EN ISO 10993-12:2012

Biological evaluation of medical devices - Part 12: Sample preparation and reference materials (ISO 10993-12:2012)

ISO 10993-12:2012 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 ISO 10993. Specifically, ISO 10993-12:2012 addresses the following:
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.
ISO 10993-12:2012 is not applicable to live cells, but can be relevant to the material or device components of combination products containing live cells.

Biologische Beurteilung von Medizinprodukten - Teil 12: Probenvorbereitung und Referenzmaterialien (ISO 10993-12:2012)

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 10993-12:2012)

Biološko ovrednotenje medicinskih pripomočkov - 12. del: Priprava vzorcev in referenčni materiali (ISO 10993-12:2012)

Ta del standarda ISO 10993 določa zahteve in smernice za postopke, ki jih je treba upoštevati pri pripravi vzorcev in izbiranju referenčnih materialov za preskušanje medicinskih pripomočkov v bioloških sistemih v skladu z enim ali več deli standarda ISO 10993. Ta del standarda ISO 10993 obravnava zlasti naslednje: – izbiranje preskusnega vzorca; – izbiranje reprezentativnih delov pripomočka, – pripravo preskusnega vzorca; – kontrole preskusa; –izbiranje referenčnih materialov in zahteve zanje; – pripravo izvlečkov. Ta del standarda ISO 10993 se ne uporablja za žive celice, vendar je lahko ustrezen za materiale ali sestavne dele kombiniranih izdelkov, ki vsebujejo žive celice.

General Information

• Status: Withdrawn
• Publication Date: 30-Jun-2012
• Withdrawal Date: 13-Apr-2025
• ICS: 11.100.20 - Biological evaluation of medical devices
• Technical Committee: CEN/TC 206 - Biocompatibility of medical and dental materials and devices
• Drafting Committee: CEN/TC 206 - Biocompatibility of medical and dental materials and devices
• Current Stage: 9960 - Withdrawal effective - Withdrawal
• Start Date: 16-Jun-2021
• Completion Date: 14-Apr-2025
• 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: SIST EN ISO 10993-12:2012 - Biological evaluation of medical devices - Part 12: Sample preparation and reference materials (ISO 10993-12:2012)

Relations

• Replaces: EN ISO 10993-12:2009 - Biological evaluation of medical devices - Part 12: Sample preparation and reference materials (ISO 10993-12:2007)
• Effective Date: 08-Jun-2022
• Replaced By: EN ISO 10993-12:2021 - Biological evaluation of medical devices - Part 12: Sample preparation and reference materials (ISO 10993-12:2021)
• Effective Date: 02-May-2018

Standards Content (Sample)

SLOVENSKI STANDARD
01-oktober-2012
1DGRPHãþD
SIST EN ISO 10993-12:2009
%LRORãNRRYUHGQRWHQMHPHGLFLQVNLKSULSRPRþNRYGHO3ULSUDYDY]RUFHYLQ
UHIHUHQþQLPDWHULDOL,62
Biological evaluation of medical devices - Part 12: Sample preparation and reference
materials (ISO 10993-12:2012)
Biologische Beurteilung von Medizinprodukten - Teil 12: Probenvorbereitung und
Referenzmaterialien (ISO 10993-12:2012)
Évaluation biologique des dispositifs médicaux - Partie 12: Préparation des échantillons
et matériaux de référence (ISO 10993-12:2012)
Ta slovenski standard je istoveten z: EN ISO 10993-12:2012
ICS:
11.100.20 %LRORãNRRYUHGQRWHQMH Biological evaluation of
PHGLFLQVNLKSULSRPRþNRY medical devices
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EUROPEAN STANDARD
EN ISO 10993-12
NORME EUROPÉENNE
EUROPÄISCHE NORM
July 2012
ICS 11.100.20 Supersedes EN ISO 10993-12:2009
English Version
Biological evaluation of medical devices - Part 12: Sample
preparation and reference materials (ISO 10993-12:2012)
Évaluation biologique des dispositifs médicaux - Partie 12: Biologische Beurteilung von Medizinprodukten - Teil 12:
Préparation des échantillons et matériaux de référence Probenvorbereitung und Referenzmaterialien (ISO 10993-
(ISO 10993-12:2012) 12:2012)
This European Standard was approved by CEN on 30 June 2012.

