SIST EN ISO 10993-12:2009

Biological evaluation of medical devices - Part 12: Sample preparation and reference materials (ISO 10993-12:2007)

This part of ISO 10993 specifies requirements and gives guidance on the procedures to be followed in the preparation of samples and the selection of reference materials for medical device testing in biological systems in accordance with one or more parts of the ISO 10993 series. Specifically this part of ISO 10993 addresses: - test sample selection; - selection of representative portions from a device; - test sample preparation; - experimental controls; - selection of and requirements for reference materials; - preparation of extracts.

Biologische Beurteilung von Medizinprodukten - Teil 12: Proben-vorbereitung und Referenzmaterialien (ISO 10993-12:2007)

Der vorliegende Teil der ISO 10993 legt Anforderungen fest und gibt Anleitungen zu Verfahren, die bei der Probenvorbereitung und bei der Auswahl von Referenzmaterialien für die biologische Prüfung von Medizinprodukten nach einem oder mehreren Teilen der ISO 10993-Reihe zu befolgen sind.
Dieser Teil der ISO 10993 bezieht sich insbesondere auf die:
-   Auswahl der Prüfmuster,
-   Auswahl repräsentativer Teile eines Medizinproduktes,
-   Probenvorbereitung,
-   Kontrollen der Prüfmethoden,
-   Auswahl und Anforderungen an die Referenzmaterialien und
-   Herstellung der Extrakte.
Dieser Teil der ISO 10993 ist nicht anwendbar auf Materialien oder Produkte, die lebende Zellen enthalten.

Évaluation biologique des dispositifs médicaux - Partie 12 : Préparation des échantillons et matériaux de référence (ISO 10993-12:2007)

L'ISO 10993-12:2007 spécifie les exigences et fournit des directives relatives aux modes opératoires à suivre pour la préparation des échantillons et le choix des matériaux de référence dans le cadre d'essais relatifs aux dispositifs médicaux dans des systèmes biologiques, conformément à une ou plusieurs parties de l'ISO 10993. L'ISO 10993‑12:2007 traite spécifiquement les points suivants:
le choix des échantillons;
le choix des parties représentatives d'un dispositif;
la préparation des échantillons;
les contrôles expérimentaux;
le choix des matériaux de référence et les exigences qui s'y rapportent;
la préparation des extraits.
L'ISO 10993-12:2007 n'est pas applicable aux matériaux ou aux dispositifs contenant des cellules vivantes.

Biološko ovrednotenje medicinskih pripomočkov - 12. del: Priprava vzorcev in referenčni materiali (ISO 10993-12:2007)

General Information

• Status: Withdrawn
• Public Enquiry End Date: 09-Mar-2009
• Publication Date: 31-May-2009
• Withdrawal Date: 19-Sep-2012
• ICS: 11.100.20 - Biological evaluation of medical devices
• Technical Committee: VAZ - Healthcare
• Current Stage: 9900 - Withdrawal (Adopted Project)
• Start Date: 14-Sep-2012
• Due Date: 07-Oct-2012
• Completion Date: 20-Sep-2012
• 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 10993-12:2009 - Biological evaluation of medical devices - Part 12: Sample preparation and reference materials (ISO 10993-12:2007)

Relations

• Replaces: SIST EN ISO 10993-12:2008 - Biological evaluation of medical devices - Part 12: Sample preparation and reference materials (ISO 10993-12:2007)
• Effective Date: 01-Jul-2009
• Replaced By: SIST EN ISO 10993-12:2012 - Biological evaluation of medical devices - Part 12: Sample preparation and reference materials (ISO 10993-12:2012)
• Effective Date: 01-Oct-2012
• Replaced By: SIST EN ISO 10993-12:2012 - Biological evaluation of medical devices - Part 12: Sample preparation and reference materials (ISO 10993-12:2012)
• Effective Date: 08-Jun-2022

Standards Content (Sample)

SLOVENSKI STANDARD
SIST EN ISO 10993-12:2009
01-julij-2009
1DGRPHãþD
SIST EN ISO 10993-12:2008
%LRORãNRRYUHGQRWHQMHPHGLFLQVNLKSULSRPRþNRYGHO3ULSUDYDY]RUFHYLQ
UHIHUHQþQLPDWHULDOL,62
Biological evaluation of medical devices - Part 12: Sample preparation and reference
materials (ISO 10993-12:2007)
Biologische Beurteilung von Medizinprodukten - Teil 12: Proben-vorbereitung und
Referenzmaterialien (ISO 10993-12:2007)
Évaluation biologique des dispositifs médicaux - Partie 12 : Préparation des échantillons
et matériaux de référence (ISO 10993-12:2007)
Ta slovenski standard je istoveten z: EN ISO 10993-12:2009
ICS:
11.100.20 %LRORãNRRYUHGQRWHQMH Biological evaluation of
PHGLFLQVNLKSULSRPRþNRY medical devices
SIST EN ISO 10993-12:2009 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 10993-12:2009

