SIST EN ISO 10993-3:2015

SLOVENSKI STANDARD
01-januar-2015
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SIST EN ISO 10993-3:2009
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Biological evaluation of medical devices - Part 3: Tests for genotoxicity, carcinogenicity
and reproductive toxicity (ISO 10993-3:2014)
Biologische Beurteilung von Medizinprodukten - Teil 3: Prüfungen auf Gentoxizität,
Karzinogenität und Reproduktionstoxizität (ISO 10993-3:2014)
Évaluation biologique des dispositifs médicaux - Partie 3: Essais concernant la
génotoxicité, la cancérogénicité et la toxicité sur la reproduction (ISO 10993-3:2014)
Ta slovenski standard je istoveten z: EN ISO 10993-3:2014
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
October 2014
ICS 11.100.20 Supersedes EN ISO 10993-3:2009
English Version
Biological evaluation of medical devices - Part 3: Tests for
genotoxicity, carcinogenicity and reproductive toxicity (ISO
10993-3:2014)
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 10993-3:2014) Reproduktionstoxizität (ISO 10993-3:2014)
This European Standard was approved by CEN on 6 September 2014.

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-CENELEC 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-CENELEC Management Centre has the same
status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United
Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2014 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 10993-3:2014 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 on medical devices .4
Annex ZB (informative) Relationship between this European Standard and the Essential
Requirements of EU Directive 90/385/EEC on active implantable medical devices .6

Foreword
This document (EN ISO 10993-3:2014) has been prepared by Technical Committee ISO/TC 194 "Biological
and clinical 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 April 2015, and conflicting national standards shall be withdrawn at the
latest by April 2015.
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 10993-3:2009.
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 EU Directives.
For relationship with EU 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, Croatia, Cyprus, Czech
Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece,
Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,
Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom.
Endorsement notice
The text of ISO 10993-3:2014 has been approved by CEN as EN ISO 10993-3:2014 without any modification.
Annex ZA
(informative)
Relationship between this European Standard and the Essential
Requirements of EU Directive 93/42 EEC on medical devices
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 Union 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 Directive 93/42/EEC on medical
devices
Essential Requirements (ERs) Clause(s)/sub-clause(s) of
Qualifying remarks/Notes
of Directive 93/42/EEC this EN
ER 7.1 is only partly covered by
ISO 10993-3, since the
standard does not provide
requirements on design and
manufacture. However, this
7.1 (First and second indent) 4, 5, 6 and 7
standard provides a means to
evaluate genotoxicity,
carcinogenicity or reproductive
toxicity risks associated with the
materials which are used.
ER 7.2 is not covered by
ISO 10993-3, since the
standard does not provide
requirements on design and
manufacture and does not
oblige to minimize risk.
However, this standard
7.2 4, 5, 6 and 7
provides a means to evaluate
genotoxicity, carcinogenicity or
reproductive toxicity. This
evaluation can be a preliminary
step for risk minimization. Other
forms of toxicity and
flammability are not dealt with in
this standard.
ER 7.5 is not covered by
ISO 10993-3, since the
standard does not provide
7.5 (First paragraph) 4, 5, 6 and 7 requirements on design,
manufacture and packaging
and does not oblige to minimize
risk.
However, this standard
provides a means to evaluate
genotoxicity, carcinogenicity or
reproductive toxicity. This
evaluation can be a preliminary
step for risk minimization. Other
forms of toxicity and
flammability are not dealt with in
this standard.
General Note: Presumption of conformity depends on also complying with all relevant clauses/subclauses of
ISO 10993-1.
WARNING — Other requirements and other EU Directives may be applicable to the product(s) falling within
the scope of this standard.
Annex ZB
(informative)
Relationship between this European Standard and the Essential
Requirements of EU Directive 90/385/EEC on active implantable medical
devices
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 90/385 EEC on active implantable medical devices.
Once this standard is cited in the Official Journal of the European Union 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 European Standard and Directive 90/385/EEC on active
implantable medical devices
Essential Requirements (ERs) Clause(s)/sub-clause(s) of
Qualifying remarks/Notes
of Directive 90/385/EEC this EN
ER 9 is only partly covered by
, since the
ISO 10993-3
standard does not provide
requirements on design and
manufacture. However, this
9 (First and second indent) 4, 5, 6 and 7 standard provides a means to
assess genotoxicity,
carcinogenicity or reproductive
toxicity used in the manufacture
of medical devices. Other forms
of toxicity are not covered.
General Note: Presumption of conformity depends on also complying with all relevant clauses/subclauses of
ISO 10993-1.
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
Third edition
2014-10-01
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 10993-3:2014(E)
©
ISO 2014
ISO 10993-3:2014(E)
© ISO 2014
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form
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Published in Switzerland
ii © ISO 2014 – All rights reserved