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

Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2012 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 10993-12:2012: E
worldwide for CEN national Members.

Contents Page
Foreword .3
Annex ZA (informative) Relationship between this European Standard and the Essential
Requirements of EU Directive 93/42/EEC on medical devices .4
Annex ZB (informative) Relationship between this European Standard and the Essential
Requirements of EU Directive 90/385/EEC on active implantable medical devices .5

Foreword
This document (EN ISO 10993-12:2012) has been prepared by Technical Committee ISO/TC 194 "Biological
evaluation of medical devices" in collaboration with Technical Committee CEN/TC 206 “Biological evaluation
of medical devices” the secretariat of which is held by NEN.
This European Standard shall be given the status of a national standard, either by publication of an identical
text or by endorsement, at the latest by January 2013, and conflicting national standards shall be withdrawn at
the latest by January 2013.
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 10993-12:2009.
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 an 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 and the United Kingdom.
Endorsement notice
The text of ISO 10993-12:2012 has been approved by CEN as a EN ISO 10993-12:2012 without any
modification.
Annex ZA
(informative)
Relationship between this European Standard and the Essential
Requirements of EU Directive 93/42/EEC on medical devices
This European Standard has been prepared under a mandate given to CEN by the European Commission
and the European Free Trade Association to provide a means of conforming to Essential Requirements of the
New Approach Directive 93/42/EEC on medical devices.
Once this standard is cited in the Official Journal of the European Union under that Directive and has been
implemented as a national standard in at least one Member State, compliance with the clauses of this
standard given in Table ZA.1 confers, within the limits of the scope of this standard, a presumption of
conformity with the corresponding Essential Requirements of that Directive and associated EFTA regulations.
Table ZA.1 — Correspondence between this European Standard
and Directive 93/42/EEC on medical devices
Clauses of this Essential Requirements (ERs) Qualifying remarks/Notes
European Standard of Directive 93/42/EEC
This standard provides a means of preparing
samples to assess conformity with this part of
this ER in conjunction with other relevant parts
4, 5, 6, 7, 8, 9, 10, 11 7.2 first sentence only
of EN ISO 10993 for the design and
manufacture of medical devices. Packaging is
not covered.
This standard provides a means of preparing
samples of medical devices to assess
4, 5, 6, 7, 8, 9, 10 , 11 7.5 first paragraph only conformity with this part of this ER in
conjunction with other relevant parts of EN ISO
10993.
WARNING — Other requirements and other EU Directives may be applicable to the product(s) falling
within the scope of this standard.
Annex ZB
(informative)
Relationship between this European Standard
and the Essential Requirements of EU Directive 90/385/EEC
on active implantable medical devices
This European Standard has been prepared under a mandate given to CEN by the European Commission
and the European Free Trade Association to provide a means of conforming to Essential Requirements of the
New Approach Directive 90/385/EEC on active implantable medical devices.
Once this standard is cited in the Official Journal of the European Union under that Directive and has been
implemented as a national standard in at least one Member State, compliance with the clauses of this
standard given in Table ZB.1 confers, within the limits of the scope of this standard, a presumption of
conformity with the corresponding Essential Requirements of that Directive and associated EFTA regulations.
Table ZB.1 — Correspondence between this European Standard
and Directive 90/385/EEC on active implantable medical devices
Clauses of this Essential Requirements (ERs) Qualifying remarks/Notes
European Standard of Directive 90/385/EEC
This standard provides a means of preparing
samples of medical devices to assess
4, 5, 6, 7, 8, 9, 10, 11 9 (first and second indents only) conformity with these parts of this ER in
conjunction with other relevant parts of EN ISO
10993.
WARNING — Other requirements and other EU Directives may be applicable to the product(s) falling
within the scope of this standard.

