SIST EN ISO 10993-6:2017

Biological evaluation of medical devices - Part 6: Tests for local effects after implantation (ISO 10993-6:2016)

ISO 10993-6:2016 specifies test methods for the assessment of the local effects after implantation of biomaterials intended for use in medical devices.
ISO 10993-6:2016 applies to materials that are
-      solid and non-absorbable,
-      non-solid, such as porous materials, liquids, gels, pastes, and particulates, and
-      degradable and/or absorbable, which may be solid or non-solid.
The test sample 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 sample in terms of mechanical or functional loading. This part of ISO 10993 can also be applied to medical devices that are intended to be used topically in clinical indications where the surface or lining might 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 sample to that caused by control materials used in medical devices whose 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 absorption/degradation of the material. In particular for degradable/absorbable materials, the degradation characteristics of the material and the resulting tissue response should be determined.
ISO 10993-6:2016 does not deal with systemic toxicity, carcinogenicity, teratogenicity or mutagenicity. However, the long-term implantation studies intended for evaluation of local biological effects might provide insight into some of these properties. Systemic toxicity studies conducted by implantation might satisfy the requirements of this part of ISO 10993. When conducting combined studies for evaluating local effects and systemic effects, the requirements of both standards is to be fulfilled.

Biologische Beurteilung von Medizinprodukten - Teil 6: Prüfungen auf lokale Effekte nach Implantationen (ISO 10993-6:2016)

Évaluation biologique des dispositifs médicaux - Partie 6: Essais concernant les effets locaux après implantation (ISO 10993-6:2016)

ISO 10993-6:2016 spécifie les méthodes d'essai pour l'évaluation des effets locaux après une implantation de biomatériaux destinés à être utilisés dans des dispositifs médicaux.
ISO 10993-6:2016 s'applique aux matériaux qui sont
-      solides et non absorbables,
-      non solides, comme les matériaux poreux, liquides, en gel, pâteux et particulaires, et
-      dégradables et/ou absorbables, qui peuvent être solides ou non solides.
L'échantillon d'essai est implanté dans un site et dans une espèce animale appropriés à l'évaluation de la sécurité biologique du matériau. Ces essais d'implantation ne sont pas destinés à évaluer ou à déterminer les performances de l'échantillon d'essai en matière de charge mécanique ou fonctionnelle. La présente partie de l'ISO 10993 peut également être appliquée à des dispositifs médicaux destinés à une utilisation topique dans des indications cliniques où la surface ou le revêtement pourrait perdre son intégrité, afin d'évaluer les réponses tissulaires locales.
Les effets locaux sont évalués par comparaison de la réponse tissulaire induite par un échantillon d'essai avec celle induite par des matériaux de contrôle utilisés dans des dispositifs médicaux dont les caractéristiques d'acceptabilité clinique et de biocompatibilité ont été établies. L'objectif des méthodes d'essai est de caractériser l'historique et l'évolution de la réponse tissulaire après l'implantation d'un dispositif médical ou biomatériau, y compris l'intégration finale ou l'absorption/dégradation du matériau. Particulièrement pour les matériaux dégradables/absorbables, il convient de déterminer les caractéristiques de dégradation du matériau et la réponse tissulaire résultante.
ISO 10993-6:2016 ne traite pas de la toxicité systémique, de la cancérogénicité, de la tératogénicité ou de la mutagénicité. Les études d'implantation à long terme destinées à l'évaluation des effets biologiques locaux pourraient, cependant, apporter des informations sur certaines de ces propriétés. Des études de toxicité systémique effectuées par implantation pourraient répondre aux exigences de la présente partie de l'ISO 10993. Lors de la réalisation d'études combinées pour l'évaluation des effets locaux et systémiques, les exigences des deux normes appliquées doivent être satisfaites.

