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ISO
STANDARD
10993-3
First edition
1992-12-15
Biological evaluation of medical devices -
Part 3:
Tests for genotoxicity, carcinogenicity and
reproductive toxicity
haha tion biologique des dispositifs medicaux -
Partie 3: Essais concernant Ia ghotoxicit6, Ia canc&og&icitk et Ia toxicit6
sur Ia reproduction
Reference number
ISO 10993-3:1992(E)
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ISO 10993=3:1992(E)
Contents
Page
1
Scope .
............................. 1
Normative references .
2
Definitions . .*.
2
.....................................................................
Genotoxicity tests
................................................................. 3
Carcinogenicity tests
4
........................................................
Reproductive toxicity tests
Annex
5
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A Bibliography
0 ISO 1992
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International Organization for Standardization
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Printed in Switzerland
ii
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ISO 10993=3:1992(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. Esch 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.
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.
International Standard ISO 10993-3 was prepared by Technical Committee
ISO/TC 194, Biological evalua tion of medical devices.
ISO 10993 consists of the following Parts, under the general title Biological
evaluation of medical devices:
- Part 1: Guidance on selection of tests
- 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 cytotoxicity: in vitro methods
- Part 6: Tests for local effects after implantation
- Part 7: Ethylene Oxide sterilization residuals
- Part 8: Clinical investigation
- Part 9: Degradation of ma terials rela ted to biological testing
- Part YO: Tests for irrita tion and sensitization
- Part 11: Tests for s ys temic toxicity
- Part 12: Sample preparation and reference materials
Future Parts will deal with other relevant aspects of biological testing.
Annex A of this part of ISO 10993 is for information only.
.‘ .
Ill
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ISO 10993=3:1992(E)
Introduction
The basis for biocompatibility evaluation of medical devices is often em-
pirical and driven by the relevant concerns for human safety. Not all test
methods for the assessment of genotoxicity, carcinogenicity or repro-
ductive toxicity are equally weil developed, nor is their validity weil estab-
lished for the testing of medical devices.
Significant issues in test Sample size and preparation, scientific under-
standing of disease processes and test Validation tan be cited as limi-
tations of available methods. For example the biological significance of
solid state carcinogenesis is poorly understood. lt is expected that ongoing
scientific and medical advances will alter our understanding and ap-
proaches to these important toxicity test methods. At the time the docu-
ment was prepared, the test methods proposed were those most
acceptable. Sound scientific alternatives to the proposed testing should
be acceptable insofar as they address relevant matters of safety assess-
ment.
In the selection of tests needed to evaluate a particular device, there is
no Substitute for a careful assessment of expected human uses and po-
tential interactions of the 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 therefore maximum test sensitivity is required. The
interpretation of findings and implications for human health effects are
beyond the scope of this part of ISO 10993. Because of the multitude of
possible outcomes and the importante of such factors as extent of expo-
Sure, species differentes and mechanical or physical considerations, risk
assessment has to be performed on a case-by-case basis.
IV
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INTERNATIONAL STANDARD
ISO 10993=3:1992(E)
Biological evaluation of medical devices -
Part 3:
Tests for genotoxicity, carcinogenicity and reproductive
toxicity
of this International Standard. At the time of publica-
1 Scope
tion, the editions indicated were valid. All Standards
are subject to revision, and Parties to agreements
This part of ISO 10993 specifies tests for the follow-
based on this International Standard are encouraged
ing biological aspects:
to investigate the possibility of applying the most re-
cent editions of the Standards indicated below.
- genotoxicity,
Members of IEC and ISO maintain registers of cur-
rently valid International Standards.
- carcinogenicity, and
ISO 10993-1: 1992, Biological evaluation of medical
- reproductive and developmental toxicity.
devices - Part 1: Guidance on selection of tests.