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SIST EN ISO 10993-12:2009
EUROPEAN STANDARD
EN ISO 10993-12
NORME EUROPÉENNE
EUROPÄISCHE NORM
April 2009
ICS 11.100.20 Supersedes EN ISO 10993-12:2007
English Version
Biological evaluation of medical devices - Part 12: Sample
preparation and reference materials (ISO 10993-12:2007)
Évaluation biologique des dispositifs médicaux - Partie 12 : Biologische Beurteilung von Medizinprodukten - Teil 12:
Préparation des échantillons et matériaux de référence Proben-vorbereitung und Referenzmaterialien (ISO 10993-
(ISO 10993-12:2007) 12:2007)
This European Standard was approved by CEN on 12 April 2009.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European
Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national
standards may be obtained on application to the CEN Management Centre or to any CEN member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by translation
under the responsibility of a CEN member into its own language and notified to the CEN Management Centre has the same status as the
official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland,
France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,
Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2009 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 10993-12:2009: E
worldwide for CEN national Members.

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SIST EN ISO 10993-12:2009
EN ISO 10993-12:2009 (E)
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 .5

2

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SIST EN ISO 10993-12:2009
EN ISO 10993-12:2009 (E)
Foreword
The text of ISO 10993-12:2007 has been prepared by Technical Committee ISO/TC 194 “Biological evaluation
of medical devices” of the International Organization for Standardization (ISO) and has been taken over as EN
ISO 10993-12:2009 by Technical Committee CEN/TC 206 “Biological evaluation of medical devices” the
secretariat of which is held by NEN.
This European Standard shall be given the status of a national standard, either by publication of an identical
text or by endorsement, at the latest by October 2009, and conflicting national standards shall be withdrawn at
the latest by March 2010.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights.
This document supersedes EN ISO 10993-12:2007.
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 93/42/EEC on
Medical Devices and 90/385/EEC on Active Implantable Medical Devices.
For relationship with EU Directives, see informative Annexes ZA and ZB, which is an integral part of this
document.
According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following
countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Cyprus, Czech
Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia,
Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain,
Sweden, Switzerland and the United Kingdom.
Endorsement notice
The text of ISO 10993-12:2007 has been approved by CEN as a EN ISO 10993-12:2009 without any
modification.
3

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SIST EN ISO 10993-12:2009
EN ISO 10993-12:2009 (E)
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 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 that Directive and associated EFTA regulations.
Table ZA — Correspondence between this European Standard and Directive 93/42/EEC on medical
devices
Clause(s)/sub-clause(s) of this Essential Requirements (ERs) of Qualifying remarks/Notes
EN Directive 93/42/EEC
4, 5, 6, 7, 8, 9, 10, 11
Annex I:
7.1, 7.2, 7.5


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













4

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SIST EN ISO 10993-12:2009
EN ISO 10993-12:2009 (E)
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 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 ZB 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 — Correspondence between this European Standard and Directive 90/385/EEC on active
implantable medical devices
Clause(s)/sub-clause(s) of this Essential Requirements (ERs) of Qualifying remarks/Notes
EN Directive 90/385/EEC
4, 5, 6, 7, 8, 9, 10, 11
Annex I :
9

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



5

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SIST EN ISO 10993-12:2009

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SIST EN ISO 10993-12:2009

INTERNATIONAL ISO
STANDARD 10993-12
Third edition
2007-11-15
Corrected version
2008-02-15


Biological evaluation of medical
devices —
Part 12:
Sample preparation and reference
materials
Évaluation biologique des dispositifs médicaux —
Partie 12: Préparation des échantillons et matériaux de référence




Reference number
ISO 10993-12:2007(E)
©
ISO 2007

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SIST EN ISO 10993-12:2009
ISO 10993-12:2007(E)
PDF disclaimer
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©  ISO 2007
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means,
electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or
ISO's member body in the country of the requester.
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Published in Switzerland

ii © ISO 2007 – All rights reserved

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SIST EN ISO 10993-12:2009
ISO 10993-12:2007(E)
Contents Page
Foreword. iv
Introduction . vi
1 Scope. 1
2 Normative references. 1
3 Terms and definitions. 1
4 General requirements. 3
5 Reference materials. 4
5.1 General. 4
5.2 Certification of RMs for biological safety testing. 4
6 Use of RMs as experimental controls. 4
7 Test sample selection. 5
8 Test sample and RM preparation . 5
9 Selection of representative portions from a device. 5
10 Preparation of extracts of samples. 6
10.1 General. 6
10.2 Containers for extraction . 6
10.3 Extraction conditions and methods. 6
10.4 Extraction conditions for hazard identification and risk estimation in the exaggerated-use
condition. 8
11 Records. 9
Annex A (informative) Experimental controls . 10
Annex B (informative) General principles on and practices of test sample preparation and sample
selection . 12
Annex C (informative) Principles of test sample extraction . 14
Bibliography . 17

