EN ISO 10993-5:1999

SLOVENSKI STANDARD
01-januar-2000
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Biological evaluation of medical devices - Part 5: Tests for in vitro cytotoxicity (ISO 10993
-5:1999)
Biologische Beurteilung von Medizinprodukten - Teil 5: Prüfungen auf Zytotoxizität: In
vitro-Methoden (ISO 10993-5:1999)
Evaluation biologique des dispositifs médicaux - Partie 5: Essais concernant la
cytotoxicité in vitro (ISO 10993-5:1999)
Ta slovenski standard je istoveten z: EN ISO 10993-5:1999
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.

INTERNATIONAL ISO
STANDARD 10993-5
Second edition
1999-05-15
Biological evaluation of medical devices —
Part 5:
Tests for in vitro cytotoxicity
Évaluation biologique des dispositifs médicaux —
Partie 5: Essais concernant la cytotoxicité in vitro
A
Reference number
ISO 10993-5:1999(E)
ISO 10993-5:1999(E)
Contents
1 Scope .1
2 Normative references .1
3 Terms and definitions .1
4 Sample preparation .2
5 Cell lines .4
6 Culture medium.4
7 Preparation of cell stock culture .4
8 Test procedures.5
9 Test report .8
10 Assessment of results.8
©  ISO 1999
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 the publisher.
International Organization for Standardization
Case postale 56 • CH-1211 Genève 20 • Switzerland
Internet iso@iso.ch
Printed in Switzerland
ii
© ISO
ISO 10993-5:1999(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 3.
Draft International Standards adopted by the technical committees are circulated to the member bodies for voting.
Publication as an International Standard requires approval by at least 75 % of the member bodies casting a vote.
International Standard ISO 10993-5 was prepared by Technical Committee ISO/TC 194, Biological evaluation of
medical devices.
This second edition cancels and replaces the first edition (ISO 10993-5:1992), 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 8: Guidance for reference materials
 Part 9: Framework for the identification and quantification of potential degradation products
 Part 10: Tests for irritation and sensitization
 Part 11: Tests for systemic toxicity
 Part 12: Sample preparation and reference materials
 Part 13: Identification and quantification of degradation products from polymers
 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: Methods for establishment of allowable limits for leachable substances using health-based risk
assessment
 Part 18: Chemical characterization
Future parts will deal with other relevant aspects of biological testing.
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© ISO
ISO 10993-5:1999(E)
Introduction
Due to the general applicability of in vitro cytotoxicity tests and their widespread use in evaluating a large range of
medical devices and materials, it is the purpose of this part of ISO 10993, rather than to specify a single test, to
define a scheme for testing which requires decisions to be made in a series of steps. This should lead to the
selection of the most appropriate test.
Three categories of test are listed: extract test, direct-contact test, indirect-contact test.
The choice of one or more of these categories depends upon the nature of the sample to be evaluated, the potential
site of use and the nature of the use.
This choice then determines the details of the preparation of the samples to be tested, the preparation of the
cultured cells, and the way in which the cells are exposed to the samples or their extracts.
At the end of the exposure time, the evaluation of the presence and extent of the cytotoxic effect is undertaken. It is
the intention of this part of ISO 10993 to leave open the choice of type of evaluation. Such a strategy makes
available a battery of tests, which reflects the approach of many groups which advocate in vitro biological tests.
The numerous methods used and end-points measured in cytotoxicity determination can be grouped into categories
of evaluation type:
a) assessments of cell damage by morphological means;
b) measurements of cell damage;
c) measurements of cell growth;
d) measurements of specific aspects of cellular metabolism.
There are, therefore, several alternative means of producing results in each of these four categories. The
investigator should be aware of the categories of test and into which a particular technique fits, in order that
comparisons may be made with other results on similar medical devices or materials, and in order that
interlaboratory tests may be conducted.
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INTERNATIONAL STANDARD  © ISO ISO 10993-5:1999(E)
Biogical evaluation of medical devices —
Part 5:
Tests for in vitro cytotoxicity
1 Scope
This part of ISO 10993 describes test methods to assess the in vitro cytotoxicity of medical devices.
These methods specify the incubation of cultured cells either directly or through diffusion
a) with extracts of a device, and/or
b) in contact with a device.
These methods are designed to determine the biological response of mammalian cells in vitro using appropriate
biological parameters.
2 Normative references
The following normative documents contain provisions which, through reference in this text, constitute provisions of
this part of ISO 10993. For dated references, subsequent amendments to, or revisions of, any of these publications
do not apply. However, parties to agreements based on this part of ISO 10993 are encouraged to investigate the
possibility of applying the most recent editions of the normative documents indicated below. For undated
references, the latest edition of the normative document referred to applies. Members of ISO and IEC maintain
registers of currently valid International Standards.
ISO 10993-1, Biological evaluation of medical devices — Part 1: Evaluation and testing.
ISO 10993-12:1996, Biological evaluation of medical devices — Part 12: Sample preparation and reference
materials.
3 Terms and definitions
For the purposes of this part of ISO 10993, the terms and definitions given in ISO 10993-1 and the following apply.
3.1
negative control material
material which, when tested in accordance with this part of ISO 10993, does not produce a cytotoxic response
NOTE The purpose of the negative control is to demonstrate background response. For example, high-density poly-
1)
ethylene for synthetic polymers, and aluminium oxide ceramic rods for dental material, have been used as negative controls.