INTERNATIONAL ISO
STANDARD 10993-12
Fourth edition
2012-07-01
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 10993-12:2012(E)
©
ISO 2012
ISO 10993-12:2012(E)
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. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail [e-mail]
Web www.iso.org
Published in Switzerland
ii © ISO 2012 – All rights reserved

ISO 10993-12:2012(E)
Contents Page
Foreword .iv
Introduction . v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 General requirements . 3
5 Reference materials (RMs) . 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 (points to consider in relation to Annex D) . 9
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
Annex D (informative) Exhaustive extraction of polymeric materials for biological evaluation .17
Bibliography .19
ISO 10993-12:2012(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 fourth edition cancels and replaces the third edition (ISO 10993-12:2007), which has been technically revised.
ISO 10993 consists of the following parts, under the general title Biological evaluation of medical devices:
— Part 1: Evaluation and testing within a risk management process
— Part 2: Animal welfare requirements
— Part 3: Tests for genotoxicity, carcinogenicity and reproductive toxicity
— Part 4: Selection of tests for interactions with blood
— Part 5: Tests for in vitro cytotoxicity
— Part 6: Tests for local effects after implantation
— Part 7: Ethylene oxide sterilization residuals
— Part 9: Framework for identification and quantification of potential degradation products
— Part 10: Tests for irritation and skin sensitization
— Part 11: Tests for systemic toxicity
— Part 12: Sample preparation and reference materials
— Part 13: Identification and quantification of degradation products from polymeric medical devices
— Part 14: Identification and quantification of degradation products from ceramics
— Part 15: Identification and quantification of degradation products from metals and alloys
— Part 16: Toxicokinetic study design for degradation products and leachables
— Part 17: Establishment of allowable limits for leachable substances
— Part 18: Chemical characterization of materials
— Part 19: Physico-chemical, morphological and topographical characterization of materials [Technical
Specification]
— Part 20: Principles and methods for immunotoxicology testing of medical devices [Technical Specification]
iv © ISO 2012 – All rights reserved

ISO 10993-12:2012(E)
Introduction
This part of ISO 10993 specifies methods of sample preparation and provides requirements and guidance for
the selection of reference materials for the biological evaluation of medical devices.
It is important that sample preparation methods 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.
INTERNATIONAL STANDARD ISO 10993-12:2012(E)
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 ISO 10993. Specifically, this part of ISO 10993 addresses the following:
— 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 live cells, but can be relevant to the material or device components
of combination products 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 (all parts), Biological evaluation of medical devices
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
accelerated extraction
extraction that provides a measure of the leachable or extractable 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
EXAMPLE Elevated temperature, agitation, changing of the extraction vehicle.
3.2
blank
extraction vehicle not containing the test material, which is retained in a vessel identical to that holding the test
sample and subjected to conditions identical to the ones the test sample is subjected to 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 10993-12:2012(E)
3.3
CRM
certified reference material
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 30:1992, definition 2.2]
3.4
exaggerated extraction
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 It is important to ensure that the exaggerated extraction does not result in a chemical change of the material.
3.5
exhaustive extraction
extraction conducted until the amount of extractable material in a subsequent extraction is less than 10 % by
gravimetric analysis of that detected in the initial extraction
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.
3.6
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
3.7
extract
liquid that results from extraction of the test sample or control
3.8
extractables
substances that can be released from a medical device or material using extraction solvents and/or extraction
conditions that are expected to be at least as aggressive as the conditions of clinical use
3.9
homogeneous
property of a material and its relationship to a biological endpoint, meaning that it is of uniform structure or
composition, thereby consistently rendering, 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 extracted.
3.10
leachables
substances that can be released from a medical device or material during clinical use
3.11
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 can include blanks and extraction vehicles/solvents.
3.12
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
2 © ISO 2012 – All rights reserved

