SIST EN ISO 10993-6:2007

SLOVENSKI STANDARD
SIST EN ISO 10993-6:2007
01-september-2007
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SIST EN 30993-6:2000
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Biological evaluation of medical devices - Part 6: Tests for local effects after implantation
(ISO 10993-6:2007)
Biologische Beurteilung von Medizinprodukten - Teil 6: Prüfungen auf lokale Effekte nach
Implantationen (ISO 10993-6:2007)
Évaluation biologique des dispositifs médicaux - Partie 6: Essais concernant les effets
locaux apres implantation (ISO 10993-6:2007)
Ta slovenski standard je istoveten z: EN ISO 10993-6:2007
ICS:
11.100.20 %LRORãNRRYUHGQRWHQMH Biological evaluation of
PHGLFLQVNLKSULSRPRþNRY medical devices
SIST EN ISO 10993-6:2007 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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EUROPEAN STANDARD
EN ISO 10993-6
NORME EUROPÉENNE
EUROPÄISCHE NORM
April 2007
ICS 11.100.20 Supersedes EN 30993-6:1994
English Version
Biological evaluation of medical devices - Part 6: Tests for local
effects after implantation (ISO 10993-6:2007)
Évaluation biologique des dispositifs médicaux - Partie 6: Biologische Beurteilung von Medizinprodukten - Teil 6:
Essais concernant les effets locaux après implantation Prüfungen auf lokale Effekte nach Implantationen (ISO
(ISO 10993-6:2007) 10993-6:2007)
This European Standard was approved by CEN on 13 March 2007.
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: rue de Stassart, 36  B-1050 Brussels
© 2007 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 10993-6:2007: E
worldwide for CEN national Members.

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EN ISO 10993-6:2007 (E)







Foreword


This document (EN ISO 10993-6:2007) has been prepared by Technical Committee ISO/TC 194
"Biological evaluation of medical devices" in collaboration with Technical Committee CEN/TC 206
"Biocompatibility of medical and dental materials and 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 October 2007, and conflicting national standards shall
be withdrawn at the latest by October 2007.

This document supersedes EN 30993-6:1994.

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 Directive(s).

For relationship with EU Directive(s), 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 United Kingdom.


Endorsement notice

The text of ISO 10993-6:2007 has been approved by CEN as EN ISO 10993-6:2007 without any
modifications.

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EN ISO 10993-6:2007 (E)


Annex ZA

(informative)

Clauses of this International Standard addressing essential requirements
or other provisions of EU Directives 90/385/EEC for Active Implantable
Medical Devices and 93/42/EEC for Medical Devices


This International Standard 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 90/385/EEC for Active Implantable Medical Devices and 93/42/EEC for 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 confers, within the limits of the scope of this standard, a
presumption of conformity with the corresponding Essential Requirements of the Directives and
associated EFTA regulations.


Table ZA 1— Correspondence between this International Standard and Directives 90/385/EEC
and 93/42/EEC

Clause(s)/Sub-clause(s) of Essential requirements (ERs) of Qualifying remarks/Notes
this International Standard Directive 90/385/EEC and 93/42/EEC
4,5,6 & Annexes B, C, D 9 of Annex I of 90/385/EEC The test methods do not include
pass/fail criteria
7.1 of Annex I of 93/42/EEC

WARNING: Other requirements and other EU Directives may be applicable to the product(s) falling
within the scope of this International Standard.




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INTERNATIONAL ISO
STANDARD 10993-6
Second edition
2007-04-15

Biological evaluation of medical
devices —
Part 6:
Tests for local effects after implantation
Évaluation biologique des dispositifs médicaux —
Partie 6: Essais concernant les effets locaux après implantation




Reference number
ISO 10993-6:2007(E)
©
ISO 2007

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ISO 10993-6:2007(E)
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ii © ISO 2007 – All rights reserved