ISO 10993-3:2014(E)
Contents Page
Foreword .iv
Introduction .vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 2
4 Requirements for test strategies . 2
4.1 General . 2
4.2 Additional requirements for carcinogenicity testing . 3
4.3 Additional requirements for reproductive toxicity testing . 3
5 Genotoxicity tests . 4
5.1 General . 4
5.2 Test strategy . 4
5.3 Sample preparation . 6
6 Carcinogenicity tests . 7
6.1 General . 7
6.2 Evaluation strategy . 7
6.3 Sample preparation . 8
6.4 Test methods . 8
7 Reproductive and developmental toxicity tests . 9
7.1 General . 9
7.2 Test strategy . 9
7.3 Sample preparation .10
7.4 Test methods .10
8 Test report .11
Annex A (informative) Guidance on selecting an appropriate sample preparation procedure in
genotoxicity testing .12
Annex B (informative) Flowchart for follow-up evaluation .20
Annex C (informative) Rationale of test systems .21
Annex D (informative) Cell transformation test systems .23
Annex E (normative) Considerations for carcinogenicity studies performed as
implantation studies .24
Annex F (informative) In vitro tests for embryo toxicity.25
Bibliography .27
ISO 10993-3:2014(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.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2. www.iso.org/directives
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. Details of any
patent rights identified during the development of the document will be in the Introduction and/or on
the ISO list of patent declarations received. www.iso.org/patents
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
The committee responsible for this document is ISO/TC 194.
This third edition of ISO 10993-3 cancels and replaces the second edition (ISO 10993-3:2003), which has
been technically revised.
The major technical changes are the following:
a) test strategy changed by inclusion of a in vivo test and a follow-up evaluation;
b) new Annex A ” Guidance on selecting an appropriate sample preparation procedure in genotoxicity
testing” included;
c) Inclusion of further in vitro and in vivo test for evaluating the genotoxic potential of medical devices;
d) new Annex B “Flowchart for follow-up evaluation” included;
e) Annex E changed to “Considerations for carcinogenicity studies performed as implantation studies”
and made normative;
f) new Annex F “In vitro tests for embryo toxicity” included.
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
iv © ISO 2014 – All rights reserved

ISO 10993-3:2014(E)
— 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
— 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]
The following part is under preparation:
— Part 33: Supplement to ISO 10993-3:— Guidance on tests to evaluate genotoxicity [Technical Report]
The following definitions apply in understanding how to implement an ISO International Standard and
other normative ISO deliverables (TS, PAS, IWA):
— “shall” indicates a requirement;
— “should” indicates a recommendation;
— “may” is used to indicate that something is permitted;
— “can” is used to indicate that something is possible, for example, that an organization or individual
is able to do something.
ISO/IEC Directives, Part 2 (sixth edition, 2011), 3.3.1, defines a requirement as an “expression in the
content of a document conveying criteria to be fulfilled if compliance with the document is to be claimed
and from which no deviation is permitted.”
ISO/IEC Directives, Part 2 (sixth edition, 2011), 3.3.2, defines a recommendation as an “expression in the
content of a document conveying that among several possibilities one is recommended as particularly
suitable, without mentioning or excluding others, or that a certain course of action is preferred but not
necessarily required, or that (in the negative form) a certain possibility or course of action is deprecated
but not prohibited.”
ISO 10993-3:2014(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 minimised 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 with 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 on-going scientific and
medical advances will improve our understanding of and approaches to these important toxicological
effects. At the time this document 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 managing toxicological risks associated with exposure to medical
device materials but, where it is appropriate, it is important that maximum test sensitivity is achieved.
In view 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 have to be performed on
a case-by-case basis.
vi © ISO 2014 – All rights reserved