ISO 10993-12:2012(E)
3.13
RM
reference material
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
NOTE 1 Adapted from ISO Guide 30:1992, definition 2.1.
NOTE 2 For the purpose of this part of ISO 10993, an RM 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.
3.14
simulated-use extraction
extraction conducted 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 possible for the duration of exposure and takes into
consideration both the tissue(s) exposed and the temperature of exposure.
3.15
stability
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 30:1992, definition 2.7.
3.16
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
4 General requirements
4.1 When identifying hazards and estimating risk in relation to 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 test samples, as described in ISO 14971. Particular attention shall be given to
residues, e.g. trace elements and cleaning and disinfection agents, of manufacturing processes.
4.2 ISO 10993 describes many different biological assay systems. Therefore, the individual parts shall be
consulted to ascertain whether these are appropriate for specific test systems.
4.3 Experimental controls shall be used in biological evaluations carried out in order 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, depending on what 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 might be affected by animal welfare regulations.
ISO 10993-12:2012(E)
5 Reference materials (RMs)
5.1 General
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 RM.
NOTE 1 See also ISO Guide 35.
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 stating 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 RM shall identify the biological
characterization necessary to qualify a RM for a specific test or procedure. Commercially available materials
may be used as RM, provided they are certified and qualified.
5.2.3 Certification of a 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, i.e. 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 The use of an RM will facilitate the comparability of the response between laboratories and help assess
reproducibility of the 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 in relation to source, manufacturer, grade and type.
RMs are processed as described in Clause 8.
6.3 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.
4 © ISO 2012 – All rights reserved

ISO 10993-12:2012(E)
7 Test sample selection
7.1 Testing shall be performed on the final product, representative samples from the final product, 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 residue from the
manufacturing processes shall be considered 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 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 Test samples from devices not required to be sterile in use shall be used as supplied and handled
aseptically throughout the test sample preparation. If sterile test samples are required for a test procedure,
e.g. for cytotoxicity testing, the effect of the sterilization or resterilization process on the test sample and RM
shall be considered.
8.3 When test samples and RMs need to be cut into pieces, as described in 10.3.3, the influence of previously
unexposed surfaces, e.g. lumens or cut surfaces, shall be considered. Tools used for cutting medical devices
into representative portions for testing shall be cleaned between uses to prevent contamination.
9 Selection of representative portions from a device
9.1 If a device cannot be tested as a whole, each individual material in the final product shall be represented
proportionally in the test sample.
a) The test sample of devices with surface coatings shall include both coating material and the substrate,
even if the substrate has no tissue contact.
b) The test sample shall include a representative portion of the joint and/or seal if adhesives, radio-frequency
(RF) seals or solvent seals are used in the manufacture of a portion of the device which comes into contact
with patients.
9.2 Composite materials shall be tested as finished materials.
9.3 When different materials are present in a single device, the potential for synergies and interactions shall
be considered in the choice of test sample.
9.4 The test sample shall be chosen to maximize the exposure of the test system to the components of a
device that are known to have potential for a biological response.
ISO 10993-12:2012(E)
10 Preparation of extracts of samples
10.1 General
If extracts of the device are required for a test procedure, the extraction vehicles and conditions of extraction
used shall be appropriate to the nature and use of the final product and to the purpose of the test, e.g. hazard
identification, risk estimation or risk assessment. The physico-chemical properties of the device materials,
leachable substances or residues shall be considered when choosing the extraction conditions.
NOTE For additional guidance on the extraction of samples, see Annex C.
10.2 Containers for extraction
10.2.1 The extraction shall be performed in clean, chemically inert, closed containers with minimum dead space.
10.2.2 To ensure that the extraction vessels do not adulterate the extract of the test sample, the extraction
vessels shall be:
a) borosilicate glass tubes with caps having an inert liner, e.g. polytetrafluoroethylene;
b) other inert extraction vessels, as required for specific materials and/or extraction procedures.
10.3 Extraction conditions and methods
10.3.1 Extraction conditions are based on common practice and are justified on the basis of providing a
standardized approach that is, in many ways, an appropriate exaggeration of product use. Extraction shall be
conducted under one of the following conditions (see also C.5):
a) (37 ± 1) °C for (72 ± 2) h;
b) (50 ± 2) °C for (72 ± 2) h;
c) (70 ± 2) °C for (24 ± 2) h;
d) (121 ± 2) °C for (1 ± 0,1) h.
NOTE Extraction at (37 ± 1) °C for (24 ± 2) h in tissue culture media is acceptable for cytotoxicity testing. For medical
devices which are in short-term contact with intact skin or mucosa and which are not implanted, extraction times of less
than 24 h, but not less than 4 h, are acceptable (see ISO 10993-5). Extraction temperatures greater than (37 ± 1) °C can
adversely impact chemistry and/or stability of the serum and other constituents in the culture medium.
The extraction conditions described above, which have been used to provide a measure of the hazard potential
for risk estimation of the device or material, are based on historical precedent. Other conditions that simulate
the leachables occurring during clinical use, or that provide an adequate measure of the hazard potential, may
be used but shall be described and justified.
Extraction is a complex process influenced by time, temperature, surface-area-to-volume ratio, the extraction
1)
vehicle and the phase equilibrium of the material. The effects of higher temperatures or other conditions on
extraction kinetics and the identity of the extraction vehicle(s) should be considered carefully if accelerated or
exaggerated extraction is used.
1) The phase equilibrium of a material during extraction controls the relative amounts of amorphous and crystalline phases
present. For the amorphous phase, the glass transition temperature, T , dictates the polymer chain mobility and the diffusion
g
rate in the phase. Usually, at temperatures higher than T , the diffusion rate is considerably higher compared with that below
g
T . The diffusion rate is lowest in the crystalline phase. The extraction conditions should not alter the phase equilibrium of
g
the material. Phase alteration can affect the amount and type of extractables.
6 © ISO 2012 – All rights reserved