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ISO 10993-6:2007(E)
Contents Page
Foreword. iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions. 2
4 Common provisions for implantation test methods . 2
4.1 General. 2
4.2 Preparation of specimens for implantation .2
5 Test methods, general aspects . 3
5.1 Tissue and implantation site . 3
5.2 Animals . 3
5.3 Test periods. 4
5.4 Surgery and testing conditions. 5
5.5 Evaluation. 6
6 Test report . 8
Annex A (informative) General considerations regarding implantation periods and tissue
responses to degradable/resorbable materials. 9
Annex B (normative) Test methods for implantation in subcutaneous tissue . 10
Annex C (normative) Test method for implantation in muscle. 12
Annex D (normative) Test method for implantation in bone . 14
Annex E (informative) Examples of evaluation of local biological effects after implantation. 17
Bibliography . 19

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ISO 10993-6:2007(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 10993-6 was prepared by Technical Committee ISO/TC 194, Biological evaluation of medical devices.
This second edition cancels and replaces the first edition (ISO 10993-6:1994) 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 system
⎯ 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 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
⎯ Part 15: Identification and quantification of degradation products from metals and alloys
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ISO 10993-6:2007(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
⎯ Part 20: Principles and methods for immunotoxicology testing of medical devices
For the purposes of this part of ISO 10993 the CEN annex regarding fulfilment of European Council Directives
will be removed at publication stage.
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INTERNATIONAL STANDARD ISO 10993-6:2007(E)