INTERNATIONAL STANDARD ISO 10993-3:2014(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 risk estimation, selection of hazard identification tests
and risk management, with respect to the possibility of the following potentially irreversible biological
effects arising as a result of exposure to medical devices:
— genotoxicity;
— carcinogenicity;
— reproductive and developmental toxicity.
This part of ISO 10993 is applicable when the need to evaluate a medical device for potential genotoxicity,
carcinogenicity, or reproductive toxicity has been established.
NOTE Guidance on selection of tests is provided in ISO 10993-1.
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.
ISO 10993-1, Biological evaluation of medical devices — Part 1: Evaluation and testing within a risk
management process
ISO 10993-2, Biological evaluation of medical devices — Part 2: Animal welfare requirements
ISO 10993-6, Biological evaluation of medical devices — Part 6: Tests for local effects after implantation
ISO 10993-12, 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
OECD 414, Prenatal Development Toxicity Study
OECD 415, One-Generation Reproduction Toxicity Study
OECD 416, Two-generation Reproduction Toxicity
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
ISO 10993-3:2014(E)
OECD 476, In vitro Mammalian Cell Gene Mutation Test
OECD 487, In Vitro Mammalian Cell Micronucleus 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 carcinogenic potential of medical devices, materials, and/or extracts using multiple
exposures for a major portion of the life span of the test animal
3.2
energy-depositing medical device
device intended to exert its therapeutic or diagnostic effect by the delivery of electromagnetic radiation,
ionising radiation or ultrasound
Note 1 to entry: 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, fungi or whole animals to determine
whether gene mutations, changes in chromosome structure, or other DNA or gene changes are caused
by the test samples
3.4
maximum tolerated dose
MTD
maximum dose that a test animal can tolerate without any adverse 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
3.6
test sample preparation
residual, extractables, leachables or biodegradable device materials that are resuspended in a vehicle
compatible with the test system
4 Requirements for test strategies
4.1 General
ISO 10993-1 indicates circumstances where the potential for genotoxicity, carcinogenicity and
reproductive toxicity is a relevant hazard for consideration in an overall biological safety evaluation.
Testing to investigate these hazards shall be justified on the basis of a risk assessment. In determining if
genotoxicity, carcinogenicity and reproductive toxicity testing of the device is warranted an assessment
of risk shall address the following factors
— an analysis of the chemical constituents of the device material(s), including manufacturing process
residues and degradation products or metabolites, to identify causes of concern on the basis of
structure-activity relationships or previous demonstration of relevant toxicity in the chemical
class,
— the mechanistic basis of the toxic response under consideration, if available,
2 © ISO 2014 – All rights reserved