ISO 10993-12:2012(E)
For example, two possibilities exist when elevated temperatures are used:
— the energy of the increased temperature may cause increased cross-linking and/or polymerization of the
polymer and, therefore, decrease the amount of free monomer that is available to migrate from the polymer;
— the increased temperature could cause degradation products to form that are not typically found in the
finished device under conditions of use.
10.3.2 For materials that dissolve or resorb under conditions of use, follow the extraction conditions described in
10.3.1. Perform extraction using the appropriate extraction vehicle and time/temperature conditions to simulate
exaggerated exposure wherever possible. Complete dissolution may be appropriate.
10.3.3 The standard surface area can be used to determine the volume of extraction vehicle needed. This area
includes the combined area of both sides of the sample and excludes indeterminate surface irregularities. When
the surface area cannot be determined due to configuration of the sample, a mass/volume of extracting fluid
shall be used. See Table 1.
Other surface-area-to-volume extraction ratios, e.g. those related to evaluation of porous materials, can be
used if they simulate the conditions during clinical use or result in a measure of the hazard potential.
Materials shall be cut into small pieces before extraction to enhance submersion in the extract media, except
when otherwise inappropriate (see 10.3.4). For example, for polymers, pieces approximately 10 mm × 50 mm
or 5 mm × 25 mm are appropriate.
Table 1 — Standard surface areas and extract liquid volumes
Thickness Extraction ratio
(surface area or mass/volume)
Examples of forms of materials