© ISO 2007 – All rights reserved iii

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SIST EN ISO 10993-12:2009
ISO 10993-12:2007(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 10993-12 was prepared by Technical Committee ISO/TC 194, Biological evaluation of medical devices.
This third edition cancels and replaces the second edition (ISO 10993-12:2002), which has been technically
revised.
ISO 10993 consists of the following parts, under the general title Biological evaluation of medical devices:
⎯ Part 1: Evaluation and testing
⎯ Part 2: Animal welfare requirements
⎯ Part 3: Tests for genotoxicity, carcinogenicity and reproductive toxicity
⎯ Part 4: Selection of tests for interactions with blood
⎯ Part 5: Tests for in vitro cytotoxicity
⎯ Part 6: Tests for local effects after implantation
⎯ Part 7: Ethylene oxide sterilization residuals
⎯ Part 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
iv © ISO 2007 – All rights reserved

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SIST EN ISO 10993-12:2009
ISO 10993-12:2007(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 [TS]
⎯ Part 20: Principles and methods for immunotoxicology testing of medical devices [TS]
Future parts will deal with other relevant aspects of biological testing.
This corrected version of ISO 10993-12 contains changes to definition 3.10 on page 3 and changes to
footnote references in A.3 on page 11.
© ISO 2007 – All rights reserved v

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SIST EN ISO 10993-12:2009
ISO 10993-12:2007(E)
Introduction
This part of ISO 10993 specifies methods of sample preparation and the selection of reference materials in the
biological evaluation of medical devices.
Sample preparation methods should be appropriate for both the biological evaluation methods and the materials
being evaluated. Each biological test method requires the selection of materials, extraction solvents and
conditions.
This part of ISO 10993 is based on existing national and international specifications, regulations and standards
wherever possible. It is periodically reviewed and revised.

vi © ISO 2007 – All rights reserved

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SIST EN ISO 10993-12:2009
INTERNATIONAL STANDARD ISO 10993-12:2007(E)

Biological evaluation of medical devices —
Part 12:
Sample preparation and reference materials
1 Scope
This part of ISO 10993 specifies requirements and gives guidance on the procedures to be followed in the
preparation of samples and the selection of reference materials for medical device testing in biological
systems in accordance with one or more parts of the ISO 10993 series. Specifically this part of ISO 10993
addresses:
⎯ test sample selection;
⎯ selection of representative portions from a device;
⎯ test sample preparation;
⎯ experimental controls;
⎯ selection of and requirements for reference materials;
⎯ preparation of extracts.
This part of ISO 10993 is not applicable to materials or devices containing live cells.
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
ISO 14971, Medical devices — Application of risk management to medical devices
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
blank
extraction vehicle not containing the test material, retained in a vessel identical to that which holds the test
sample and subjected to identical conditions to which the test sample is subjected during its extraction
NOTE The purpose of the blank is to evaluate possible confounding effects due to the extraction vessel, extraction
vehicle and extraction process.
© ISO 2007 – All rights reserved 1

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SIST EN ISO 10993-12:2009
ISO 10993-12:2007(E)
3.2
certified reference material
(CRM)
reference material, accompanied by a certificate, one or more of whose property values are certified by a
procedure which establishes its traceability to an accurate realization of the unit in which the property values
are expressed, and for which each certified value is accompanied by an uncertainty at a stated level of
confidence
[ISO Guide 30, definition 2.2]
3.3
experimental control
substance with well characterized responses, which is used in a specific test system to assist in evaluating if
the test system has responded in a reproducible and appropriate manner
3.4
extract
liquid that results from extraction of the test sample or control
3.5
homogeneous
property of a material and its relationship to a biological endpoint such that it is of uniform structure or
composition to consistently render or not a specific biological response
NOTE A reference material is said to be homogeneous if the biological response to a specific test is found to lie
within the specified uncertainty limits of the test, irrespective of the batch or lot of material from which the test sample is
removed.
3.6
negative control
any well characterized material and/or substance, which, when tested by a specific procedure, demonstrates
the suitability of the procedure to yield a reproducible, appropriately negative, non-reactive or minimal
response in the test system
NOTE In practice, negative controls are reference materials but may include blanks and extraction vehicles/solvents.
3.7
positive control
any well characterized material and/or substance, which, when evaluated by a specific test method,
demonstrates the suitability of the test system to yield a reproducible, appropriately positive or reactive
response in the test system
3.8
reference material
(RM)
material with one or more property values that are sufficiently reproducible and well established to enable use
of the material or substance for the calibration of an apparatus, the assessment of a measurement method, or
for the assignment of values to materials
[ISO Guide 30, definition 2.1]
NOTE For the purpose of this part of ISO 10993, a reference material is any well characterized material or substance,
which, when tested by the procedure described, demonstrates the suitability of the procedure to yield a reproducible,
predictable response. The response may be negative or positive.
3.9
stability
〈of property values〉 ability of a material, when stored under specified conditions, to maintain a specific stated
biological response, within specified limits, for a specific period of time
NOTE Adapted from ISO Guide 30, definition 2.7.
2 © ISO 2007 – All rights reserved

