DIN EN ISO 10993-5-2009 Biological evaluation of medical devices - Part 5 Tests for in vitro cytotoxicity (ISO 10993-5 2009) English version of DIN EN ISO 10993-5 2009-10《医疗设备的生物评估.pdf

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1、October 2009DEUTSCHE NORM English price group 18No part of this standard may be reproduced without prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).ICS 11.100.20!$Z=v“1552683

2、www.din.deDDIN EN ISO 10993-5Biological evaluation of medical devices Part 5: Tests for in vitro cytotoxicity (ISO 10993-5:2009)English version of DIN EN ISO 10993-5:2009-10Biologische Beurteilung von Medizinprodukten Teil 5: Prfungen auf In-vitro-Zytotoxizitt (ISO 10993-5:2009)Englische Fassung DIN

3、 EN ISO 10993-5:2009-10SupersedesDIN EN ISO 10993-5:1999-11www.beuth.deDocument comprises pages442 National oreword DIN EN ISO 10993-5:2009-10 This standard has been prepared by Technical Committee ISO/TC 194 “Biological evaluation of medical devices“ in collaboration with Technical Committee CEN/TC

4、 206 “Biological evaluation of medical devices“ (Secretariat: NEN, Netherlands). The responsible German body involved in its preparation was the Normenausschuss Feinmechanik und Optik (Optics and Precision Mechanics Standards Committee), Technical Committee NA 027-02-12 AA Biologische Beurteilung vo

5、n Medizinprodukten. 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 system Part 2: Animal welfare requirements Part 3: Tests for genotoxicity, carcinogenicity and reproductive toxicit

6、y 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

7、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 cer

8、amics 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: Phy

9、sico-chemical, morphological and topographical characterization of materials Technical Specification Part 20: Principles and methods for immunotoxicology testing of medical devices Technical Specification f3 DIN EN ISO 10993-5:2009-10 The DIN Standards corresponding to the International and European

10、 Standards referred to in clause 2 of this document are as follows: ISO 10993-1 DIN EN ISO 10993-1 ISO 10993-12 DIN EN ISO 10993-12 Amendments This standard differs from DIN EN ISO 10993-5:1999-11 as follows: a) The standard has been editorially revised. b) The preparation of controls has been inclu

11、ded in clause 4. c) The extraction conditions have been revised (4.2.3.2). d) In clause 8.5, Tables 1 and 2 have been added with regard to the qualitative evaluation of cytotoxicity. e) Annex A “Neutral red uptake (NRU) cytotoxicity test” has been added. f) Annex B “Colony formation cytotoxicity tes

12、t” has been added. g) Annex C “MTT cytotoxicity test” has been added. h) Annex D “XTT cytotoxicity test” has been added. i) Annex ZA (informative) concerning the relationship between this European Standard and the essential requirements of EU Directive 93/42/EEC has been added. j) Annex ZB (informat

13、ive) concerning the relationship between this European Standard and the essential requirements of EU Directive 90/385/EEC has been added. Previous editions DIN EN 30993-5: 1994-08 DIN EN ISO 10993-5: 1999-11 4 National Annex NA (informative) Bibliography DIN EN ISO 10993-1, Biological evaluation of

14、medical devices Part 1: Evaluation and testing DIN EN ISO 10993-12, Biological evaluation of medical devices Part 12: Sample preparation and reference materials DIN EN ISO 10993-5:2009-10 EUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN ISO 10993-5June 2009ICS 11.100.20 Supersedes EN ISO 10993-5:19

15、99 English VersionBiological evaluation of medical devices - Part 5: Tests for invitro cytotoxicity (ISO 10993-5:2009)valuation biologique des dispositifs mdicaux - Partie 5:Essais concernant la cytotoxicit in vitro 5:2009)Biologische Beurteilung von Medizinprodukten - Teil 5:Prfungen auf In-vitro-Z

16、ytotoxizitt This European Standard was approved by CEN on 17 April 2009.CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibliograph

17、ical references concerning such nationalstandards 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 translationunder the responsibility of a CEN

18、member into its own language and notified to the CEN Management Centre has the same status as theofficial 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,

19、 Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR NORMUNGManagement Centre: Avenue Marnix 17, B-1000 Brusse

20、ls 2009 CEN All rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN ISO 10993-5:2009: E(ISO 10993- (ISO 10993-5:2009)2 Contents Page DIN EN ISO 10993-5:2009-10 EN ISO 10993-5:2009 (E) Foreword .3 Introduction.4 1 Scope5 2 Normative references5 3

21、 Terms and definitions .5 4 Sample and control preparation 6 4.1 General .6 4.2 Preparation of liquid extracts of material .7 4.3 Preparation of material for direct-contact tests .8 4.4 Preparation of controls.9 5 Cell lines.9 6 Culture medium .9 7 Preparation of cell stock culture.10 8 Test procedu

22、res.10 8.1 Number of replicates.10 8.2 Test on extracts .10 8.3 Test by direct contact .11 8.4 Test by indirect contact 11 8.5 Determination of cytotoxicity.13 9 Test report14 10 Assessment of results 15 Annex A (informative) Neutral red uptake (NRU) cytotoxicity test 16 Annex B (informative) Colony

23、 formation cytotoxicity test .23 Annex C (informative) MTT cytotoxicity test28 Annex D (informative) XTT cytotoxicity test.33 Bibliography38 Annex ZA (informative) Relationship between this European Standard and the Essential Requirements of EU Directive 93/42/EEC .39 Annex ZB (informative) Relation