mm ±10 %
˂0,5 6 cm /ml Film, sheet, tubing wall
0,5 to 1,0 3 cm /ml Tubing wall, slab, small moulded items
>1,0 3 cm /ml Larger moulded items
>1,0 1,25 cm /ml Elastomeric closures
Powder, pellets, foam,
Irregularly shaped solid devices 0,2 g/ml
non-absorbent moulded items
Irregularly shaped porous devices
0,1 g/ml Membranes, textiles
(low-density materials)
NOTE While there are no standardized methods available at present for testing absorbents and hydrocolloids, a suggested
protocol is as follows:
— determine the volume of extraction vehicle that each 0,1 g or 1,0 cm of material absorbs;
— then, in performing the material extraction, add this additional volume to each 0,1 g or 1,0 cm in an extraction mixture.
10.3.4 Elastomers, coated materials, composites, laminates, etc. shall be tested intact whenever possible
because of potential differences in extraction characteristics between the intact and cut surfaces.
NOTE As a result of manufacturing processes, many elastomers might have surface properties that are different from
those of the bulk material.
10.3.5 Extraction using both polar and non-polar extraction vehicles shall be performed. The following are
examples of extraction vehicles:
a) polar extraction vehicle: water, physiological saline, culture media without serum;
b) non-polar extraction vehicle: freshly refined vegetable oil (e.g. cottonseed or sesame oil) of the quality
defined in various pharmacopoeias;
ISO 10993-12:2012(E)
c) additional extraction vehicles: ethanol/water, ethanol/saline, polyethylene glycol 400 (diluted to a
physiological osmotic pressure), dimethyl-sulfoxide and culture media with serum.
NOTE 1 Other extraction vehicles appropriate to the nature and use of the device or to the methods for hazard
identification may also be used if their effects on the material and the biological system are known (see Annex D).
NOTE 2 The use of a culture medium with serum is preferred for extraction in testing for cytotoxicity because of its
ability to support cellular growth as well as extract both polar and non-polar substances.
10.3.6 Extractions shall be performed with agitation or circulation. When extraction under static conditions is
considered to be appropriate, the method shall be justified, specified and reported.
10.3.7 Liquid extracts shall, if possible, be used immediately after preparation to prevent sorption on to the
extraction container or other changes in composition. If an extract is stored for longer than 24 hours, then the
stability and homogeneity of the extract under the storage conditions shall be verified.
10.3.8 Extract pH shall not be adjusted unless a rationale is provided.
10.3.9 The extract shall not routinely be processed by filtration, centrifugation or other methods to remove
suspended particulates. However, if such processing is necessary, the rationale shall be documented.
10.3.10 For hazard identification of polymeric devices, exhaustive extraction conditions shall be considered.
The extraction vehicle and conditions of extraction shall be selected on the basis of physico-chemical properties
of the material and/or predicted low-molecular-weight chemicals that might be extracted.
10.3.11 For materials or devices not expected to dissolve or resorb under conditions of use, any solvents used
in the extraction of a polymeric material or device shall not cause dissolution of the polymer formulation. No
more than a slight softening of the polymeric material shall occur in the presence of the volatile solvent (e.g. less
than 10 % dissolution). The solvent shall be removed (prior to use in a bioassay) to the extent that any residues
do not adversely affect the biological assay (e.g. cause protein denaturation or skin irritation). For materials or
devices expected to dissolve or resorb under conditions of use, see 10.3.12.
10.3.12 For solution and soluble materials, the standard extraction methods used for insoluble materials might
be inappropriate. The following guidance should be considered in addition to information contained in Table 1.
a) Factors such as test system compatibility, route of administration and extent of dissolution or degradation
should be considered in the final preparation for testing. Use an appropriate vehicle and conditions to
simulate exaggerated exposure wherever possible. A pre-test can help to determine appropriate conditions.
b) If the material completely dissolves, in a vehicle or diluent that is compatible with the material and the test
system, the resulting solution can be evaluated neat, provided the solution properties are also compatible
with the test system, e.g. pH, osmolarity, solute concentrations.
c) If the material is an aqueous solution and used in this form, it shall be tested directly and not extracted,
provided the solution properties are compatible with the test system [see also a) and b) above].
d) OECD Guidelines for the Testing of Chemicals, or similar chemical testing standards, can be used as
guidance in determining maximum concentrations of test substances used for specific test methods.
10.3.13 Where fluids circulate through the device under normal conditions of use, e.g. extra-corporeal devices,
extraction via re-circulation may be used. When possible, one or more of the conditions shall be exaggerated,
e.g. temperature, time, volume, flow rate. The rationale for the extraction chosen shall be reported.
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ISO 10993-12:2012(E)
10.4 Extraction conditions for hazard identification and risk estimation in the exaggerated-
use condition (points to consider in relation to Annex D)
10.4.1 Hazards that arise from changes in the manufacturing process or insufficient control of the manufa
...