gene Mutation Test
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 10993-1, ISO 10993-12 and the
following apply.
3.1
carcinogenicity test
test to determine the tumorigenic potential of medical devices, materials and/or extracts using either single or
multiple exposures over a major portion of the life span of the test animal
NOTE These tests may be designed to examine both chronic toxicity and tumorigenicity in a single experimental study.
When chronic toxicity and carcinogenicity are evaluated within a single study, care in study design with emphasis on dose
selection should be exercised. This will help to ensure that premature mortality from chronic/cumulative toxicity does not
compromise the statistical evaluation of animals that survive until scheduled study termination (i.e. normal life-span).
3.2
energy-depositing medical device
device intended to exert its therapeutic or diagnostic effect by the delivery of electromagnetic radiation,
ionizing radiation or ultrasound
NOTE This does not include medical devices that deliver simple electrical current, such as electrocautery medical
devices, pacemakers or functional electrical stimulators.
3.3
genotoxicity test
test using mammalian or non-mammalian cells, bacteria, yeasts or fungi to determine whether gene mutations,
changes in chromosome structure, or other DNA or gene changes are caused by the test samples
NOTE These tests can include whole animals.
3.4
maximum tolerated dose
MTD
maximum dose that a test animal can tolerate without any adverse physical effects
3.5
reproductive and developmental toxicity test
test to evaluate the potential effects of test samples on reproductive function, embryonic morphology
(teratogenicity), and prenatal and early postnatal development
2 © ISO 2003 — All rights reserved

ISO 10993-3:2003(E)
4 Genotoxicity tests
4.1 General
Before a decision to perform a genotoxicity test is made, ISO 10993-1 and the chemical characterization of
materials (ISO 10993-18) shall be taken into account. The rationale for a test programme, taking into
consideration all relevant factors, shall be documented.
ISO 10993-1 indicates circumstances where the potential for genotoxicity is a relevant hazard for
consideration in an overall biological safety evaluation (see ISO 10993-1:1997, Table 1). Testing for
genotoxicity, however, is not necessary for medical devices, and components thereof, made only from
materials known to show no genotoxicity. Testing for genotoxicity is indicated where a review of the
composition of the materials reveals the possible presence in the final medical device of compounds that
might interact with genetic material, or when the chemical composition of the medical device is unknown. In
such circumstances, the genotoxic potential of suspect chemical components should be assessed, bearing in
mind the potential for synergy, in preference to carrying out genotoxicity tests on the material or medical
device as a whole.
When the genotoxicity of a medical device has to be experimentally assessed, a series of in vitro tests shall
be used. This series shall include either two tests if 4.2.1.2 is performed which uses the mouse lymphoma
assay incorporating colony number and size determination, or three tests if 4.2.1.1 is performed. When tests
are performed, at least two tests, investigating different end-points, shall use mammalian cells.
4.2 Test strategy
4.2.1 Genotoxicity testing shall be performed on the basis of an initial decision to test in accordance with
either Option 1 (4.2.1.1) or Option 2 (4.2.1.2).
4.2.1.1 Option 1
a) a test for gene mutations in bacteria (OECD 471); and
b) a test for gene mutations in mammalian cells (OECD 476); and
c) a test for clastogenicity in mammalian cells (OECD 473)
4.2.1.2 Option 2
a) a test for gene mutations in bacteria (OECD 471); and
b) a test for gene mutations in mammalian cells (OECD 476), specifically a mouse lymphoma assay
incorporating colony number and size determination in order to cover both endpoints (clastogenicity and
gene mutations).
4.2.2 If the results of all in vitro tests performed in accordance with 4.2.1 are negative, further genotoxicity
testing in animals is not normally justified and should not be performed, in the interest of preventing undue use
of animals.
In vivo testing shall be performed in accordance with ISO 10993-2.
4.2.3 If any of the in vitro tests is positive, either in vivo mutagenicity tests shall be performed (see 4.2.4) or
the presumption shall be made that the compound is mutagenic.