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SIST EN ISO 10993-12:2009
ISO 10993-12:2007(E)
3.10
test sample
medical device, component or material (or a representative sample thereof, manufactured and processed by
equivalent methods) or an extract or portion thereof that is subjected to biological or chemical testing or
evaluation
3.11
simulated-use extraction
extraction to demonstrate compliance with the requirements of this part of ISO 10993, by evaluating leachable
material levels available to the patient or user from devices during the routine use of a device using an
extraction method that simulates product use
NOTE The burden of validation on the analytical laboratory is to demonstrate that the simulated-use extraction is
carried out under conditions that provide the greatest challenge to the intended use. Product-use simulation is carried out
assuming the device is assigned to the most stringent category probable for duration of exposure and takes into
consideration both tissue(s) exposed and temperature of exposure.
3.12
exaggerated extraction
any extraction that is intended to result in a greater amount of a chemical constituent being released as
compared to the amount generated under the simulated conditions of use
NOTE Exaggerated extraction might result in a chemical change of the material or the substances being extracted.
3.13
accelerated extraction
extraction that provides a measure of the leachable materials of the device or material using conditions that
shorten the time for leaching of the substances into the extraction vehicle but do not result in a chemical
change of the substances being extracted
NOTE Examples of accelerated extraction conditions are as follows: elevated temperature, agitation, changing of the
extraction vehicle, etc.
3.14
exhaustive extraction
extraction until the amount of leachable material in a subsequent extraction is less than 10 % of that detected
in the initial extraction, or until there is no analytically significant increase in the cumulative leachable material
levels detected
NOTE As it is not possible to demonstrate the exhaustive nature of residual recovery, the definition of exhaustive
extraction adopted is as above. See also Annex C.
4 General requirements
4.1 As described in ISO 14971, in the identification of hazard and risk estimation for medical devices,
hazards that arise from changes in the manufacturing process, or insufficient control of the manufacturing
process, shall be considered in the design and preparation of samples for test and preparation of extracts of
those devices. Particular attention shall be given to residues, e.g., trace elements and cleaning and
disinfection agents, of those manufacturing processes.
4.2 Because ISO 10993 describes many different biological assay systems, the individual standards shall
be consulted to ascertain if these recommendations are appropriate for specific test systems.
4.3 Experimental controls shall be used in biological evaluations to validate a test procedure and/or to
compare the results between materials. Depending on the biological test, negative controls, blanks and/or
positive controls shall be used as is appropriate to the test.
NOTE The same type of control can be applicable to different tests and may allow cross-reference to other
established materials and test methods. Additional guidance on the selection of experimental controls is given in Annex A.
Use of positive controls for in-vivo testing may be affected by animal welfare regulations.
© ISO 2007 – All rights reserved 3

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SIST EN ISO 10993-12:2009
ISO 10993-12:2007(E)
5 Reference materials
5.1 General
Reference materials (RMs) are established by individual laboratories. The extent of chemical, physical and
biological characterization is determined by the individual laboratory. Commercially available articles may be
used as reference materials.
NOTE 1 See also ISO Guide 35.
Certified reference materials (CRMs) are selected for their high purity, critical characteristics, suitability for the
intended purpose and general availability. The critical chemical, physical and biological characteristics shall be
determined by collaborative testing in three or more laboratories, and made available to the investigator by the
distributor.
NOTE 2 It is desirable for users to obtain a commitment from suppliers of RMs or CRMs that these materials will be
available to the user for at least five years. A second, but less desirable, option is for the source of the RM or CRM to
publish an “open formulation” for the material, i.e., publication of the source materials and details of the processing needed
to ensure uniform batches of the RM.
5.2 Certification of RMs for biological safety testing
5.2.1 Qualification of an RM is a procedure that establishes the numerical or qualitative value of the
biological response of the material under specified test conditions, ensuring reproducibility of the response
within and/or between laboratories. The range of biological responses associated with the material shall be
established through laboratory tests.
NOTE See also ISO Guide 34.
5.2.2 Suppliers of RMs shall certify the materials. The supplier determines the extent of chemical and
physical characterization that is performed. The individual laboratories that use the RMs shall identify the
biological characterization necessary to qualify an RM for a specific test or procedure. Commercially available
materials may be used as RMs provided they are certified and qualified.
5.2.3 Certification of an RM is a procedure that establishes the numerical or qualitative value of the
biological response of the material under the specified test conditions. This process serves to validate the
testing of the material for that particular response and results in the issuance of a certificate. The biological
response of the material shall be established through interlaboratory tests.
6 Use of RMs as experimental controls
6.1 RMs or CRMs shall be used in biological tests as control materials to demonstrate the suitability of a
procedure to yield a reproducible response, such as either positive and/or negative. Any material used in this
way shall be characterized with each biological test procedure for which the use of the material is desired. A
material characterized and then certified for one reference test method or response, e.g., delayed-type
hypersensitivity, shall not be used as an RM for another, e.g., cytotoxicity, without additional validation.
NOTE Use of an RM will facilitate the comparability of the response between laboratories and assist in assessing
reproducibility of test performance within individu
...