Biološko ovrednotenje medicinskih pripomočkov - 6. del: Preskusi, povezani z lokalnimi učinki po implantaciji (ISO 10993-6:2016)

Ta del standarda ISO 10993 določa preskusne metode za oceno lokalnih učinkov po implantaciji biomaterialov, namenjenih za uporabo v medicinskih pripomočkih.
Ta del standarda ISO 10993 se uporablja za materiale, ki:
- so trdni in niso vpojni;
- niso trdni, npr. porozni materiali, tekočine, geli, paste in delci; ter
- so razgradljivi in/ali vpojni, ki so lahko trdni ali netrdni.
Preskusni vzorec je vsajen v mesto in živalsko vrsto, primerno za ovrednotenje
biološke varnosti materiala. Ti implantacijski preskusi niso namenjeni ovrednotenju ali določanju uspešnosti preskusnih vzorcev v smislu mehanske ali funkcionalne obremenitve. Ta del standarda ISO 10993 je mogoče uporabljati tudi za medicinske pripomočke, ki so namenjeni za lokalni nanos pri kliničnih indikacijah, pri katerih je površina ali sluznica morda predrta, za ovrednotenje odzivov lokalnega tkiva.
Lokalni učinki so ovrednoteni s primerjavo odziva tkiva, ki ga povzroči preskusni vzorec, in odziva, ki ga povzroči kontrolni material, ki se uporablja v medicinskih pripomočkih, katerih lastnosti klinične sprejemljivosti in biološke združljivosti so že bile ugotovljene. Namen preskusnih metod je opredelitev zgodovine in razvoja odzivov tkiva po implantaciji medicinskega pripomočka/biomateriala, vključno s končno integracijo ali absorpcijo/degradacijo materiala. Zlasti pri razgradljivih/vpojnih materialih je treba določiti degradacijske značilnosti materiala in posledični odziv tkiva.
Ta del standarda ISO 10993 ne zajema sistemske toksičnosti, kancerogenosti, teratogenosti ali mutagenosti. Kljub temu bi dolgoročne študije o implantaciji, namenjene ovrednotenju lokalnih bioloških učinkov, lahko zagotovile vpogled v nekatere od teh lastnosti. Študije o sistemski toksičnosti, opravljene z implantacijo, morda izpolnjujejo zahteve tega dela standarda ISO 10993. Pri izvajanju kombiniranih študij za ovrednotenje lokalnih in sistemskih učinkov, morajo biti izpolnjene zahteve obeh standardov.

General Information

• Status: Published
• Public Enquiry End Date: 29-Jan-2015
• Publication Date: 09-Jan-2017
• ICS: 11.100.20 - Biological evaluation of medical devices
• Technical Committee: VAZ - Healthcare
• Current Stage: 6060 - National Implementation/Publication (Adopted Project)
• Start Date: 03-Jan-2017
• Due Date: 10-Mar-2017
• Completion Date: 10-Jan-2017
• Directive: 2007/47/EC - Directive 2007/47/EC of the European Parliament and of the council of 5 September 2007 amending Council Directive 90/385/EEC on the approximation of the laws of the Member States relating to active implantable medical devices, Council Directive 93/42/EEC concerning medical devices and Directive 98/8/EC concerning the placing of biocidal products on the market
• Directive: 90/385/EEC - Council Directive 90/385/EEC of 20 June 1990 on the approximation of the laws of the Member States relating to active implantable medical devices
• Directive: 93/42/EEC - Council Directive 93/42/EEC of 14 June 1993 concerning medical devices
• Mandate: M/BC/CEN/89/9 - Joint Standardization request to CEN/CENELEC concerning harmonized standards relating to horizontal aspects in the field of medical devices
• Ref Project: EN ISO 10993-6:2016 - Biological evaluation of medical devices - Part 6: Tests for local effects after implantation (ISO 10993-6:2016)

Relations

• Replaces: SIST EN ISO 10993-6:2009 - Biological evaluation of medical devices - Part 6: Tests for local effects after implantation (ISO 10993-6:2007)
• Effective Date: 01-Feb-2017
• Revised: kSIST FprEN ISO 10993-6:2025 - Biological evaluation of medical devices - Part 6: Tests for local effects after implantation (ISO/FDIS 10993-6:2025)
• Effective Date: 28-Jan-2023

Standards Content (Sample)

SLOVENSKI STANDARD
01-februar-2017
1DGRPHãþD
SIST EN ISO 10993-6:2009
%LRORãNRRYUHGQRWHQMHPHGLFLQVNLKSULSRPRþNRYGHO3UHVNXVLSRYH]DQL]
ORNDOQLPLXþLQNLSRLPSODQWDFLML,62
Biological evaluation of medical devices - Part 6: Tests for local effects after implantation
(ISO 10993-6:2016)
Biologische Beurteilung von Medizinprodukten - Teil 6: Prüfungen auf lokale Effekte nach
Implantationen (ISO 10993-6:2016)
Évaluation biologique des dispositifs médicaux - Partie 6: Essais concernant les effets
locaux après implantation (ISO 10993-6:2016)
Ta slovenski standard je istoveten z: EN ISO 10993-6:2016
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.