24、ship between this European Standard and the Essential Requirements of EU Directive 90/385/EEC .40 3 Foreword This document (EN ISO 10993-5:2009) has been prepared by Technical Committee ISO/TC 194 “Biological evaluation of medical devices“ in collaboration with Technical Committee CEN/TC 206 “Biolog

25、ical 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 December 2009, and conflicting national standards shall be withdrawn at

26、 the latest by December 2009. 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 10993-5:1999. This docum

27、ent has been prepared under a mandate given to CEN by the European Commission and the European Free Trade Association, and supports essential requirements of EC Directives. For relationship with EC Directives, see informative Annex ZA and ZB, which are integral parts of this document. According to t

28、he 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, Lithua

29、nia, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. Endorsement notice The text of ISO 10993-5:2009 has been approved by CEN as EN ISO 10993-5:2009 without any modifications. DIN EN ISO 10993-5:2009-10 EN ISO

30、10993-5:2009 (E) Introduction Due to the general applicability of in vitro cytotoxicity tests and their widespread use in evaluating a large range of 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 require

31、s 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 evalua

32、ted, 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 e

33、valuation 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 that advocate in vitro biological t

34、ests. The numerous methods used and endpoints measured in cytotoxicity determination can be grouped into the following categories of evaluation: assessments of cell damage by morphological means; measurements of cell damage; measurements of cell growth; measurements of specific aspects of cellular m

35、etabolism. There are several means of producing results in each of these four categories. The investigator should be aware of the test categories and into which category a particular technique fits, in order that comparisons be able to be made with other results on similar devices or materials both

36、at the intra- and interlaboratory level. Examples of quantitative test protocols are given in annexes. Guidance for the interpretation of the results is given in this part of ISO 10993. DIN EN ISO 10993-5:2009-10 EN ISO 10993-5:2009 (E) 41 Scope This part of ISO 10993 describes test methods to asses

37、s the in vitro cytotoxicity of medical devices. These methods specify the incubation of cultured cells in contact with a device and/or extracts of a device either directly or through diffusion. These methods are designed to determine the biological response of mammalian cells in vitro using appropri

38、ate biological parameters. 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.

39、 ISO 10993-1, Biological evaluation of medical devices Part 1: Evaluation and testing within a risk management system ISO 10993-12, Biological evaluation of medical devices Part 12: Sample preparation and reference materials 3 Terms and definitions For the purposes of this document, the terms and de

40、finitions given in ISO 10993-1 and the following apply. 3.1 culture vessels vessels appropriate for cell culture including glass petri dishes, plastic culture flasks or plastic multiwells and microtitre plates NOTE These can be used interchangeably in these methods provided that they meet the requir

41、ements of tissue culture grade and are suitable for use with mammalian cells. 3.2 positive control material material which, when tested in accordance with this part of ISO 10993, provides a reproducible cytotoxic response DIN EN ISO 10993-5:2009-10 EN ISO 10993-5:2009 (E) 5NOTE The purpose of the po

42、sitive control is to demonstrate an appropriate test system response. For example, an organotin-stabilized polyurethane1)has been used as positive control for solid materials and extracts. Dilutions of phenol, for example, have been used as a positive control for extracts. In addition to a material,

43、 pure chemicals can also be used to demonstrate the performance of the test system. 3.3 blank extraction vehicle not containing the test sample, retained in a vessel identical to that which holds the test sample and subjected to conditions identical to those to which the test sample is subjected dur

44、ing its extraction NOTE The purpose of the blank is to evaluate the possible confounding effects due to the extraction vessel, vehicle and extraction process. 3.4 negative control material material which, when tested in accordance with this part of ISO 10993, does not produce a cytotoxic response NO

45、TE The purpose of the negative control is to demonstrate background response of the cells. For example, high-density polyethylene2)for synthetic polymers, and aluminium oxide ceramic rods for dental material have been used as negative controls. 3.5 test sample material, device, device portion, compo

46、nent, extract or portion thereof that is subjected to biological or chemical testing or evaluation 3.6 subconfluency approximately 80 % confluency, i.e. the end of the logarithmic phase of growth 4 Sample and control preparation 4.1 General The test shall be performed on a) an extract of the test sa

47、mple and/or b) the test sample itself. Sample preparation shall be in accordance with ISO 10993-12. Negative and positive controls shall be included in each assay. 1) The ZDEC and ZDBC polyurethanes are available from the Food and Drug Safety Center, Hatano Research Institute, Ochiai 729-5, Hadanosh

48、i, Kanagawa 257, Japan. 2) High-density polyethylene can be obtained from the U.S. Pharmacopeia (Rockville, MD, USA) and from the Food and Drug Safety Center, Hatano Research Institute (Ochiai 729-5, Hadanoshi, Kanagawa 257, Japan). The information given in 1) and 2) is for the convenience of the us

49、er of this part of ISO 10993 and does not constitute an endorsement by ISO of these products. Equivalent products may be used if they can be shown to lead to the same results. DIN EN ISO 10993-5:2009-10 EN ISO 10993-5:2009 (E) 64.2 Preparation of liquid extracts of material 4.2.1 Principles of extraction Extracting conditions should attempt to simulate or exaggerate the clinical use conditions so as to determine the potential toxicological hazard without causing significant changes in the test sample, such as fus