1) High-density polyethylene can be obtained from the U.S. Pharmacopeia (Rockville, Maryland, USA) and Food and Drug
Safety Center, Hatano Research Institute (Ochiai 729-5, Hadanoshi, Kanagawa 257 - Japan). This information is given for the
convenience of the user of this part of ISO 10993 and does not constitute an endorsement by ISO of these products.
© ISO
ISO 10993-5:1999(E)
3.2
positive control material
material which, when tested in accordance with this part of ISO 10993, provides a reproducible cytotoxic response
NOTE The purpose of the positive control is to demonstrate appropriate test system response. For example, an organo-tin
2)
stabilized poly(vinylchloride) has been used as a positive control for solid materials and extracts. Dilutions of phenol, for
example, have been used as a positive control for extracts.
3.3
reagent control
extraction vehicle without test material subjected to extraction conditions and test procedures
NOTE For the purposes of this part of ISO 10993, this definition replaces that given in 3.1 of ISO 10993-12:1996.
3.4
culture vessels
vessels appropriate for cell culture, including glass Petri dishes, plastic culture flasks or plastic multiwells and
microtitre plates
NOTE These vessels may be used interchangeably in these methods provided that they meet the requirements of tissue
culture grade and are suitable for use with mammalian cells.
3.5
subconfluency
approximately 80 % confluency, i.e. the end of the logarithmic phase of growth
4 Sample preparation
4.1 General
The test shall be performed on either
a) an extract of the material; and/or
b) the material itself.
Sample preparation shall be in accordance with ISO 10993-12.
4.2 Preparation of liquid extracts of material
4.2.1 Principles of extraction
Extraction conditions should attempt to simulate or exaggerate the conditions of clinical use so as to determine the
potential toxicological hazard, without causing significant changes in the test material such as fusion, melting or
alteration of the chemical structure.
NOTE The concentration of any endogenous or extraneous substances in the extract, and hence the amount of exposure
to the test cells, depends on the interfacial area, extraction volume, pH, chemical solubility, diffusion rate, osmolarity, agitation,
temperature, time and other factors.

2) Organo-tin poly(vinyl chloride) positive control material is available from SIMS Portex Ltd, Hythe, Kent, CT21 6JL, UK,
(product No 499-300-000). The ZDEC and ZDBC polyurethanes are available from Food and Drug Safety Center, Hatano
Research Institute, (Ochiai 729-5, Hadanoshi, Kanagawa 257, Japan). This information is given for the convenience of the user
of this part of ISO 10993 and does not constitute an endorsement by ISO of the product.
© ISO
ISO 10993-5:1999(E)
4.2.2 Extraction vehicle
For mammalian cell assays, one or more of the following solvents shall be used. The choice of the extraction
vehicule(s) shall be justified:
a) culture medium with serum;
b) culture medium without serum;
c) physiological saline solution;
d) other suitable solvent.
NOTE The choice of solvent should reflect the aim of the extraction and consideration shall be given to the use of both a
polar and a nonpolar solvent. Suitable solvents include purified water, vegetable oil and dimethyl sulfoxide (DMSO). DMSO is
known to be cytotoxic in selected assay systems at greater than 0,5 % (volume fraction) concentrations.
4.2.3 Extraction conditions
4.2.3.1  The extraction shall be performed in sterile, chemically inert closed containers using aseptic techniques, in
general accordance with ISO 10993-12.
4.2.3.2  Recommended extraction conditions are:
a) not less than 24 h at (37 + 2) °C;
b) (72 + 2) h at (50 + 2) °C;
c) (24 + 2) h at (70 + 2) °C;
d) (1 + 0,2) h at (121 + 2) °C.
The recommended conditions may be applied according to the device characteristics and specific conditions for
use.
Extraction procedures using culture medium with serum can only be used under the conditions specified in
4.2.3.2 a).
4.2.3.3  If the extract is filtered, centrifuged or processed by other methods prior to being applied to the cells, this
shall be included in the final report (see clause 9). Any pH adjustment of the extract shall be reported. Manipulation
of the extract, such as by pH adjustment, could influence the
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