EN ISO 10993-6
EUROPEAN STANDARD
NORME EUROPÉENNE
December 2016
EUROPÄISCHE NORM
ICS 11.100.20 Supersedes EN ISO 10993-6:2009
English Version
Biological evaluation of medical devices - Part 6: Tests for
local effects after implantation (ISO 10993-6:2016)
Évaluation biologique des dispositifs médicaux - Partie Biologische Beurteilung von Medizinprodukten - Teil 6:
6: Essais concernant les effets locaux après Prüfungen auf lokale Effekte nach Implantationen (ISO
implantation (ISO 10993-6:2016) 10993-6:2016)
This European Standard was approved by CEN on 4 November 2016.

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
© 2016 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 10993-6:2016 E
worldwide for CEN national Members.

Contents Page
European foreword . 3
Annex ZA (informative)  Relationship between this European Standard and the essential
requirements of Directive 93/42/EEC [OJ L 169] aimed to be covered . 5
Annex ZB (informative)  Relationship between this European Standard and the essential
requirements of Directive 90/385/EEC [OJ L 189] aimed to be covered . 7

European foreword
This document (EN ISO 10993-6:2016) 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 and clinical evaluation of medical devices” the secretariat of which is held by
DIN.
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 June 2017, and conflicting national standards shall be
withdrawn at the latest by June 2017.
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 10993-6:2009.
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 10993-6:2016 has been approved by CEN as EN ISO 10993-6:2016 without any
modification.
The following referenced documents are indispensable for the application of this document. For
undated references, the latest edition of the referenced document (including any amendments) applies.
For dated references, only the edition cited applies. However, for any use of this standard ‘within the
meaning of Annex ZA’, the user should always check that any referenced document has not been
superseded and that its relevant contents can still be considered the generally acknowledged state-of-
art.
When an IEC or ISO standard is referred to in the ISO standard text, this shall be understood as a
normative reference to the corresponding EN standard, if available, and otherwise to the dated version
of the ISO or IEC standard, as listed below.
NOTE The way in which these referenced documents are cited in normative requirements determines the
extent (in whole or in part) to which they apply.
Table 1 — Correlations between undated normative references and dated EN and ISO standards
Normative references Equivalent dated standard
as listed in Clause 2 of
EN ISO or IEC
the ISO standard
ISO 10993-1 EN ISO 10993-1:2009 ISO 10993-1:2009
ISO 10993-2 EN ISO 10993-2:2006 ISO 10993-2:2006
ISO 10993-4 EN ISO 10993-4:2009 ISO 10993-4:2002
ISO 10993-12 EN ISO 10993-12:2012 ISO 10993-12:2012
ISO 10993-16 EN ISO 10993-16:2010 ISO 10993-16:2010