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Biologische Beurteilung von Medizinprodukten - Teil 12: Proben-vorbereitung und Referenzmaterialien (ISO 10993-12:2007)Évaluation biologique des dispositifs médicaux - Partie 12 : Préparation des échantillons et matériaux de référence (ISO 10993-12:2007)Biological evaluation of medical devices - Part 12: Sample preparation and reference materials (ISO 10993-12:2007)11.100.20Biological evaluation of medical devicesICS:Ta slovenski standard je istoveten z:prEN ISO 10993-12kSIST prEN ISO 10993-12:2009en01-marec-2009kSIST prEN ISO 10993-12:2009SLOVENSKI
STANDARD



kSIST prEN ISO 10993-12:2009



EUROPEAN STANDARDNORME EUROPÉENNEEUROPÄISCHE NORMFINAL DRAFTprEN ISO 10993-12December 2008ICS 11.100.20Will supersede EN ISO 10993-12:2007
English VersionBiological evaluation of medical devices - Part 12: Samplepreparation and reference materials (ISO 10993-12:2007)Évaluation biologique des dispositifs médicaux - Partie 12:Préparation des échantillons et matériaux de référence(ISO 10993-12:2007)Biologische Beurteilung von Medizinprodukten - Teil 12:Proben-vorbereitung und Referenzmaterialien (ISO 10993-12:2007)This draft European Standard is submitted to CEN members for unique acceptance procedure. It has been drawn up by the TechnicalCommittee CEN/TC 206.If this draft becomes a European Standard, CEN members are bound to comply with the CEN/CENELEC Internal Regulations whichstipulate the conditions for giving this European Standard the status of a national standard without any alteration.This draft European Standard was established by CEN in three official versions (English, French, German). A version in any other languagemade by translation under the responsibility of a CEN member into its own language and notified to the CEN Management Centre has thesame 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.Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without notice andshall not be referred to as a European Standard.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMITÉ EUROPÉEN DE NORMALISATIONEUROPÄISCHES KOMITEE FÜR NORMUNGManagement Centre: rue de Stassart, 36
B-1050 Brussels© 2008 CENAll rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. prEN ISO 10993-12:2008: EkSIST prEN ISO 10993-12:2009



prEN ISO 10993-12:2008 (E) 2 Contents Page Foreword .3Annex ZA (informative)
Relationship between this
European
Standard and the Essential Requirements
of EU Directive 93/42/EEC on Medical Devices .4Annex ZB (informative)
Relationship between this
European
Standard and the Essential Requirements
of EU Directive 90/385/EEC on Active Implantable Medical Devices .5 kSIST prEN ISO 10993-12:2009



prEN ISO 10993-12:2008 (E) 3 Foreword The text of ISO 10993-12:2007 has been prepared by Technical Committee ISO/TC 194 “Biological evaluation of medical devices” of the International Organization for Standardization (ISO) and has been taken over as prEN ISO 10993-12:2008 by Technical Committee CEN/TC 206 “Biological evaluation of medical devices” the secretariat of which is held by NEN. This document is currently submitted to the Unique Acceptance Procedure. This document will supersede EN ISO 10993-12:2007. 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 93/42/EEC on Medical Devices and 90/385/EEC on Active Implantable Medical Devices. For relationship with EU Directives, see informative Annexes ZA and ZB, which is an integral part of this document. Endorsement notice The text of ISO 10993-12:2007 has been approved by CEN as a prEN ISO 10993-12:2008 without any modification. kSIST prEN ISO 10993-12:2009



prEN ISO 10993-12:2008 (E) 4 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 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 that Directive and associated EFTA regulations. Table ZA — Correspondence between this European Standard and Directive 93/42/EEC on medical devices Clause(s)/sub-clause(s) of this EN Essential Requirements (ERs) of Directive 93/42/EEC Qualifying remarks/Notes 4, 5, 6, 7, 8, 9, 10, 11
Annex I: 7.1, 7.2, 7.5
WARNING — Other requirements and other EU Directives may be applicable to the product(s) falling within the scope of this standard.
kSIST prEN ISO 10993-12:2009