4.2.4 Any in vivo test shall be chosen on the basis of the most appropriate endpoint identified by the in vitro
tests. An attempt shall be made to demonstrate that the test substance has reached the target organ. If this
cannot be demonstrated, a second in vivo test in another target organ may be required to verify the lack of in
vivo genotoxicity.
ISO 10993-3:2003(E)
In vivo tests commonly used are:
a) micronucleus test in rodents (OECD 474) or
b) metaphase analysis in rodent bone marrow (OECD 475) or
c) unscheduled DNA synthesis test with mammalian liver cells (OECD 486).
The decision as to the most appropriate test system shall be justified and documented.
4.2.5 If other in vivo test systems to investigate genotoxicity are used in order to obtain additional
information, the rationale for this shall be justified and documented.
4.3 Sample preparation
4.3.1 Where genotoxicity tests are carried out on the material or a medical device or as a whole, sample
preparation shall be in accordance with ISO 10993-12. Tests shall be performed on extracts, exaggerated
extracts or the individual chemical compounds of the material/medical device. The highest test concentration
shall be within OECD guidelines. If exaggerated extraction conditions are used, care shall be taken that this
does not alter the chemical characteristics.
4.3.2 An appropriate solvent shall be chosen on the basis of its compatibility with the test system and its
ability to maximize extraction of the material or medical device. The rationale for the choice of solvent shall be
documented.
4.3.3 Where relevant, two appropriate extractants shall be used, one of which is a polar solvent, the second
a non-polar solvent or liquid appropriate to the nature and use of the medical device, both of which are
compatible with the test system.
4.4 Test methods
4.4.1 In vitro genotoxicity tests
Test methods for in vitro genotoxicity tests shall be chosen from the OECD Guidelines for Testing of
Chemicals.
Preferred test methods are: OECD 471, OECD 473, OECD 476, OECD 479 and OECD 482. It may be
necessary to consider, in the design and selection of tests, that a number of materials or substances can
influence the test, e.g. antibiotics and antiseptics. If this is relevant, the rationale for the decision shall be
documented.
4.4.2 In vivo genotoxicity tests
Test methods for in vivo genotoxicity tests shall be chosen from the OECD Guidelines for Testing of
Chemicals.
Preferred test methods are: OECD 474, OECD 475, OECD 478, OECD 483, OECD 484, OECD 485 and
OECD 486.
NOTE Recently, transgenic animal test systems have been developed for genotoxicity testing. These tests may prove
valuable for medical device testing, but their use has not been validated at the time of publication of this part of ISO 10993.
References on test systems are given in the bibliography for transgenic animals.
4 © ISO 2003 — All rights reserved

ISO 10993-3:2003(E)
5 Carcinogenicity tests
5.1 General
Before a decision to perform a carcinogenicity test is made, ISO 10993-1 and ISO 10993-18 shall be taken
into account. The decision to perform a test shall be justified on the basis of an assessment of the risk of
carcinogenesis arising from the use of the medical device. Carcinogenicity testing shall not be performed
when risks can be adequately assessed or managed without generating new carcinogenicity test data.
NOTE There are suitable in vitro cell transformation systems that may be used for carcinogenicity prescreening. Cell
transformation tests have so far not been described in International Standards. Additional information on cell
transformation test systems are given in Annex A.
5.2 Test strategy
5.2.1 In the absence of evidence to rule out carcinogenic risks, situations in which the need for
carcinogenicity testing shall be considered may include the following:
a) resorbable materials and medical devices for which the resorption time is greater than 30 days, unless
there are significant and adequate data on human use or exposure;
b) materials and medical devices introduced in the body and/or its cavities with a permanent or cumulative
contact of greater than 30 days, except when significant and adequate human-use history is available.
Carcinogenicity testing of genotoxic materials is not scientifically justified. For genotoxic materials, a
carcinogenic hazard shall be presumed and the risk managed accordingly.
5.2.2 When in accordance with ISO 10993-1, chronic toxicity and carcinogenicity have been con
...