NOTE This part of EN ISO 10993 refers to ISO 10993-1 which itself refers to ISO 14971. In Europe, it should
be assumed that the reference to ISO 14971 is to EN ISO 14971:2012.
Annex ZA
(informative)
Relationship between this European Standard and the essential
requirements of Directive 93/42/EEC [OJ L 169] aimed to be covered
This European Standard has been prepared under a Commission’s joint standardization request
M/BC/CEN/89/9 concerning harmonized standards relating to horizontal aspects in the field of medical
devices to provide one voluntary means of conforming to essential requirements of Council Directive
93/42/EEC of 14 June 1993 concerning medical devices [OJ L 169].
Once this standard is cited in the Official Journal of the European Union under that Directive,
compliance with the normative 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.
NOTE 1 Where a reference from a clause of this standard to the risk management process is made, the risk
management process needs to be in compliance with Directive 93/42/EEC as amended by 2007/47/EC. This
means that risks have to be reduced ‘as far as possible’, ‘to a minimum’, ‘to the lowest possible level’, ‘minimized’
or ‘removed’, according to the wording of the corresponding essential requirement.
NOTE 2 The manufacturer’s policy for determining acceptable risk must be in compliance with Essential
Requirements 1, 2, 5, 6, 7, 8, 9, 11 and 12 of the Directive.
NOTE 3 This Annex ZA is based on normative references according to the table of references in the European
foreword, replacing the references in the core text.
NOTE 4 When an Essential Requirement does not appear in Table ZA.1, it means that it is not addressed by this
European Standard.
Table ZA.1 — Correspondence between this European Standard and Annex I of Directive
93/42/EEC [OJ L 169]
Essential Requirements of Clause(s)/sub-clause(s) of Remarks/Notes
Directive 93/42/EEC this EN
This part of ISO 10993 specifies
test methods for the assessment
4, 5, 6 and Annex A, Annex B,
of the local effects after
7.1 (First and second indent)
Annex C and Annex D
implantation of biomaterials
intended for use in medical
devices.
These implantation tests are not
intended to evaluate or
4, 5, 6 and Annex A, Annex B,
determine the performance of
7.2
Annex C and Annex D
the test sample in terms of
mechanical or functional
loading.
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
4, 5, 6 and Annex A, Annex B,
implantation may satisfy the
7.5 (First paragraph)
Annex C and Annex D
requirements of this part of
ISO 10993. When conducting
combined studies for evaluating
local effects and systemic effects,
the requirements of this part of
ISO 10993 and ISO 10993-11
shall be fulfilled.
For ER 7.1 (first and second
indent), flammability is not
covered
General Note: Presumption of conformity depends on also complying with all relevant
clauses/subclauses of ISO 10993-1.
WARNING 1 — Presumption of conformity stays valid only as long as a reference to this European
Standard is maintained in the list published in the Official Journal of the European Union. Users of this
standard should consult frequently the latest list published in the Official Journal of the European
Union.
WARNING 2 — Other Union legislation may be applicable to the products falling within the scope of this
standard.
Annex ZB
(informative)
Relationship between this European Standard and the essential
requirements of Directive 90/385/EEC [OJ L 189] aimed to be covered
This European Standard has been prepared under a Commission’s joint standardization request
M/BC/CEN/89/9 concerning harmonized standards relating to horizontal aspects in the field of medical
devices to provide one voluntary means of conforming to essential requirements of Council Directive
90/385/EEC of 20 June 1990 on the approximation of the laws of the Member States relating to active
implantable medical devices [OJ L 189].
Once this standard is cited in the Official Journal of the European Union under that Directive,
compliance with the normative 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.
NOTE 1 Where a reference from a clause of this standard to the risk management process is made, the risk
management process needs to be in compliance with Directive 90/385/EEC as amended by 2007/47/EC. This
means that risks have to be reduced ‘as far as possible’, ‘to a minimum’, ‘to the lowest possible level’, ‘minimized’
or ‘removed’, according to the wording of the corresponding essential requirement.
NOTE 2 The manufacturer’s policy for determining acceptable risk must be in compliance with Essential
Requirements 1, 4, 5, 8, 9 and 10 of the Directive.
NOTE 3 This Annex ZB is based on normative references according to the table of references in the European
foreword, replacing the references in the core text.
NOTE 4 When an Essential Requirement does not appear in Table ZB.1, it means that it is not addressed by this
European Standard.
Table ZB.1 — Correspondence between this European Standard and Annex I of Directive
90/385/EEC [OJ L 189]
Essential Requirements of Clause(s)/sub-clause(s) Remarks/Notes
Directive 90/385/EEC of this EN
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.
These implantation tests are not
intended to evaluate or determine
the performance of the test sample
in terms of mechanical or
functional loading.
This part of ISO 10993 does not
deal with systemic toxicity,
4, 5, 6 and Annex A,
carcinogenicity, teratogenicity or
9 (only first and second
Annex B, Annex C and
mutagenicity. However, the long-
indent)
Annex D
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. When conducting
combined studies for evaluating
local effects and systemic effects,
the requirements of this part of
ISO 10993 and ISO 10993-11 shall
be fulfilled.
General Note: Presumption of conformity depends on also complying with all relevant
clauses/subclauses of ISO 10993-1.
WARNING 1 — Presumption of conformity stays valid only as long as a reference to this European
Standard is maintained in the list published in the Official Journal of the European Union. Users of this
standard should consult frequently the latest list published in the Official Journal of the European
Union.
WARNING 2 — Other Union legislation may be applicable to the products falling within the scope of this
standard.
INTERNATIONAL ISO
STANDARD 10993-6
Third edition
2016-12-01
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 10993-6:2016(E)
©
ISO 2016
ISO 10993-6:2016(E)
© ISO 2016, Published in Switzerland
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form
or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior
written permission. Permission can be requested from either ISO at the address below or ISO’s member body in the country of
the requester.
ISO copyright office
Ch. de Blandonnet 8 • CP 401
CH-1214 Vernier, Geneva, Switzerland
Tel. [phone]
Fax [phone]
[e-mail]
www.iso.org
ii © ISO 2016 – All rights reserved

