DIN EN 14675-2015 en 7257 Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of virucidal activity of chemical disinfectants and antiseptics u.pdf

1、June 2015Translation by DIN-Sprachendienst.English price group 15No part of this translation 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 71

2、.100.35!%CZd“2325565www.din.deDDIN EN 14675Chemical disinfectants and antiseptics Quantitative suspension test for the evaluation of virucidal activity ofchemical disinfectants and antiseptics used in the veterinary area Test method and requirements (Phase 2, step 1);English version EN 14675:2015,En

3、glish translation of DIN EN 14675:2015-06Chemische Desinfektionsmittel und Antiseptika Quantitativer Suspensionsversuch zur Bestimmung der viruziden Wirkung chemischerDesinfektionsmittel und Antiseptika fr den Veterinrbereich Prfverfahren und Anforderungen (Phase 2, Stufe 1);Englische Fassung EN 146

4、75:2015,Englische bersetzung von DIN EN 14675:2015-06Antiseptiques et dsinfectants chimiques Essai quantitatif de suspension pour lvaluation de lactivit virucide des antiseptiques etdes dsinfectants chimiques utiliss dans le domaine vtrinaire Mthode dessai et prescriptions (phase 2, tape 1);Version

5、anglaise EN 14675:2015,Traduction anglaise de DIN EN 14675:2015-06SupersedesDIN EN 14675:2006-04www.beuth.deIn case of doubt, the German-language original shall be considered authoritative.Document comprises 36 pages06.15 DIN EN 14675:2015-06 2 A comma is used as the decimal marker. National forewor

6、d This document (EN 14675:2015) has been prepared by Technical Committee CEN/TC 216 “Chemical dis-infectants and antiseptics” (Secretariat: AFNOR, France), Working Group WG 2 “Veterinary use” (Secretariat: BSI, United Kingdom). The responsible German body involved in its preparation was the DIN-Norm

7、enausschuss Lebensmittel und landwirtschaftliche Produkte (DIN Standards Committee Food and Agricultural Products), Working Committee NA 057-02-03 AA Desinfektionsmittel Tierhaltung/Lebensmittelbereich (DTL). Amendments This standard differs from DIN EN 14675:2006-04 as follows: a) Clause 2 “Normati

8、ve references” has been added; b) Clause 3 “Terms and definitions” has been revised; c) Clause 4 “Requirements” has been revised, Table 1 “Obligatory and additional test conditions” has been added; d) Subclause 5.6.3 “Virus titration on monolayers of cells on microtiter plates” has been added; e) Su

9、bclause 5.8 “Control of efficiency for suppression of disinfectant activity” has been added; f) Annex B has been revised and Subclause B.4 “Detoxification of test mixtures by molecular sieving” has been added; g) the former Annex E (informative) “Information on the application and interpretation of

10、European Standards on chemical disinfectants and antiseptics” has been deleted; h) Annex E (informative) “Presentation of test results of one active concentration” has been added; i) the standard has been editorially revised. Previous editions DIN EN 14675: 2006-04 EN 14675 April 2015 ICS 71.100.35

11、Supersedes EN 14675:2006English Version Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of virucidal activity of chemical disinfectants and antiseptics used in the veterinary area - Test method and requirements (Phase 2, step 1) Antiseptiques et dsinfectants

12、chimiques - Essai quantitatif de suspension pour lvaluation de lactivit virucide des antiseptiques et des dsinfectants chimiques utiliss dans le domaine vtrinaire - Mthode dessai et prescriptions (phase 2, tape 1) Chemische Desinfektionsmittel und Antiseptika - Quantitativer Suspensionsversuch zur B

13、estimmung der viruziden Wirkung chemischer Desinfektionsmittel und Antiseptika fr den Veterinrbereich - Prfverfahren und Anforderungen (Phase 2, Stufe 1) This European Standard was approved by CEN on 14 February 2015. CEN members are bound to comply with the CEN/CENELEC Internal Regulations which st

14、ipulate 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 Europea

15、n 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 t

16、he 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, Roman

17、ia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey andUnited Kingdom. CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels 2015 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 14675:2015 EEUROPEAN STANDARDNORME