These are relevant in the biological evaluation of some
ISO 10993-2: -l) Biological evaluation of medical de-
categories of medical devices (see note 1). Guidance
vices - Part 2: Animal welfare requirements.
on selection of tests is provided in ISO 10993-1.
Where the need for the evaluation of the potential for
OECD Guidelines for testing of chemicals - Se-
genotoxicity, carcinogenicity or reproductive toxicity
lected assays
has been identified, they should be evaluated in ac-
cordante with this part of ISO 10993.
- ln vitro genotoxicity tests
Most tests included in this part of the International
471 Genetic Toxicology: Salmonella typhimurium,
Standard refer to the OECD guidelines for testing of
Reverse Mutation Assay.
chemicals. Reference to these tests is made by the
term “OECD guideline(s)” followed by the appropriate
472 Genetic Toxicology: Escheric iia coli, Reverse
test number(s).
Mutation Assay.
At the time of testing, these tests are to be per-
473 Genetic Toxicology: In v tro Mammalian
formed according to current OECD guidelines.
Cytogene tic Test.
NOTE 1 The term “devices” corresponds to the defi- 476 Genetic Toxicology: In vitro Mammalian Cell
nition given in ISO 10993-1 and covers materials, as weil as
Gene Mutation Test.
dental materials and devices. The definition is in accordance
with the CEN Standard document. 479 Genetic Toxicology: I n vitro Sis ter Chroma tid
Exchange Assay in Mammalian Cells.
480
Genetic Toxicology: Saccharomyces cerevisiae,
2 Normative references
Gene Mutation Assay.
481 Genetic Toxicology: Saccharomyces cerevisiae,
The following Standards contain provisions which,
Mito tic Recombina tion Assa y.
through reference in this text, constitute provisions
1) To be published.
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ISO 10993=3:1992(E)
DNA Darnage and
482 Genetic Toxicology: 3.3 reproductive and developmental toxicity
ßepair/ Unscheduled DNA Synthesis in tests: Tests to evaluate the potential effects of de-
Mammalian Cells I n vitro. vices, materials, and/or extracts on reproductive
function, embryonie development (teratogenicity), and
- In vivo genotoxicity tests
prenatal and early postnatal development.
474 Genetic Toxicology: Micronucleus Test.
3.4 maximum implantable dose (MID): Maximum
amount of implant material (dose) that a test animal
475 Genetic Toxicology: In vivo Mammalian Bone
tan tolerate without any adverse physical or mechan-
Marrow Cytogenetic Test - Chromosomal
ical effects.
Analysis.
478 Gene tic Toxicology: Roden t Dominant Le thal NOTE 4 To avoid unnecessary morbidity in animals on a
long-term test, preliminary testing may be necessary.
Test.
483 Gene tic Toxicology: Mammalian Germ-Cell
3.5 energy-depositing device: Device intended to
Cytogenetic Assay.
exert its therapeutic or diagnostic effect by the ab-
sorption of electromagnetic, ionic or ultrasonic radi-
484 Genetic Toxicology: Mouse Spot Test.
ation.
485 Genetic Toxicology: Mouse Heritabie Translo-
NOTE 5 This does not include devices which deliver
ca tion Assay.
simple electrical current, such as electrocautery devices,
Pacemakers or functional electrical stimulators.
- Carcinogenicity tests
451 Carcinogenicity Studies.
4 Genotoxicity tests
453 Combined Chronic Toxicityl Carcinogenicity
Studies.
- Tests for reproductive toxicity
4.1 General
4 14
Tera togenicity.
When the genetic toxicity of a medical device has to
be experimentally assessed, a series of in vitro tests
4 i 5 One-Genera tion Reproduction Taxicity Study.
shall be used. This series shall include at least three
assays. At least two of these should preferably use
ßules Governing Medicinal Products in the European
mammalian cells as a target. The tests should prefer-
Community. Volume 3. Guidelines on the Quality,
ably cover the three levels of genotoxic effects: DNA
Safety and Efficacy of Medicinal Products for Human
effects, gene mutations and chromosomal aber-
Use. Commission of the European Community 1989.
rations.