ISO 10993-6:2016(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
5 Test methods, general aspects. 4
6 Test report . 9
6.1 General . 9
6.2 Test laboratory . 9
6.3 Implant samples . 9
6.4 Animals and implantation . 9
6.5 Retrieval and histological procedure .10
6.6 Macroscopic and microscopic evaluation .10
6.7 Final evaluation .10
Annex A (normative) Test methods for implantation in subcutaneous tissue .11
Annex B (normative) Test method for implantation in muscle .13
Annex C (normative) Test method for implantation in bone .15
Annex D (normative) Test method for implantation in brain tissue .18
Annex E (informative) Examples of evaluation of local biological effects after implantation .23
Bibliography .27
ISO 10993-6:2016(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 (see 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 (see 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.
For an explanation on the meaning of ISO specific terms and expressions related to conformity
assessment, as well as information about ISO’s adherence to the WTO principles in the Technical
Barriers to Trade (TBT) see the following URL: Foreword - Supplementary information
The committee responsible for this document is ISO/TC 194, Biological and clinical evaluation of medical
devices.
This third edition cancels and replaces the second edition (ISO 10993-6:2007), which has been
technically revised with the following changes:
a) addition of guidance on biological evaluation of absorbable medical devices;
b) new Annex D.
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 6: Tests for local effects after implantation
— Part 7: Ethylene oxide sterilization residuals
— Part 9: Framework for identification and quantification of potential degradation products
— Part 10: Tests for irritation and skin sensitization
— Part 11: Tests for systemic toxicity
— Part 12: Sample preparation and reference materials
— Part 13: Identification and quantification of degradation products from polymeric medical devices
— Part 14: Identification and quantification of degradation products from ceramics
iv © ISO 2016 – All rights reserved

