BS EN 14675-2015 Chemical disinfectants and antiseptics Quantitative suspension test for the evaluation of virucidal activity of chemical disinfectants and antiseptics used in the a.pdf

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1、BSI Standards PublicationBS EN 14675:2015Chemical disinfectants andantiseptics Quantitativesuspension test for theevaluation of virucidal activityof chemical disinfectantsand antiseptics used in theveterinary area Test methodand requirements (Phase 2,step 1)BS EN 14675:2015 BRITISH STANDARDNational

2、forewordThis British Standard is the UK implementation of EN 14675:2015. Itsupersedes BS EN 14675:2006 which is withdrawn.The UK participation in its preparation was entrusted to TechnicalCommittee CH/216, Chemical disinfectants and antiseptics.A list of organizations represented on this committee c

3、an beobtained on request to its secretary.This publication does not purport to include all the necessaryprovisions of a contract. Users are responsible for its correctapplication. The British Standards Institution 2015. Published by BSI StandardsLimited 2015ISBN 978 0 580 79145 1ICS 11.080.20; 11.22

4、0Compliance with a British Standard cannot confer immunity fromlegal obligations.This British Standard was published under the authority of theStandards Policy and Strategy Committee on 30 April 2015.Amendments issued since publicationDate Text affectedBS EN 14675:2015EUROPEAN STANDARD NORME EUROPEN

5、NE EUROPISCHE NORM EN 14675 April 2015 ICS 71.100.35 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 requirem

6、ents (Phase 2, step 1) Antiseptiques et dsinfectants 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

7、 Antiseptika - Quantitativer Suspensionsversuch zur Bestimmung 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 comp

8、ly 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

9、 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 sa

10、me 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

11、, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels 20

12、15 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 14675:2015 EBS EN 14675:2015EN 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 meth

13、od 8 5.1 Principle 8 5.2 Materials 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 assessin

14、g the virucidal activity of 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 susp

15、ension on microtitre plates . 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-co

16、ld medium . 19 5.8.2 Filtration 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.

17、6 Expression of results . 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 cause

18、d by product solutions . 24 B.2 Reference inactivation test 24 BS EN 14675:2015EN 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 o

19、f TCID50determination 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 BS EN 14675:2015EN 14675:2015 (E) 4 Foreword This document (EN 1467

20、5:2015) has been prepared 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 Oct

21、ober 2015 and conflicting 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

22、 rights. This document 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, F

23、ormer 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 the United Kingdom. BS EN 14675:2015EN 14675:2015 (E) 5 In

24、troduction This 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 inclu

25、ding contact time, 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 th

26、e chemical disinfectant 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. BS EN 14675:2015EN 14675:2015 (E) 6 1 Scope This Europe

27、an Standard 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

28、 at a concentration 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 w

29、hen in the 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 N

30、ormative references 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 amendmen

31、ts) applies. 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 al

32、teration of 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 o

33、f a product 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 natio

34、nal culture 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. BS EN 14675:2015EN 14675:2015 (E) 7 3.5 stock virus suspensio

35、n virus 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

36、liquid 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 alter

37、ation 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 p

38、resent 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 poss

39、ible to 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

40、 60 min 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 a

41、ccording to practical applications. BS EN 14675:2015EN 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 mix

42、ture 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-c

43、old 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 incub

44、ation, the titre of infectivity is calculated. The titration results of quantal tests shall show dilution steps with the percentage of positive results (presence of CPE or plaques) lying between 100 % and 0 %. The values are calculated according to Spearman and Krber (see Annex C). Values of virus i

45、nactivation are calculated from differences of virus titres before and after treatment with the product. 5.1.2 Additional and optional contact times and temperatures are specified. 5.2 Materials and reagents 5.2.1 Test virus The virucidal activity shall be evaluated using the following strain: Bovin

46、e enterovirus Type 1 (Enteric Cytopathogenic Bovine Orphan Virus ECBO) ATCC VR-2481). NOTE 1 Bovine enterovirus Type 1, strain ECBO, is selected as the model virus for the large Genus Picornavirus. The Genus Picornavirus includes many clinically important virus species, for example Coxsackie A and B

47、, and enteric cytopathogenic human orphan (ECHO). Some of these viruses are of primary importance and therefore a constant risk for animals in the veterinary area. Moreover, they have a high resistance to chemicals, are acid-stable (except inter alia rhinovirus, aphtovirus) and are unaffected by lip

48、id solvents such as ether, and most detergents or quaternary ammonium products. NOTE 2 It is the model virus for all applications namely for disinfection of instruments and surfaces and post-contamination treatment of post-mortem rooms, kennels and for animal accommodation. NOTE 3 Due to large diffe

49、rences of resistance against physical and chemical influences between and within different virus groups, the testing of all viruses against any particular chemical disinfectant or antiseptic is financially impossible. Therefore, in this European Standard, testing is restricted to only one so called model virus that has been selected on the basis of the present knowledge as a representative example of virus tenacity and of important clinical relevance in the veterinary area. If a chemical disinfectant or antiseptic sho