SIST EN 14476:2005
(Main)Chemical disinfectants and antiseptics - Virucidal quantitative suspension test for chemical disinfectants and antiseptics used in human medicine - Test method and requirements (phase 2, step 1)
Chemical disinfectants and antiseptics - Virucidal quantitative suspension test for chemical disinfectants and antiseptics used in human medicine - Test method and requirements (phase 2, step 1)
This document specifies test methods and minimum requirements for virucidal activity of chemical disinfectants or antiseptic products for instruments, surfaces, laundry or hands that form a homogeneous physically stable preparation in water.
This document is applicable to a broad spectrum of viruses (see Annex B) and to areas and situations where disinfection is medically indicated. Such indications occur in patient care, for example:
3 in hospitals, in community medical facilities, and in dental institutions;
3 clinics of schools, kindergartens, and nursing homes;
and may occur in the workplace and in the home. It may also include services such as laundries and kitchens supplying products directly for the patients.
It is impossible to determine the virucidal activity of the undiluted product as some dilution is always produced by adding the inoculum. However, a disinfectant or antiseptic which is used in undiluted form is tested in 80% concentration and shall pass this test prior to further assessment.
Chemische Desinfektionsmittel und Antiseptika - Quantitativer Suspensionsversuch Viruzidie für in der Humanmedizin verwendete chemische Desinfektionsmittel und Antiseptika - Prüfverfahren und Anforderungen (Phase 2, Stufe 1)
Dieses Dokument legt Prüfverfahren und Mindestanforderungen an die viruzide Wirkung von chemischen Desinfektionsmitteln oder Antiseptika für Instrumente, Oberflächen oder Hände fest, die in Wasser als homogenes, physikalisch stabiles Präparat vorliegen.
Dieses Dokument ist auf ein breites Spektrum von Viren (siehe Anhang B) und auf Bereiche und Bedingungen anwendbar, wo eine Desinfektion aus medizinischen Gründen indiziert ist. Solche Indikationen ergeben sich bei der Versorgung von Patienten, beispielsweise
- in Krankenhäusern, kommunalen medizinischen Einrichtungen und im Dentalbereich;
- in medizinischen Bereichen in Schulen, Kindergärten und Heimen;
und können auch am Arbeitsplatz oder im privaten Bereich gegeben sein. Eingeschlossen sein können auch Einrichtungen wie Wäschereien und Küchen, die der direkten Versorgung von Patienten dienen.
Eine Bestimmung der viruziden Wirkung des unverdünnten Produktes ist nicht möglich, da durch Zugabe des Inokulums immer eine bestimmte Verdünnung bewirkt wird. Jedoch wird ein Desinfektionsmittel oder Antiseptikum, das in unverdünnter Form verwendet wird, in 80 %iger Konzentration geprüft und muss vor weiterer Bewertung diese Prüfung bestehen.
Antiseptiques et désinfectants chimiques - Essai virucide quantitatif de suspension pour les antiseptiques et désinfectants chimiques utilisés en médecine humaine - Méthode d'essai et prescriptions (phase 2, étape 1)
Kemična razkužila in antiseptiki - Kvantitativni suspenzijski preskus virucidnosti kemičnih razkužil in antiseptikov, ki se uporabljajo v humani medicini - Preskusna metoda in zahteve (faza 2, stopnja 1)
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2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Chemical disinfectants and antiseptics - Virucidal quantitative suspension test for chemical disinfectants and antiseptics used in human medicine - Test method and requirements (phase 2, step 1)Antiseptiques et désinfectants chimiques - Essai virucide quantitatif de suspension pour les antiseptiques et désinfectants chimiques utilisés en médecine humaine - Méthode d'essai et prescriptions (phase 2, étape 1)Chemische Desinfektionsmittel und Antiseptika - Quantitativer Suspensionsversuch Viruzidie für in der Humanmedizin verwendete chemische Desinfektionsmittel und Antiseptika - Prüfverfahren und Anforderungen (Phase 2, Stufe 1)Ta slovenski standard je istoveten z:EN 14476:2005SIST EN 14476:2005English
or
French or German
language11.080.20Dezinfektanti in antiseptikiDisinfectants and antisepticsICS:SLOVENSKI
STANDARDSIST EN 14476:200501-junij-2005
EUROPEAN STANDARDNORME EUROPÉENNEEUROPÄISCHE NORMEN 14476April 2005ICS 11.080.20English versionChemical disinfectants and antiseptics - Virucidal quantitativesuspension test for chemical disinfectants and antiseptics usedin human medicine - Test method and requirements (phase 2,step 1)Antiseptiques et désinfectans chimique - Essai quantitatifde suspension virucide pour les antiseptiques etdésinfectants chimiques utilisés en médecine humaine -Méthode d'essai et exigences (phase 2, étape 1)Chemische Desinfektionsmittel und Antiseptika -Quantitativer Suspensionsversuch Viruzidie für in derHumanmedizin verwendete chemische Desinfektionsmittelund Antiseptika - Prüfverfahren und Anforderungen (Phase2, Stufe 1)This European Standard was approved by CEN on 3 March 2005.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 bibliographical references concerning such nationalstandards may be obtained on application to the Central Secretariat 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 member into its own language and notified to the Central Secretariat has the same status as the officialversions.CEN members are the national standards bodies of Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France,Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia,Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMITÉ EUROPÉEN DE NORMALISATIONEUROPÄISCHES KOMITEE FÜR NORMUNGManagement Centre: rue de Stassart, 36