ISO 10993-6:2016(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
— 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]
— Part 33: Guidance on tests to evaluate genotoxicity — Supplement to ISO 10993-3 [Technical Report]
The following parts are under preparation:
— Part 5: Tests for in vitro cytotoxicity
INTERNATIONAL STANDARD ISO 10993-6:2016(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-absorbable,
— non-solid, such as porous materials, liquids, gels, pastes, and particulates, and
— degradable and/or absorbable, which may be solid or non-solid.
The test sample 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 sample in terms of mechanical or functional loading. This part of ISO 10993 can
also be applied to medical devices that are intended to be used topically in clinical indications where
the surface or lining might 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 sample to that
caused by control materials used in medical devices whose 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 absorption/degradation of the material. In particular for degradable/absorbable
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 might provide insight into some of these properties. Systemic toxicity studies conducted by
implantation might satisfy the requirements of this part of ISO 10993. When conducting combined
studies for evaluating local effects and systemic effects, the requirements of both standards is to be
fulfilled.
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 10993-4, Biological evaluation of medical devices — Part 4: Selection of tests for interactions with blood
ISO 10993-12, Biological evaluation of medical devices — Part 12: Sample preparation and reference
materials
ISO 10993-6:2016(E)
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
absorb/absorption
action of a non-endogenous (foreign) material or substance, or its decomposition products passing
through or being assimilated by cells and/or tissue over time
3.2
degradation
decomposition of a material
[SOURCE: ISO 10993-9:2009, 3.1]
3.3
degradation product
any intermediate or final by-product which results from the physical, metabolic, and/or chemical
decomposition of a material or substance
[SOURCE: ISO/TR 37137:2014, 2.2, modified]
3.4
degrade
to physically, metabolically, and/or chemically decompose a material or substance
[SOURCE: ISO/TR 37137:2014, 2.3]
3.5
biomaterial
material or substance intended to interface with biological systems to evaluate, treat, augment or
replace any tissue, organ or function of the body.
[SOURCE: 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 recognized organization and
in accordance with all appropriate regulations dealing with laboratory animal welfare to comply with
the requirements of ISO 10993-2. These studies shall be performed under good laboratory practices or
other recognized, quality assurance systems.
The provisions of this Clause shall apply to the test methods specified in Annex A, Annex B, Annex C,
and Annex D.
4.2 Preparation of samples for implantation
4.2.1 Test sample and reference or control material preparation shall be in accordance with ISO 10993-
12. The implant size and shape shall be documented and justified. Test samples for various implant sites
are described in Annex A, Annex B, Annex C, and Annex D. Physical characteristics (such as form, density,
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ISO 10993-6:2016(E)
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. Control articles should be matched
as closely as reasonably possible for physical characteristics.
4.2.2 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 samples 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.
4.2.3 For materials used as scaffolds for tissue-engineered medical products, it may be appropriate
not to use the final preparation pre-populated with cells and/or proteins, as the immune reaction of the
animal to the cellular/protein components of such products and the reaction of the cells to the animal
may interfere with the resulting local tissue response, making it difficult to interpret.
4.2.4 For composite materials (e.g. bone cements, dental materials), the components may be mixed
before use and allowed to set before implantation. For multicomponent materials designed to be cured
prior to placement, the components may be mixed before use and allowed to set before implantation.
However, materials that are designed to polymerize in situ (e.g. bone cements, many dental materials)
shall be introduced in a manner such that in situ polymerization occurs. The procedure used shall be
documented and justified
4.2.5 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.
Polyethylene (PE), polypropylene (PP), or polytetrafluoroethylene (PTFE) tubes are commonly used
for this purpose. PE tubes can be deformed by autoclaving.
4.2.6 Evaluation shall be performed by comparing the tissue reaction to that of a similar
sample/material whose clinical acceptability and biocompatibility characteristics have been established.
NOTE For further guidance, see ISO 10993-12.
4.2.7 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 samples as is practical, 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.
4.2.8 For implantation studies, the amount or size of the test and control article shall be documented.
4.3 Study design
For devices comprising/composed of two or more different materials, the test articles should be of
similar composition or multiple implants may be needed, e.g. if a device is made of HDPE and titanium
then the test article should be made of HDPE and titanium.
ISO 10993-6:2016(E)
5 Test methods, general aspects
5.1 Tissue and implantation site
5.1.1 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 Annex A, Annex B, Annex C, and
Annex D. If other implantation sites are chosen, the general scientific principles behind the test methods
described in Annex A, Annex B, Annex C, and Annex D shall still be adhered to and the justification
provided.
NOTE For some devices, there are vertical standards prescribing specific implant studies to evaluate local
[47] [12]
tissue responses, e.g. intraocular lens implant and dental usage tests . These studies can be used to satisfy
the requirements in ISO 10993-6.
5.1.2 For absorbable 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 sample is recommended for short-term
study intervals only. In most circumstances, a location marker comprised of an appropriate non-
absorbable negative control (e.g. HDPE 1 mm by 2 mm by 5 mm, PP suture, gold band, clips) may be used
to identify the location of the implant site. These location markers can be removed without inducing
artefacts to the test article-tissue interface prior to histology processing.
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.
5.2 Animals
5.2.1 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.
5.2.2 The use of larger animals may be justified based upon special scientific considerations of the
particular biomaterial under study, or if needed to accommodate implant size, with whole device testing.
5.2.3 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 samples, 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.