ISBN 92-825-9619-2.
NOTE 6 OECD tests 471 and 473 have proven useful in
3 Definitions the first instance, supported where necessary by test 476.
For the purposes of this part of ISO 10993, the defi- In vivo testing on animals shall only be carried out in
nitions given in ISO 10993-1 and the following defi- accordance with subclause 4.1 of ISO 10993-2.
nitions apply.
Medical devices shall be tested for genotoxicity as
specified in ISO 10993-1 :1992, except those made
genotoxicity test: Test that applies mammalian
3.1
only from materials known to show no genotoxicity,
or non-mammalian cells, bacteria, yeasts or fungi to
when, moreover, all major components of extracts
determine whether gene mutations, changes in chro-
tan be identified by suitable analytical methods and
mosome structure, or other DNA or gene changes are
have been shown to have no genetic toxicity (see also
caused by the test materials, devices and/or extracts
table 1 of ISO 10993-1:1992).
from materials.
NOTE 2 Tests on whole animals may also address these
endpoints. 4.2 Sample preparation
3.2 carcinogenicity test: Test to determine the Any material or device shall be in its “ready-to-use”
tumorigenic potential of devices, materials, and/or form (i.e. as a final product) Prior to any extraction or
extracts to either a Single or multiple exposures over test procedure. Tests shall be performed either on
a period of the total life-span of the test animal. extracts or the dissolved material using appropriate
media.
NOTE 3 These tests may be designed to examine both
chronic toxicity and tumorigenicity in a Single experimental Where mean ingful, two app ropriate extractant s shall
study. of which is a p hysiological mediu m, the
be used, one
2
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ISO 10993=3:1992(E)
second a solvent such as dimethylsulfoxide (DMSO), Situations suggesting the need for carcinogenicity
testing may include the following:
which is reasonably compatible with the test System.
WARNING - DMSO is known to be cytotoxic in resorbable materials and devices, unless there are
a)
selected assay Systems at greater than 5 g/l con- significant and adequate data on human use or
centrations of aqueous solvent. exposure;
materials and devices where positive results have
The highest reasonably possible surface area per vol- b)
been obtained in genetic toxicity testing on
ume of extractant (expressed in Square centimetres
mammalian cells;
per millilitre) shall be used.
Materials and devices which are cured in situ shall be
materials and devices introduced in the body
Cl
tested in the cured as well as in the non-cured state.
and/or its cavities with a permanent or cumulative
contact of 30 days or longer, except when signifi-
Extraction shall be performed in closed Containers
cant and adequate human-use history is available.
with minimum headspace.
In those cases where carcinogenicity testing is re-
To ensure comparability of results, the extraction
quired but no effects have occurred in genotoxicity
temperature should preferably be 37 OC and the ex-
tests, clinical testing may be performed concurrently
traction time at least 24 h.
with carcinogenicity testing.
When biphasic release characteristics are to be ex-
Where implantation does not represent the most ap-
pected, this shall be taken into account.
propriate route of exposure, scientifically justified al-
ternatives should be considered.
NOTE 7 A general guideline on Sample preparation is
under way (ISO 10993-12; see page iii) and may amend or
partly Substitute this section on Sample preparation.
5.2 Sample preparation
Whenever possible the d evice shall be tested in its
4.3 Test methsds
“ready-to-use” form. Ott Terwise a suitably formed
implant shall be made of the test material, with ap-
propriate consideration of potential solid state
4.3.1 ln vitro genotoxicity
carcinogenicity (Oppenhei mer effect, see annex A.3,
c15-J).
Test methods shall normally be Chosen from the
OECD Guidelines for testing of chemicals: 471, 472,
NOTE 10 A general guideline on Sample preparation is
473, 476, 479, 480, 481 and 482.
under way (
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