Biological evaluation of medical devices —
Part 6:
Tests for local effects after implantation
1 Scope
This part of ISO 10993 specifies test methods for the assessment of the local effects after implantation of
biomaterials intended for use in medical devices.
This part of ISO 10993 applies to materials that are:
⎯ solid and non-biodegradable;
⎯ degradable and/or resorbable;
⎯ non-solid, such as porous materials, liquids, pastes and particulates.
The test specimen is implanted into a site and animal species appropriate for the evaluation of the biological
safety of the material. These implantation tests are not intended to evaluate or determine the performance of
the test specimen in terms of mechanical or functional loading. This part of ISO 10993 may also be applied to
medical devices that are intended to be used topically in clinical indications where the surface or lining may
have been breached, in order to evaluate local tissue responses.
The local effects are evaluated by a comparison of the tissue response caused by a test specimen to that
caused by control materials used in medical devices of which the clinical acceptability and biocompatibility
characteristics have been established. The objective of the test methods is to characterize the history and
evolution of the tissue response after implantation of a medical device/biomaterial including final integration or
resorption/degradation of the material. In particular for degradable/resorbable materials the degradation
characteristics of the material and the resulting tissue response should be determined.
This part of ISO 10993 does not deal with systemic toxicity, carcinogenicity, teratogenicity or mutagenicity.
However, the long-term implantation studies intended for evaluation of local biological effects may provide
insight into some of these properties. Systemic toxicity studies conducted by implantation may satisfy the
requirements of this part of ISO 10993.
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 10993-1:2003, Biological evaluation of medical devices — Part 1: Evaluation and testing within a risk
management system
ISO 10993-2, Biological evaluation of medical devices — Part 2: Animal welfare requirements
ISO 10993-11, Biological evaluation of medical devices — Part 11: Tests for systemic toxicity
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ISO 10993-6:2007(E)
ISO 10993-12, Biological evaluation of medical devices — Part 12: Sample preparation and reference
materials
ISO 10993-16, Biological evaluation of medical devices — Part 16: Toxicokinetic study design for degradation
products and leachables
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 10993-1, ISO 10993-2,
ISO 10993-12, ISO 10993-16 and the following apply.
3.1
degradation
decomposition of a material
[ISO 10993-9:1999, definition 3.1]
3.2
degradation product
product of a material which is generated by the chemical breakdown or decomposition of the material
[ISO 10993-16:1997, definition 3.1]
3.3
biomaterial
material intended to interface with biological systems to evaluate, treat, augment or replace any tissue, organ
or function of the body
[Taken from European Society Biomaterials Conference II]
4 Common provisions for implantation test methods
4.1 General
It is important that the study be planned in sufficient detail such that all relevant information can be extracted
from the use of each animal and each study (see ISO 10993-2, ISO 10993-11 and ISO 10993-16).
All animal studies shall be performed in a facility approved by a nationally recognised organization and in
accordance with all appropriate regulations dealing with laboratory animal welfare. These studies shall be
performed under good laboratory practices or other recognized quality assurance systems, and comply with
the requirements of ISO 10993-2.
The provisions of this clause shall apply to the test methods described in Annexes B, C and D.
4.2 Preparation of specimens for implantation
Test sample and reference or control material preparation shall be in compliance with ISO 10993-12. The
implant size and shape shall be documented and justified. Test specimens for various implant sites are
described in Annexes B, C and D. Physical characteristics (such as form, density, hardness, surface) can
influence the character of the tissue response to the test material and shall be recorded and taken into
account when the response is characterized.
Each implant shall be manufactured, processed, cleaned of contaminants and sterilized by the method
intended for the final product and this shall be confirmed in the study documentation. After final preparation
and sterilization, the implant specimens shall be handled aseptically and in such a way as to ensure that they
are not damaged or contaminated in any way prior to or during implantation.
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ISO 10993-6:2007(E)
For materials used as scaffolds for tissue-engineered medical products, it may be appropriate not to use the
final preparation pre-populated with cells, as the immune reaction of the animal to the cellular components of
such products and the reaction of the cells to the animal, may interfere with the resulting local tissue response.
For composite materials (e.g. bone cements, dental materials), the components may be mixed before use and
allowed to set before implantation. However, materials that are designed for use in devices with in situ
polymerization shall be introduced in a manner such that in situ polymerization occurs. For certain types of
study other procedures may be used. The procedure used shall be documented and justified.
Non-solid materials (including powders) may be contained in open-ended cylindrical tubes for the purpose of
testing for local effects after implantation (see ISO 10993-12 for the selection of materials for tubes). Prepare
the test material according to the manufacturer’s instructions and insert the material into the tube until level
with the end, taking care not to contaminate the outer surface of the tube with the test material; if
contamination occurs the sample shall not be implanted. Avoid entrapment of air in the tube and ensure that
the end surfaces of the inserted material in the tube and the tube ends are smooth.
NOTE 1 Polyethylene (PE), polypropylene (PP) or polytetrafluoroethylene (PTFE) tubes are commonly used for this
purpose. PE tubes may be deformed by autoclaving. PTFE tubes are difficult to section in the microtome, and substitution
by PE or PP tubes of the same dimensions may be preferable when the tubes are to remain in the tissue blocks during
sectioning.
Evaluation shall be undertaken by comparing to the tissue reaction to that of a similar specimen/material of
which the clinical acceptability and biocompatibility characteristics have been established.
NOTE 2 For further guidance, see ISO 10993-12.
The physical characteristics such as shape, and especially the surface condition of the control(s), shall be as
similar to that of the implant test specimens as is practically possible, with any deviations being explained and
justified. When the test material is contained in a tube, the control shall be of the same material as the tube
and have the same diameter as the outer diameter of the tube. The choice of the control rod or tube shall be
documented and justified.
5 Test methods, general aspects
5.1 Tissue and implantation site