5.2.4 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.
5.2.5 Before starting an animal study with degradable materials, relevant information from in vitro
degradation studies should be considered. For absorbable materials, a pilot study in rodents may be
considered to determine the expected rate of degradation before embarking on studies on larger animals.
5.2.6 The samples 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 into an animal depends on the size of the species and the
anatomical location. Whenever possible, the reference control and the test samples should be implanted
into the same animal.
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ISO 10993-6:2016(E)
5.2.7 However, when a neuroimplantation study (see Annex D) is conducted or when the local effects
after implantation are investigated as part of a systemic toxicity study by implantation, control and test
samples shall not be placed in the same animal.
5.3 Test periods
5.3.1 The test period shall be determined by the likely clinical exposure time or be continued until or
beyond when a steady-state with respect to the biological response has been reached. The time points
selected shall be explained and justified.
5.3.2 For non-absorbable 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.
5.3.3 For absorbable materials, the test period shall be related to the estimated degradation time of
the test product at a clinically relevant implantation site. When determining the time points for sample
evaluation, an estimation of the degradation time shall be made. This can be accomplished in vitro by
real-time or accelerated degradation studies or in certain circumstances by mathematical modelling. In
general, study duration should extend up to or beyond the point of complete absorption. The evaluation
period for absorbable materials will depend in part on the degradation rate of the materials. Study
intervals should span a significant portion of the degradation time frame for the implant, and shall
include, as a minimum, the following time points:
a) early time frame (where there is no or minimal degradation) — For absorbable materials, usually a
study interval of between 1 week and 2 weeks post-implantation should be used to assess the early
tissue response.
b) mid time frame (when degradation is taking place) — Subsequent study intervals for absorbable
devices should be guided by the degradation profile of the specific absorbable material. The target
interval should allow assessment of histological response when the tissue response is expected
to be most pronounced (e.g. substantial structural disruption and/or fragmentation of the device
is most likely to occur). Implants with longer-term degradation profiles may require multiple
assessment time points, with intervals targeted in accordance with the expected pattern of
degradation.
When a device with multiple materials with differing absorption rates is implanted, implant
intervals reflecting the degradation profile of those components should be included.
c) late time frame (when the implant is essentially absorbed) — This interval is targeted to observe
when minimal amounts of the absorbable component remain at the implant site.
Gross and microscopic evaluation after complete implant absorption is highly desirable. However,
in the absence of complete absorption, the overall data collected should be sufficient to allow
characterization of the local effects after implantation if:
— the affected tissue’s response, structure, and function have achieved an acceptable steady-state
condition, and
— the absorbable material and/or its degradation products are in a state of limited visually-
identifiable presence.
ISO 10993-6:2016(E)
NOTE In vivo degradation can occur over a long period of time, sometimes more than one year.
Additional animals to extend the observation period (intervals “to-be-determined” group) can be beneficial
if the implant has not been completely absorbed within the expected investigational time period and cannot
be observed microscopically.
In those situations when the material is not fully absorbed within the late time frame, an appropriate
scientific justification can be included for ending the study and the estimated percentage (%) of
remaining absorbable material should be reported.
Long term studies that span a significant portion of the degradation time frame for the implant are
recommended. Implantation of in vitro pre-degraded material (for instance, up to 50 % weight loss
or 50 % loss of mechanical strength) may be considered on a case-by-case basis in order to more
rapidly observe late stage events after implantation. However, these studies do not replace studies
that characterize the real-time in vivo degradation profile of the absorbable device.
5.3.4 Characterization of an absorbable device’s degradation process may not be applicable to the
evaluation of the local effects of the same absorbable material when used in combination: with a drug as
carrier for drug release, a scaffold for tissue-engineered medical products, or a surface coating for non-
absorbable implants. Since combinations of devices with drugs and/or cells can introduce new issues,
the appropriate regulatory authorities should be consulted regarding study designs for absorbable
combination products.
5.3.5 Although this part of ISO 10993 does not address the issues of systemic toxicity 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.
5.3.6 For long-term studies, generally accepted observation periods for non-absorbable biomaterials
are given in Table 1. Animals should be humanely sacrificed 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 — Possible test periods for long-term implantation of biomaterials
a
Implantation period in weeks
Species
13 26 52 78 104
Mice X X X — —
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
These implantation periods are commonly used; however, other periods may be applicable
based on the specific characteristics of the test material. Depending on the intended use of the
test material, not all implantation periods may be necessary.
5.4 Surgery and testing conditions
5.4.1 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, bone or neural implantation are described in Annex A,
Annex B, Annex C, and Annex D, respectively.
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ISO 10993-6:2016(E)
5.4.2 The number of implants per animal and the number of animals per observation period are
described in Annex A, Annex B, Annex C, and Annex D. A sufficient number of test and control samples
shall be implanted to ensure that the final number of samples to be evaluated will give valid results.
5.4.3 The surgical technique may profoundly influence the result of any implantation procedure.
Surgery shall be carried out under aseptic conditions and in a manner that minimizes trauma at the
implant site. Remove the hair from the surgical area by clipping, shaving, or other mechanical means.
Disinfect the exposed area of skin with an appropriate antiseptic. Ensure that the implants or wound
surfaces do not come in contact with the hair. After surgery close the wound using either sutures or
wound cli
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