prEN ISO 10993-12:2008 (E) 5 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 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 ZB 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 — Correspondence between this European Standard and Directive 90/385/EEC on active implantable medical devices Clause(s)/sub-clause(s) of this EN Essential Requirements (ERs) of Directive 90/385/EEC Qualifying remarks/Notes 4, 5, 6, 7, 8, 9, 10, 11 Annex I : 9
WARNING — Other requirements and other EU Directives may be applicable to the product(s) falling within the scope of this standard.
kSIST prEN ISO 10993-12:2009



kSIST prEN ISO 10993-12:2009



Reference numberISO 10993-12:2007(E)© ISO 2007
INTERNATIONAL STANDARD ISO10993-12Third edition2007-11-15Corrected version2008-02-15Biological evaluation of medical devices — Part 12: Sample preparation and reference materials Évaluation biologique des dispositifs médicaux — Partie 12: Préparation des échantillons et matériaux de référence
kSIST prEN ISO 10993-12:2009



ISO 10993-12:2007(E) PDF disclaimer This PDF file may contain embedded typefaces. In accordance with Adobe's licensing policy, this file may be printed or viewed but shall not be edited unless the typefaces which are embedded are licensed to and installed on the computer performing the editing. In downloading this file, parties accept therein the responsibility of not infringing Adobe's licensing policy. The ISO Central Secretariat accepts no liability in this area. Adobe is a trademark of Adobe Systems Incorporated. Details of the software products used to create this PDF file can be found in the General Info relative to the file; the PDF-creation parameters were optimized for printing. Every care has been taken to ensure that the file is suitable for use by ISO member bodies. In the unlikely event that a problem relating to it is found, please inform the Central Secretariat at the address given below.
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ISO 2007 All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or ISO's member body in the country of the requester. ISO copyright office Case postale 56 • CH-1211 Geneva 20 Tel.
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ii © ISO 2007 – All rights reserved
kSIST prEN ISO 10993-12:2009



ISO 10993-12:2007(E) © ISO 2007 – All rights reserved iiiContents Page Foreword.iv Introduction.vi 1 Scope.1 2 Normative references.1 3 Terms and definitions.1 4 General requirements.3 5 Reference materials.4 5.1 General.4 5.2 Certification of RMs for biological safety testing.4 6 Use of RMs as experimental controls.4 7 Test sample selection.5 8 Test sample and RM preparation.5 9 Selection of representative portions from a device.5 10 Preparation of extracts of samples.6 10.1 General.6 10.2 Containers for extraction.6 10.3 Extraction conditions and methods.6 10.4 Extraction conditions for hazard identification and risk estimation in the exaggerated-use condition.8 11 Records.9 Annex A (informative)
Experimental controls.10 Annex B (informative)
General principles on and practices of test sample preparation and sample selection.12 Annex C (informative)
Principles of test sample extraction.14 Bibliography.17
kSIST prEN ISO 10993-12:2009



ISO 10993-12:2007(E) iv © ISO 2007 – All rights reserved 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 10993-12 was prepared by Technical Committee ISO/TC 194, Biological evaluation of medical devices. This third edition cancels and replaces the second edition (ISO 10993-12:2002), which has been technically revised. ISO 10993 consists of the following parts, under the general title Biological evaluation of medical devices: ⎯ Part 1: Evaluation and testing ⎯ Part 2: Animal welfare requirements ⎯ Part 3: Tests for genotoxicity, carcinogenicity and reproductive toxicity ⎯ Part 4: Selection of tests for interactions with blood ⎯ Part 5: Tests for in vitro cytotoxicity ⎯ Part 6: Tests for local effects after implantation ⎯ Part 7: Ethylene oxide sterilization residuals ⎯ Part 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 kSIST prEN ISO 10993-12:2009



ISO 10993-12:2007(E) © ISO 2007 – All rights reserved v⎯ 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 [TS] ⎯ Part 20: Principles and methods for immunotoxicology testing of medical devices [TS] Future parts will deal with other relevant aspects of biological testing. This corrected version of ISO 10993-12 contains changes to definition 3.10 on page 3 and changes to footnote references in A.3 on page 11. kSIST prEN ISO 10993-12:2009



ISO 10993-12:2007(E) vi © ISO 2007 – All rights reserved Introduction This part of ISO 10993 specifies methods of sample preparation and the selection of reference materials in the biological evaluation of medical devices. Sample preparation methods should be appropriate for both the biological evaluation methods and the materials being evaluated. Each biological test method requires the selection of materials, extraction solvents and conditions. This part of ISO 10993 is based on existing national and international specifications, regulations and standards wherever possible. It is periodically reviewed and revised.
kSIST prEN ISO 10993-12:2009