18、EUROPENNEEUROPISCHE NORMEUROPEAN COMMITTEE FOR STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR NORMUNGEN 14675:2015 (E) 2 Contents Page Foreword 4 Introduction .5 1 Scope 6 2 Normative references 6 3 Terms and definitions .6 4 Requirements .7 5 Test method 8 5.1 Principle 8 5.2 M

19、aterials and reagents 8 5.2.1 Test virus 8 5.2.2 Culture media and reagents .9 5.3 Apparatus and glassware 15 5.3.1 General . 15 5.3.2 Usual microbiological laboratory equipment and, in particular, the following: . 15 5.4 Product test solutions 16 5.5 Procedure for assessing the virucidal activity o

20、f the product . 17 5.5.1 Experimental conditions (obligatory and additional) 17 5.5.2 Preparation of the test virus suspension . 17 5.5.3 Preparation of cell line . 18 5.6 Infectivity assay 18 5.6.1 Predilution of viral suspension . 18 5.6.2 Virus titration of cells in suspension on microtitre plate

21、s . 18 5.6.3 Virus titration on monolayers of cells on microtiter plates 18 5.6.4 Plaque assay . 18 5.7 Virucidal test preparation . 19 5.7.1 General . 19 5.7.2 Test method . 19 5.8 Control of efficiency for suppression of disinfectant activity . 19 5.8.1 Dilution in ice-cold medium . 19 5.8.2 Filtr

22、ation technique. 19 6 Calculation and expression of results 20 6.1 Protocol of the CPE result . 20 6.2 Calculation of infectivity titre (TCID50) 20 6.3 Calculation of PFU 20 6.4 Verification of the methodology 20 6.5 Calculation of the virucidal activity of products . 20 6.6 Expression of results .

23、20 7 Conclusion 21 7.1 General . 21 7.2 Test report . 21 Annex A (informative) Referenced strains of national collections . 23 Annex B (normative) Cytotoxicity, reference inactivation test, test virus titration and detoxification of test mixtures . 24 B.1 Cytotoxicity caused by product solutions . 2

24、4 B.2 Reference inactivation test 24 DIN EN 14675:2015-06EN 14675:2015 (E) 3 B.3 Titration of test virus suspension 25 B.4 Detoxification of test mixtures by molecular sieving 25 Annex C (informative) Calculation of the viral infectivity titre . 28 C.1 Quantal tests - Example of TCID50determination

25、by the Spearman-Krber method 28 C.2 Plaque test . 28 Annex D (informative) Example of a typical test report 30 Annex E (informative) Presentation of test results of one active concentration . 32 Bibliography 34 DIN EN 14675:2015-06EN 14675:2015 (E) 4 Foreword This document (EN 14675:2015) has been p

26、repared by Technical Committee CEN/TC 216 “Chemical disinfectants and antiseptics”, the secretariat of which is held by AFNOR. 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 2015 and conf

27、licting national standards shall be withdrawn at the latest by October 2015. 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 docu

28、ment supersedes EN 14675:2006. 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 Rep

29、ublic 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 the United Kingdom. DIN EN 14675:2015-06EN 14675:2015 (E) 5 Introduction Thi

30、s European Standard specifies a suspension test for establishing whether a chemical disinfectant or antiseptic has or does not have a virucidal activity in the areas described in the scope. This laboratory test takes into account practical conditions of application of the product including contact t

31、ime, temperature, test organisms and interfering substances, i.e. conditions which may influence its action in practical situations. The conditions are intended to cover general purposes and to allow reference between laboratories and product types. Each utilization concentration of the chemical dis

32、infectant or antiseptic found by this test corresponds to defined experimental conditions. However, for some applications the recommendations of use of a product may differ and therefore additional test conditions need to be used. DIN EN 14675:2015-06EN 14675:2015 (E) 6 1 Scope This European Standar

33、d specifies a test method and the minimum requirements for virucidal activity of chemical disinfectant and antiseptic products that form a homogeneous, physically stable preparation when diluted with hard water or in the case of ready-to-use-products with water. Products can only be tested at a conc

34、entration of 80 % or less as some dilution is always produced by adding the test organisms and interfering substance. This European Standard applies to products that are used in the veterinary area, i.e. in the breeding, husbandry, production, transport and disposal of all animals except when in the