ISO 10993-3:2014(E)
— existing information relevant to the genotoxicity, carcinogenicity and reproductive toxicity
evaluation of the medical device,
— the extent of previous use of comparable materials in relevant applications,
— consideration of residuals from the final finished device with respect to how well they are
characterized and their potential biological activity (e.g. structure-activity relationships, or
previous demonstration of relevant outcomes).
— exposure route,
— patient population,
— extent and duration of localized (at the site of implantation or use) and systemic exposure,
— the anticipated impact of test results (or lack of testing) on risk management judgements, and
— changes in the type or amount of residuals that the patient will be exposed to, either through an
increase in device exposure, or an increase in devices size when compared to an equivalent device.
Commonly used risk assessment tools (e.g. TTC) may be helpful in evaluating these factors.
Where an analysis of the composition of device materials reveals the presence of chemical constituents
that are of concern but for which inadequate toxicity data are available, consideration shall be given to
testing individual chemical. Individual chemicals shall be tested in preference to compounded materials
or extracts, where this would improve the risk estimate. Where testing of a device material is indicated
testing shall be conducted on the final product (including sterilization if applicable), or representatives
from the final products, or materials processed in the same manner as the final product (including
sterilization if applicable). The decision to test, and the nature of the test sample, shall be justified and
documented.
Testing may be warranted for additional states of the device such as, wear debris generated from
the device or materials that cure in situ (e.g. cements, adhesives and pre-polymer mixtures) unless
toxicological risk assessment determines no cause for concern from additional device/material states.
For guidance on in situ curing devices see ISO 10993-12.
4.2 Additional requirements for carcinogenicity testing
For carcinogenicity testing, in addition to 4.1, the following factors shall be addressed:
— physical characteristics (e.g. particle size and shape, pore size, surface continuity, surface condition,
device thickness);
— results from genotoxicity, implantation and other studies.
4.3 Additional requirements for reproductive toxicity testing
For reproductive testing, in addition to 4.1, the total direct or indirect cumulative contact duration with
reproductive tissue, the embryo/foetus or the germ cells shall be addressed.
Any information from published literature on the effect of device materials on male/female reproductive
organs or from subacute/chronic study on the histopathology of reproductive system should also form
the basis before a full scale reproductive toxicity testing is performed.
ISO 10993-3:2014(E)
5 Genotoxicity tests
5.1 General
Before a decision to perform a genotoxicity test is made, ISO 10993-1 shall be taken into account. The
rationale for a test programme, taking into consideration all relevant factors given in 4.1 to 4.3, shall be
justified and documented.
Genotoxicity tests are designed to detect the two major classes of genetic damage:
— Gene mutations (point mutations);
— Chromosomal damage [structural aberrations such as translocations, small or large deletions and
insertions, and numerical chromosomal aberrations (aneuploidy)].
5.2 Test strategy
5.2.1 General
No single test is capable of detecting all relevant genotoxic agents. Therefore, the usual approach is to
conduct a battery of in vitro and under certain circumstances also in vivo tests.
Bacterial reverse mutation assays have been shown to detect relevant genetic changes produced by
the majority of genotoxic carcinogens detected by rodent assays. Certain classes of genotoxin, e.g. alkyl
halides, are not detected.
The potential of test materials to produce DNA damage in bacterial systems might not be relevant to
their likely effects in eukaryotic cells, and therefore, testing in mammalian cell test systems shall be
performed unless otherwise justified. Several mammalian cell systems are in use: systems that detect
gross chromosomal damage (in vitro tests for structural and numerical chromosomal aberrations),
systems that detect primarily gene mutations (HPRT mutation assay), and a system that detects gene
mutations and clastogenic effects [mouse lymphoma thymidine kinase (tk) assay with both colony
number and size determination]. In vitro tests for chromosomal damage and the mouse lymphoma tk
assay yield results that are equivalent. Results from both tests have a relatively high level of congruence
for compounds that are regarded as genotoxic but yield negative results in the bacterial reverse
mutation assay. Therefore, the chromosome aberration test and the mouse lymphoma tk assay are
currently considered equally acceptable when either is used with the bacterial reverse mutation assay
in a standard battery for genotoxicity testing.
5.2.2 Test battery
When genotoxicity testing is performed, the test battery shall include
a) a test for gene mutations in bacteria (OECD 471), modified for medical devices to allow, for example,
testing with extracts from devices, see ISO/TR 10993-33:—, Clause 6, and either
b) an in vitro test with cytogenetic evaluation of chromosomal damage with mammalian cells
(OECD 473), modified for medical devices, see ISO/TR 10993-33:—, Clause 7, or
c) an in vitro mouse lymphoma tk assay (OECD 476), modified for medical devices, including detection
of small (slow growing) and large colonies, see ISO/TR 10993-33:—, Clause 9, or
d) an in vitro mammalian cell micronucleus test for chromosomal damage and aneugenicity (OECD
487), modified for medical devices, see ISO/TR 10993-33:—, Clause 8.
When additional relevant factors (such as genotoxic mechanism and pharmacokinetics) that can
influence the genotoxic activity of a compound, need to be considered an in vivo test may be performed
if justified. An in vivo test for chromosomal damage in rodent haematopoietic cells could be either an
analysis of chromosomal aberrations in bone marrow cells or an analysis of micronuclei in bone marrow
4 © ISO 2014 – All rights reserved