INTERNATIONAL STANDARD ISO 10993-12:2007(E) © ISO 2007 – All rights reserved 1Biological evaluation of medical devices — Part 12: Sample preparation and reference materials 1 Scope This part of ISO 10993 specifies requirements and gives guidance on the procedures to be followed in the preparation of samples and the selection of reference materials for medical device testing in biological systems in accordance with one or more parts of the ISO 10993 series. Specifically this part of ISO 10993 addresses: ⎯ test sample selection; ⎯ selection of representative portions from a device; ⎯ test sample preparation; ⎯ experimental controls; ⎯ selection of and requirements for reference materials; ⎯ preparation of extracts. This part of ISO 10993 is not applicable to materials or devices containing live cells. 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 ISO 14971, Medical devices — Application of risk management to medical devices 3 Terms and definitions For the purposes of this document, the following terms and definitions apply. 3.1 blank extraction vehicle not containing the test material, retained in a vessel identical to that which holds the test sample and subjected to identical conditions to which the test sample is subjected during its extraction NOTE The purpose of the blank is to evaluate possible confounding effects due to the extraction vessel, extraction vehicle and extraction process. kSIST prEN ISO 10993-12:2009



ISO 10993-12:2007(E) 2 © ISO 2007 – All rights reserved 3.2 certified reference material (CRM) reference material, accompanied by a certificate, one or more of whose property values are certified by a procedure which establishes its traceability to an accurate realization of the unit in which the property values are expressed, and for which each certified value is accompanied by an uncertainty at a stated level of confidence [ISO Guide 30, definition 2.2] 3.3 experimental control substance with well characterized responses, which is used in a specific test system to assist in evaluating if the test system has responded in a reproducible and appropriate manner 3.4 extract liquid that results from extraction of the test sample or control 3.5 homogeneous property of a material and its relationship to a biological endpoint such that it is of uniform structure or composition to consistently render or not a specific biological response NOTE A reference material is said to be homogeneous if the biological response to a specific test is found to lie within the specified uncertainty limits of the test, irrespective of the batch or lot of material from which the test sample is removed. 3.6 negative control any well characterized material and/or substance, which, when tested by a specific procedure, demonstrates the suitability of the procedure to yield a reproducible, appropriately negative, non-reactive or minimal response in the test system NOTE In practice, negative controls are reference materials but may include blanks and extraction vehicles/solvents. 3.7 positive control any well characterized material and/or substance, which, when evaluated by a specific test method, demonstrates the suitability of the test system to yield a reproducible, appropriately positive or reactive response in the test system 3.8 reference material (RM) material with one or more property values that are sufficiently reproducible and well established to enable use of the material or substance for the calibration of an apparatus, the assessment of a measurement method, or for the assignment of values to materials [ISO Guide 30, definition 2.1] NOTE For the purpose of this part of ISO 10993, a reference material is any well characterized material or substance, which, when tested by the procedure described, demonstrates the suitability of the procedure to yield a reproducible, predictable response. The response may be negative or positive. 3.9 stability 〈of property values〉 ability of a material, when stored under specified conditions, to maintain a specific stated biological response, within specified limits, for a specific period of time NOTE Adapted from ISO Guide 30, definition 2.7. kSIST prEN ISO 10993-12:2009



ISO 10993-12:2007(E) © ISO 2007 – All rights reserved 33.10 test sample medical device, component or material (or a representative sample thereof, manufactured and processed by equivalent methods) or an extract or portion thereof that is subjected to biological or chemical testing or evaluation 3.11 simulated-use extraction extraction to demonstrate compliance with the requirements of this part of ISO 10993, by evaluating leachable material levels available to the patient or user from devices during the routine use of a device using an extraction method that simulates product use NOTE The burden of validation on the analytical laboratory is to demonstrate that the simulated-use extraction is carried out under conditions that provide the greatest challenge to the intended use. Product-use simulation is carried out assuming the device is assigned to the most stringent category probable for duration of exposure and takes into consideration both tissue(s) exposed and temperature of exposure. 3.12 exaggerated extraction any extraction that is intended to result in a greater amount of a chemical constituent being released as compared to the amount generated under the simulated conditions of use NOTE Exaggerated extraction might result in a chemical change of the material or the substances being extracted. 3.13 accelerated extraction extraction that provides a measure of the leachable materials of the device or material using conditions that shorten the time for leaching of the substances into the extraction vehicle but do not result in a chemical change of the substances being extracted NOTE Examples of accelerated extraction conditions are as follows: elevated temperature, agitation, changing of the extraction vehicle, etc. 3.14 exhaustive extraction extraction until the amount of leachable material in a subsequent extraction is less than 10 % of that detected in the initial extraction, or until there is no analytically significant increase in the cumulative leachable material levels detected NOTE As it is not possible to demonstrate the exhaustive nature of residual recovery, the definition of exhaustive extraction adopted is as above. See also Annex C. 4 General requirements 4.1 As described in ISO 14971, in the identification of hazard and risk estimation for medical devices, hazards that arise from changes in the manufacturing process, or insufficient control of the manufacturing process, shall be considered in the design and preparation of samples for test and preparation of extracts of those devices. Particular attention shall be given to residues, e.g., trace elements and cleaning and disinfection agents, of those manufacturing processes. 4.2 Because ISO 10993 describes many different biological assay systems, the individual standards shall be consulted to ascertain if these recommendations are appropriate for specific test systems. 4.3 Experimental controls shall be used in biological evaluations to validate a test procedure and/or to compare the results between materials. Depending on the biological test, negative controls, blanks and/or positive controls shall be used as is appropriate to the test. NOTE The same type of control can be applicable to different tests and may allow cross-reference to other established materials and test methods. Additional guidance on the selection of experimental controls is given in Annex A. Use of positive controls for in-vivo testing may be affected by animal welfare regulations. kSIST prEN ISO 10993-12:2009