35、 food chain following death and entry to the processing industry. NOTE 1 The method described is intended to determine the virucidal activity of commercial formulations or active substances under the conditions in which they are used. NOTE 2 This method corresponds to a phase 2 step 1. 2 Normative r

36、eferences The following documents, in whole or in part, are normatively referenced in this document and are indispensable for its application. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applie

37、s. EN 14885, Chemical disinfectants and antiseptics Application of European Standards for chemical disinfectants and antiseptics 3 Terms and definitions For the purposes of this document, the terms and definitions given in EN 14885 and the following apply: 3.1 cytotoxicity morphological alteration o

38、f cells and/or their destruction or their reduced sensitivity to virus multiplication caused by the product 3.2 plaque forming units PFU number of infectious virus particles per unit volume (ml) 3.3 reference test for virus inactivation test with a defined reagent (e.g. formalin) instead of a produc

39、t for the internal control of the test Note 1 to entry: Results of reference virus inactivation test should be within limits for validating the method. 3.4 reference virus suspension virus suspension of a defined virus strain which is not passaged more than 10 times, is maintained in national cultur

40、e collection centres and kept in small volumes (less than 1 ml) at a temperature of -70C or preferably at about -196 C under liquid nitrogen Note 1 to entry: Stock virus suspensions are prepared from reference virus suspensions. DIN EN 14675:2015-06EN 14675:2015 (E) 7 3.5 stock virus suspension viru

41、s suspension of a defined strain that is multiplied in a suitable cell line which produces high virus titers, to obtain a virus suspension of the same characteristics as the reference virus suspension and kept in a small volume at a temperature of below 70 C or preferably at about -196 C over liquid

42、 nitrogen 3.6 test virus suspension virus suspension that is used in the virucidal testing of the disinfectant 3.7 tissue culture infectious dose TCID50viral dose that induces a cytopathic effect (CPE) (3.8) in 50 % of inoculated cell culture 3.8 viral cytopathic effect CPE morphological alteration

43、of cells and/or their destruction as a consequence of virus multiplication 3.9 viral plaque area of lysis formed in a cell monolayer under semisolid medium due to infection by and multiplication of a single infectious virus particle 3.10 virus titre amount of infectious virus per unit volume present

44、 in a cell culture lysate or in a solution 4 Requirements The product when diluted with hard water (5.2.2.3) or in the case of ready-to-use products with water (5.2.2.2) and tested in accordance with Table 1 and Clause 5 shall demonstrate at least a lg reduction in virus titre of 4. It is possible t

45、o test also the product as delivered (highest test concentration is 80 %). Table 1 Obligatory and additional test conditions Obligatory conditions Additional conditions Test organism Bovine enterovirus Type 1 (ECBO) Test temperature a10 C 4 C, 20 C or 40 C Contact time b30 min 1 min, 5 min and 60 mi

46、n Interfering substancelow level soiling c3,0 g/l bovine albumin Interfering substance-high level soiling c10 g/l bovine albumin plus 10 g/l yeast extract aAllowed deviation 1 C. bThe allowed deviation for each chosen contact time is 10 s, except for 1 min for which it is 5 s. c To be chosen accordi

47、ng to practical applications. DIN EN 14675:2015-06EN 14675:2015 (E) 8 5 Test method 5.1 Principle 5.1.1 A sample of the product as delivered and/or diluted with hard water (or water for ready to use products) is added to a test suspension of virus in a solution of an interfering substance. The mixtu

48、re is maintained at 10 C 1 C for 30 min 10 s (obligatory test conditions). At the end of the contact time, 0,5 ml of virus/disinfectant mixture is taken. The virucidal activity is immediately suppressed by dilution in ice-cold diluent. A dilution series with a factor of ten is prepared in an ice-col

49、d medium held in an ice bath for 10 min. Pipettes shall be changed after each dilution to avoid carry-over of virus. The dilutions are transferred into cell culture units (wells of microtitre plates) containing suspended cells. Eight series units shall be inoculated with each dilution. After incubation, the titre of infectivity is calculated. The titration results of quantal tests shall show dilution steps with the percentage of