prEN ISO 10993-5

Biological evaluation of medical devices - Part 5: Tests for in vitro cytotoxicity (ISO 10993-5:1999)

20090130 - WI abandoned due to duplication under WI 00206035

Biologische Beurteilung von Medizinprodukten - Teil 5: Prüfungen auf Zytotoxizität: In vitro-Methoden (ISO 10993-5:1999)

Évaluation biologique des dispositifs médicaux - Partie 5 : Essais concernant la cytotoxicité in vitro (ISO 10993-5:1999)

Biološko ovrednotenje medicinskih pripomočkov - 5. del: Preskusi za ugotavljanje citotoksičnosti in vitro (ISO 10993-5:1999)

General Information

• Status: Not Published
• Publication Date: 28-Jun-2009
• Withdrawal Date: 28-Dec-2009
• ICS: 11.100 - Laboratory medicine
• 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: 5098 - Decision to abandon - Formal Approval
• Start Date: 28-Mar-2009
• 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: kSIST prEN ISO 10993-5:2009 - Biological evaluation of medical devices - Part 5: Tests for in vitro cytotoxicity (ISO 10993-5:1999)

Relations

• Revises: EN ISO 10993-5:1999 - Biological evaluation of medical devices - Part 5: Tests for in vitro cytotoxicity (ISO 10993-5:1999)
• Effective Date: 18-Jan-2023

Overview

prEN ISO 10993-5 / ISO 10993-5:1999 specifies principles and methods for the biological evaluation of medical devices by in vitro cytotoxicity testing. Rather than prescribing a single assay, it defines a testing scheme and decision steps to select appropriate methods that assess whether device materials or their extracts cause toxic effects on cultured mammalian cells. This document was adopted as a CEN draft (prEN) based on ISO 10993-5 and is linked to European regulatory requirements for medical devices.

Key topics and requirements

• Test categories: extract tests, direct-contact tests and indirect-contact tests - choice depends on material type, site and mode of use.
• Sample preparation: extraction procedures follow ISO 10993-12 principles. Extraction aims to simulate or exaggerate clinical exposure without altering material chemistry.
• Extraction vehicles: culture medium (with/without serum), physiological saline, and other solvents (e.g., purified water, vegetable oil, DMSO). Choice must be justified; DMSO is noted as cytotoxic in some assays at >0.5% v/v.
• Extraction conditions (recommended examples):
  • ≥24 h at (37 ± 2) °C
  • 72 ± 2 h at (50 ± 2) °C
  • 24 ± 2 h at (70 ± 2) °C
  • 1 ± 0.2 h at (121 ± 2) °C
• Controls:
  • Negative control material - demonstrates background response.
  • Positive control material - provides a reproducible cytotoxic response.
  • Reagent control - extraction vehicle processed without test material.
• Cell culture conditions: use appropriate tissue-culture–grade vessels and cells at ~80% confluency (subconfluency) unless justified otherwise.
• Endpoints and assessment: multiple endpoint categories are allowed - morphological assessment, measures of cell damage, cell growth, or specific metabolic functions. The standard permits a battery of tests to cover different mechanisms.

Practical applications and users

• Who uses it: medical device manufacturers, biocompatibility and regulatory affairs teams, contract testing laboratories, toxicologists and research groups assessing material safety.
• Why it matters: in vitro cytotoxicity testing is an early, cost-effective screen for biocompatibility. Results support risk assessments, product development decisions, and compliance with EU Directives (e.g., 93/42/EEC and 90/385/EEC) when referenced appropriately.
• Where applied: screening new polymers, coatings, adhesives, implants, disposables and other device components for cytotoxic leachables or surface-mediated toxicity.

Related standards

• ISO 10993-1 (Evaluation and testing framework)
• ISO 10993-12 (Sample preparation and reference materials)
• Other parts of the ISO 10993 series covering systemic toxicity, irritation, implantation, and chemical characterization.

Keywords: ISO 10993-5, in vitro cytotoxicity, biological evaluation of medical devices, cytotoxicity testing, medical device biocompatibility, extract test, direct-contact test, ISO 10993 series.

Standards Content (Sample)

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Biologische Beurteilung von Medizinprodukten - Teil 5: Prüfungen auf Zytotoxizität: In vitro-Methoden (ISO 10993-5:1999)Évaluation biologique des dispositifs médicaux - Partie 5 : Essais concernant la cytotoxicité in vitro (ISO 10993-5:1999)Biological evaluation of medical devices - Part 5: Tests for in vitro cytotoxicity (ISO 10993-5:1999)11.100.20Biological evaluation of medical devicesICS:Ta slovenski standard je istoveten z:prEN ISO 10993-5kSIST prEN ISO 10993-5:2009en01-marec-2009kSIST prEN ISO 10993-5:2009SLOVENSKI
STANDARD
EUROPEAN STANDARDNORME EUROPÉENNEEUROPÄISCHE NORMFINAL DRAFTprEN ISO 10993-5November 2008ICS 11.100.20Will supersede EN ISO 10993-5:1999
English VersionBiological evaluation of medical devices - Part 5: Tests for invitro cytotoxicity (ISO 10993-5:1999)Évaluation biologique des dispositifs médicaux - Partie 5:Essais concernant la cytotoxicité in vitro (ISO 10993-5:1999)Biologische Beurteilung von Medizinprodukten - Teil 5:Prüfungen auf Zytotoxizität: In vitro-Methoden (ISO 10993-5:1999)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 10993-5:2008: EkSIST prEN ISO 10993-5:2009