ISO 10993-3:2014(E)
or peripheral blood erythrocytes [see ISO/TR 10993-33:—, Clause 10 (OECD 474) or ISO/TR 10993-
33:—, Clause 11 (OECD 475)].
Where applicable, the in vivo test for chromosomal damage in rodent haemapoietic cells shall be
performed using two extracts (see ISO 10993-12 or Annex A). The preferred application route of polar
vehicles is intravenously. The preferred application route for the non-polar vehicles is intraperitoneally.
An in vivo assay is not necessary, if the user can demonstrate that the quantities of extractables from
the test article are less than the amount of material that would induce a positive response with a potent
well-characterized in vivo micronucleus genotoxin.
An example is cisplatin (CAS no. 15663-27-1), which was shown a positive response at 0,3 mg/kg, see
[35]
Reference.
5.2.3 Follow-up evaluation
If genotoxicity testing is performed in accordance with 5.2.2 and if the results of the two in vitro tests
are negative, further genotoxicity testing in animals is unnecessary.
If any test is positive, the following step-wise procedure is applicable (see also Annex B).
Step 1: Identification of confounding factors in results from the initial set of genotoxicity tests, if
available.
a) Identification of confounding factors (e.g. non-physiological conditions, interaction of test article
with culture medium, auto-oxidation and cytotoxicity).
b) Identification of metabolic effects (e.g. nature of the exogenous metabolic system, nature of the
metabolic profile, unique metabolites).
c) Identification of impurities by chemical characterization (i.e. materials ingredient research or
analytical testing).
Step 2: Weight of evidence (WOE) assessment with mechanism and mode of action (MOA) to be
considered.
a) Direct DNA reactive versus non direct DNA reactive mode of action.
b) Aneuploidy and polyploidy issues. Is an aneuploidy mechanism involved?
Step 3: Decision point.
Determine whether the extract from the medical device or chemical of concern is a genotoxin and if,
a) the interpretation of results and WOE/MOA analysis within a toxicological risk assessment
framework present a low/negligible concern for patients under the expected usage, or
b) the interpretation of results and WOE/MOA analysis within a toxicological risk assessment
framework suggest there may be potential risks for patients under expected usage.
If the determination is a) no further additional tests or evaluation are needed.
If the decision is b), then continue to step 4.
Step 4: Perform risk management.
Either manage risks assuming a genotoxic hazard or select the appropriate in vitro and/or in vivo follow-
up testing.
Step 5: Select and run additional in vitro and/or in vivo test.
Any in vivo test shall be chosen on the basis of the most appropriate end point identified by the in vitro
tests.
ISO 10993-3:2014(E)
in vivo tests commonly used are
— micronucleus test in rodents (OECD 474),
— metaphase analysis in rodent bone marrow (OECD 475),
— transgenic mutagenicity tests (OECD 488).
The decision as to the most appropriate test system shall be justified and documented.
NOTE Recently, a draft OECD guideline for the testing of chemicals on rodent alkaline single cell gel
electrophoresis (Comet) assay is under development for genotoxicity testing. This test might prove valuable for
medical device testing, but at the time of publication of this International Standard the OECD Guideline was not
1)
published.
An attempt shall be made to demonstrate that the test substance has reached the target organ. For
micronucleus test in rodents or metaphase analysis in rodent bone marrow, the bioavailability can be
proved by one of the following approaches
— analytical quantification of specific extract compounds in the blood or serum,
— test extract induced cytotoxicity to the bone marrow cells,
— intravenous route of exposure (for polar vehicles).
If the target organ exposure cannot be demonstrated, a second in vivo test in another target organ shall
be performed to verify the lack of in vivo genotoxicity.
Step 6: Reinterpret all of the accumulated data and determine if the test article is genotoxic.
In some cases, positive in vitro tests may not be relevant. The following should be considered in
determining the overall relevance of the in vitro results. This list is not exhaustive but is given as an aid
for decision making process.
a) Only one of the original two in vitro tests performed had a positive result.
b) Further in vitro investigation using similar mechanistic end points do not confirm the positive
result.
c) Mechanistic information indicates that positive in vitro results are not relevant to in vivo situations
(e.g. high cytotoxicity, osmolality, etc).
d) In vivo testing including evidence that the test sample reached the target organ did not demonstrate
a genotoxic effect.
The overall WOE and interpretation of the entire data set shall be documented with the final conclusion.
In some cases, site-specific or genetic end point specific tests might be necessary. In most cases, these
tests do not have internationally recognized protocols.
5.3 Sample preparation
Unless the sample can be dissolved in a solvent compatible with the test system, appropriate extraction
solvents shall be chosen on the basis of its ability to maximize extraction of the material or medical
device to a level at which the concentration of genotoxic residues would be sufficient to produce a positive
response in the test system, but without degradation of the device or the test sample. The test system
vehicle(s) shall be chosen on the basis of its compatibility with the genotoxicity test system. Tests shall
be performed on solutions, suspensions (e.g. Method A in Annex A), extracts (e.g. Method C in Annex A)
or exaggerated extracts (e.g. Method B in Annex A) of the finished device (including sterilization if
applicable), device material, device component or the individual chemicals of the device.

1) OECD Draft-Guideline for the testing of chemicals – In vivo Mammalian Alkaline Comet Assay, available at:
http://www.oecd.org/
6 © ISO 2014 – All rights reserved

ISO 10993-3:2014(E)
Device materials should include all final formulation and processing, unless otherwise justified. It is
generally not appropriate to conduct testing on raw materials, as formulation and processing could
change the potential for toxicity of the final device.
The rationale for choosing to test individual chemicals shall be justified and documented. The rationale
shall include considerations of interactions and synergistic effects.
Where relevant, the test material should be extracted with the two solvents (see ISO 10993-12 or
Annex A).
Any decision to omit testing with one class of solvent shall be justified and documented.
6 Carcinogenicity tests
6.1 General
Before a decision to perform a carcinogenicity test is made, ISO 10993-1 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.
These tests may be designed to examine simultaneously in a single study
...