ISO 10993-12:2007(E) 4 © ISO 2007 – All rights reserved 5 Reference materials 5.1 General Reference materials (RMs) are established by individual laboratories. The extent of chemical, physical and biological characterization is determined by the individual laboratory. Commercially available articles may be used as reference materials. NOTE 1 See also ISO Guide 35. Certified reference materials (CRMs) are selected for their high purity, critical characteristics, suitability for the intended purpose and general availability. The critical chemical, physical and biological characteristics shall be determined by collaborative testing in three or more laboratories, and made available to the investigator by the distributor. NOTE 2 It is desirable for users to obtain a commitment from suppliers of RMs or CRMs that these materials will be available to the user for at least five years. A second, but less desirable, option is for the source of the RM or CRM to publish an “open formulation” for the material, i.e., publication of the source materials and details of the processing needed to ensure uniform batches of the RM. 5.2 Certification of RMs for biological safety testing 5.2.1 Qualification of an RM is a procedure that establishes the numerical or qualitative value of the biological response of the material under specified test conditions, ensuring reproducibility of the response within and/or between laboratories. The range of biological responses associated with the material shall be established through laboratory tests. NOTE See also ISO Guide 34. 5.2.2 Suppliers of RMs shall certify the materials. The supplier determines the extent of chemical and physical characterization that is performed. The individual laboratories that use the RMs shall identify the biological characterization necessary to qualify an RM for a specific test or procedure. Commercially available materials may be used as RMs provided they are certified and qualified. 5.2.3 Certification of an RM is a procedure that establishes the numerical or qualitative value of the biological response of the material under the specified test conditions. This process serves to validate the testing of the material for that particular response and results in the issuance of a certificate. The biological response of the material shall be established through interlaboratory tests. 6 Use of RMs as experimental controls 6.1 RMs or CRMs shall be used in biological tests as control materials to demonstrate the suitability of a procedure to yield a reproducible response, such as either positive and/or negative. Any material used in this way shall be characterized with each biological test procedure for which the use of the material is desired. A material characterized and then certified for one reference test method or response, e.g., delayed-type hypersensitivity, shall not be used as an RM for another, e.g., cytotoxicity, without additional validation. NOTE Use of an RM will facilitate the comparability of the response between laboratories and assist in assessing reproducibility of test performance within individual laboratories. For comparison of the biological response, it is desirable to use RMs having a range of responses, e.g., minimum, intermediate or severe. 6.2 RMs used as experimental controls shall meet the established quality assurance procedures of the manufacturer and test laboratory. They shall be identified as to source, manufacturer, grade and type. RMs are processed as described in Clause 8. kSIST prEN ISO 10993-12:2009



ISO 10993-12:2007(E) © ISO 2007 – All rights reserved 56.3 When RMs are used as experimental controls, they shall be in the same material class as the test sample, i.e., polymer, ceramic, metal, colloid, etc. However, pure chemicals may be used as experimental controls for mechanistically-based test procedures, e.g., genotoxicity and immune delayed-type hypersensitivity assays. 7 Test sample selection 7.1 Testing shall be performed on the final product, or representative samples from the final products or materials processed in the same manner as the final product (see ISO 10993-1) or on appropriate extracts of any of these. The choice of test sample shall be justified. NOTE In the case of materials that cure in-situ, different test samples representative of the cured material versus the uncured state of the material might be needed. 7.2 The same test sample selection procedure applies when an extract is required. 8 Test sample and RM preparation 8.1 Test samples and RMs shall be handled with care to prevent contamination. Any residues from the manufacturing processes shall be considered to be integral to the device, device portion or component. NOTE For additional guidance on preparation see Annex B. a) Test samples from sterilized devices and RMs shall be handled aseptically if appropriate to the test procedure. b) Test samples from a device which is normally supplied non-sterile, but which requires sterilization prior to use, shall be sterilized by the method recommended by the manufacturer and handled aseptically if appropriate to the test procedure. c) If test samples are cleaned prior to sterilization, the influence of the cleaning process and cleaning agent shall be considered in the selection and handling of the test sample. 8.2 If sterile test samples are required for the test procedure, the effect of the sterilization or rester
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