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 10993-5:2009

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
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.
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 Annex I : 9
WARNING — Other requirements and other EU Directives may be applicable to the product(s) falling within the scope of this standard.
AReference numberISO 10993-5:1999(E)INTERNATIONALSTANDARDISO10993-5Second edition1999-05-15Biological evaluation of medical devices —Part 5:Tests for in vitro cytotoxicityÉvaluation biologique des dispositifs médicaux —Partie 5: Essais concernant la cytotoxicité in vitrokSIST prEN ISO 10993-5:2009

ISO 10993-5:1999(E)©
ISO 1999All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means, electronicor mechanical, including photocopying and microfilm, without permission in writing from the publisher.International Organization for StandardizationCase postale 56 · CH-1211 Genève 20 · [e-mail] in SwitzerlandiiContents1 Scope.12 Normative references.13 Terms and definitions.14 Sample preparation.25 Cell lines.46 Culture medium.47 Preparation of cell stock culture.48 Test procedures.59 Test report.810 Assessment of results.8kSIST prEN ISO 10993-5:2009

© ISOISO 10993-5:1999(E)iiiForewordISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISOmember bodies). The work of preparing International Standards is normally carried out through ISO technicalcommittees. Each member body interested in a subject for which a technical committee has been established hasthe right to be represented on that committee. International organizations, governmental and non-governmental, inliaison with ISO, also take part in the work. ISO collaborates closely with the International ElectrotechnicalCommission (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 ofmedical 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:¾ Part 1: Evaluation and testing¾ Part 2: Animal welfare requirements¾ Part 3: Tests for genotoxicity, carcinogenicity and reproductive toxicity¾ Part 4: Selection of tests for interactions with blood¾ Part 5: Tests for in vitro cytotoxicity¾ Part 6: Tests for local effects after implantation¾ Part 7: Ethylene oxide sterilization residuals¾ Part 8: Guidance for reference materials¾ Part 9: Framework for the identification and quantification of potential degradation products¾ Part 10: Tests for irritation and sensitization¾ Part 11: Tests for systemic toxicity¾ Part 12: Sample preparation and reference materials¾ Part 13: Identification and quantification of degradation products from polymers¾ Part 14: Identification and quantification of degradation products from ceramics¾ Part 15: Identification and quantification of degradation products from metals and alloys¾ Part 16: Toxicokinetic study design for degradation products and leachables¾ Part 17: Methods for establishment of allowable limits for leachable substances using health-based riskassessment¾ Part 18: Chemical characterizationFuture parts will deal with other relevant aspects of biological testing.kSIST prEN ISO 10993-5:2009

ISO 10993-5:1999(E)© ISOivIntroductionDue to the general applicability of in vitro cytotoxicity tests and their widespread use in evaluating a large range ofmedical devices and materials, it is the purpose of this part of ISO 10993, rather than to specify a single test, todefine a scheme for testing which requires decisions to be made in a series of steps. This should lead to theselection 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 potentialsite 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 thecultured 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 isthe intention of this part of ISO 10993 to leave open the choice of type of evaluation. Such a strategy makesavailable 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 categoriesof 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. Theinvestigator should be aware of the categories of test and into which a particular technique fits, in order thatcomparisons may be made with other results on similar medical devices or materials, and in order thatinterlaboratory tests may be conducted.kSIST prEN ISO 10993-5:2009

INTERNATIONAL STANDARD
© ISOISO 10993-5:1999(E)1Biogical evaluation of medical devices —Part 5:Tests for in vitro cytotoxicity1 ScopeThis 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 diffusiona) with extracts of a device, and/orb) in contact with a device.These methods are designed to determine the biological response of mammalian cells in vitro using appropriatebiological parameters.2 Normative referencesThe following normative documents contain provisions which, through reference in this text, constitute provisions ofthis part of ISO 10993. For dated references, subsequent amendments to, or revisions of, any of these publicationsdo not apply. However, parties to agreements based on this part of ISO 10993 are encouraged to investigate thepossibility of applying the most recent editions of the normative documents indicated below. For undatedreferences, the latest edition of the normative document referred to applies. Members of ISO and IEC maintainregisters 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 referencematerials.3 Terms and definitionsFor the purposes of this part of ISO 10993, the terms and definitions given in ISO 10993-1 and the following apply.3.1negative control materialmaterial which, when tested in accordance with this part of ISO 10993, does not produce a cytotoxic responseNOTE The purpose of the negative control is to demonstrate background response. For example, high-density poly-ethylene1) 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 DrugSafety Center, Hatano Research Institute (Ochiai 729-5, Hadanoshi, Kanagawa 257 - Japan). This information is given for theconvenience of the user of this part of ISO 10993 and does not constitute an endorsement by ISO of these products.kSIST prEN ISO 10993-5:2009

ISO 10993-5:1999(E)© ISO23.2positive control materialmaterial which, when tested in accordance with this part of ISO 10993, provides a reproducible cytotoxic responseNOTE The purpose of the positive control is to demonstrate appropriate test system response. For example, an organo-tinstabilized poly(vinylchloride)2) has been used as a positive control for solid materials and extracts. Dilutions of phenol, forexample, have been used as a positive control for extracts.3.3reagent controlextraction vehicle without test material subjected to extraction conditions and test proceduresNOTE
For the purposes of this part of ISO 10993, this definition replaces that given in 3.1 of ISO 10993-12:1996.3.4culture vesselsvessels appropriate for cell culture, including glass Petri dishes, plastic culture flasks or plastic multiwells andmicrotitre platesNOTE These
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