The test sample shall be implanted into the tissues most relevant to the intended clinical use of the material.
The justification for the choice of sample numbers, tissue and implantation sites shall be documented. Test
methods for various implantation sites are given in Annexes B, C and D. If other implantation sites are chosen,
the general scientific principles behind the test methods described in Annexes B, C and D shall still be
adhered to and the justification provided.
NOTE 1 For special dental usage test, see ISO 7405.
For degradable/resorbable materials, the implantation site shall be marked in a manner suitable for
identification of the site at the end of the designated time periods. The use of a non-invasive permanent skin
marker and/or a template marking the placement of the specimen is recommended. In certain circumstances
an appropriate negative control may be used as a marker for the location of the implant site. Exceptionally, a
sham surgical procedure might be used to evaluate the impact of the procedure on the tissue involved; in
these cases the specific justification shall be provided.
NOTE 2 Markers for identification of the implant site of resorbable test specimens may be non-absorbable sutures or
skin paints.
5.2 Animals
All aspects of animal care and accommodation shall be in accordance with ISO 10993-2. In general, small
laboratory animals such as mice, rats, hamsters or rabbits are preferred.
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ISO 10993-6:2007(E)
The use of larger animals may be justified based upon special scientific considerations of the particular
biomaterial under study.
Select an animal species in line with the principles set out in ISO 10993-2, giving due consideration to the size
of the implant test specimens, the number of implants per animal, the intended duration of the test in relation
to the expected lifespan of the animals, as well as potential species' differences regarding biological response
(see Annex B).
For short-term testing, animals such as rodents or rabbits are commonly used. For long-term testing, animals
such as rodents, rabbits, dogs, sheep, goats, pigs and other animals with a relatively long life expectancy are
suitable.
Before starting an animal study with degradable materials, relevant information from in vitro degradation
studies should be considered. For biodegradable materials a pilot study in rodents should be undertaken to
determine the expected rate of degradation before embarking on studies on larger animals.
The specimens of test and control materials shall be implanted under the same conditions in animals of the
same species and of the same age, sex and strain in corresponding anatomical sites. The number and size of
implants inserted in an animal depends on the size of the species and the anatomical location. Whenever
possible, the reference control and the test specimens should be implanted into the same animal.
However, when the local effects after implantation are investigated as part of a systemic toxicity study by
implantation, control and test samples should not be placed in the same animal.
5.3 Test periods
The test period shall be determined by the likely clinical exposure time or be continued until or beyond a
steady state has been reached with respect to the biological response. The time points selected shall be
explained and justified.
For non-degradable and non-resorbable materials the short-term responses are normally assessed from
1 week up to 4 weeks and the long-term responses in tests exceeding 12 weeks. The local biological
response to implanted materials depends both on the properties of the materials and on the response to the
associated trauma of surgery. The tissue configuration in the vicinity of an implant changes with the time
elapsed after surgery. During the first two weeks after implantation the reaction due to the surgical procedure
itself may be difficult to distinguish from the tissue reaction evoked by the implant. In muscle and connective
tissue, depending on the species, and the severity of the surgical trauma, a steady state is seen in the cell
population after 9 weeks to 12 weeks. Implantation in bone tissue may need longer observation periods before
a steady state is reached. In general, it is expected that experiments that go up to or beyond the point of
absorption are needed for the evaluation of degradable materials.
For degradable/resorbable materials the test period shall be related to the estimated degradation time of the
test product. Annex A gives general considerations regarding degradable/absorbable materials. Before
starting with animal studies and determining the time points for sample evaluation, an estimation of the
degradation time shall be made. This can be done in vitro by real-time or accelerated degradation studies or in
certain circumstances by mathematical modelling. In general, experiments that extend up to or beyond the
point of absorption are needed for the evaluation of degradable materials. The evaluation of degradable
materials will depend in part on the degradation rate of the materials.
Local tissue responses shall be evaluated relative to the degradation process of the implant at various time
points:
⎯ where there is no or minimal degradation, usually to be evaluated at 1 week to 12 weeks after
implantation;
⎯ when degradation is taking place;
⎯ when a steady state has been reached resulting in tissue restoration or degradation nearing completion.
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ISO 10993-6:2007(E)
In the absence of complete degradation, absorption, or restoration to normal tissue structure and function, the
overall data collected may be sufficient to allow characterization of the local effects after implantation.
NOTE In vivo degradation may need a rather long period of time, sometimes more than one year. Additional animals
may be beneficial to extend the observation period when the implant has not been degraded completely within the
expected investigational time period.
Although this part of ISO 10993 does not address the issues of systemic toxicity as given in ISO 10993-11, it
is recommended that the information required to meet this part of ISO 10993 be obtained from any systemic
toxicity studies using implantation.
For long-term implantation studies, generally accepted observation periods are given in Table 1.
Animals should be killed at each time point, in line with ISO 10993-2. Serial harvest under general
anaesthesia with recovery may be acceptable under special circumstances, which shall be documented and
justified.
Table 1 — Selection of test periods for long-term implantation
Species Implantation period in weeks
a
12 26 52 78
(104)
Rats X X X
Guinea pigs X X X
Rabbits X X X X X
Dogs X X X X X
Sheep X X X X X
Goats X X X X X
Pigs X X X X X
a
Depending on the intended use of the test material, not all implantation
periods may be necessary (see ISO 10993-12). An observation period of 104
weeks may be of interest in selected instances.
5.4 Surgery and testing conditions
Surgery shall be performed under general anaesthesia. If another type of anaesthesia is used, this shall be
justified and shall be in compliance with ISO 10993-2. The specific insertion or implantation procedures for
subcutaneous, intramuscular or bone implantation are described in Annexes B, C and D, respectively.
The number of implants per
...