B-1050 Brussels© 2005 CENAll rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 14476:2005: E
EN 14476:2005 (E) 2 Contents Page Foreword.3 1 Scope.4 2 Normative references.4 3 Terms and definitions.4 4 Requirements.6 5 Materials and reagents.6 6 Test method (general).11 7 Test protocols with specific test viruses for specified intended use.15 8 Calculation and expression of results.18 9 Expression of results.19 Annex A (normative)
Detoxification of test mixtures by molecular sieving.21 Annex B (informative)
Examples of viruses which may contaminate human medical instruments, hands, surfaces.23 Annex C (informative)
Calculation of the viral infectivity titre.25 Annex D (informative)
Presentation of test results of one active concentration.27 Annex E (informative)
Quantitative determination of formaldehyde concentrations.30 Annex F (informative)
Information on the application and interpretation of European standards on chemical disinfectants and antiseptics.31 Bibliography.33
EN 14476:2005 (E) 3 Foreword This document (EN 14476:2005) 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 October 2005, and conflicting national standards shall be withdrawn at the latest by October 2005. 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. Other methods to evaluate the efficacy of chemical disinfectants and antiseptics for different applications in the medical field are in preparation. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.
EN 14476:2005 (E) 4 1 Scope This document specifies a test method and the minimum requirements for virucidal activity of chemical disinfectants or antiseptic products for instruments, surfaces or hands that form a homogeneous physically stable preparation when diluted with hard water – or in the case of ready-to-use products – with water. This document is applicable to a broad spectrum of viruses (Annex B) and to areas and situations where disinfection is medically indicated. Such indications occur in patient care, for example: in hospitals, in community medical facilities, and in dental institutions; in clinics of schools, of kindergartens, and of nursing homes; and may occur in the workplace and in the home. It may also include services such as laundries and kitchens supplying products directly for the patients. It is impossible to determine the virucidal activity of the undiluted product as some dilution is always produced by adding the inoculum and interfering substance. However, a disinfectant or antiseptic which is used in undiluted form is tested in 80% concentration and shall pass this test prior to further assessment. NOTE 1 The method described is intended to determine the 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 test (see Annex F). 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. EN 14820, Single-use containers for human venous blood specimen collection 3 Terms and definitions For the purposes of this document, the following terms and definitions apply. 3.1 clean conditions conditions representative of surfaces which have received a satisfactory cleaning programme and/or are known to contain minimal levels of organic and/or inorganic substances
3.2 cytotoxicity morphological alteration of cells and/or their destruction or their reduced sensitivity to virus multiplication caused by the product
3.3 dirty conditions conditions representative of surfaces which are known to or may contain organic and/or inorganic substances
3.4 inactivation of viruses reduction of infectivity of a virus by a product NOTE Alteration of antigenic reactivity or of any viral component does not necessarily mean reduction of infectivity of a virus.
EN 14476:2005 (E) 5 3.5 interfering substances protein solutions and erythrocytes that are added to a test virus suspension before addition of the product test solution to demonstrate any influence of protein and erythrocytes on the virucidal activity of the product test solution 3.6 plaque forming units (PFU) number of infectious virus particles per unit volume (ml) 3.7 product
chemical agent or formulation used as a chemical disinfectant or antiseptic 3.8 reference virus inactivation test test with a defined product (e.g. formaldehyde) in parallel with a product under test for the internal control of the test 3.9 TCID50 50 % infecting dose of a virus suspension or that dilution of the virus suspension that induce a CPE (see 3.10) in 50 % of cell culture units 3.10 viral cytopathic effect (CPE)
morphological alteration of cells and/or their destruction as a consequence of virus multiplication 3.11 viral infectivity ability of a virus to produce infectious progeny in a sensitive cell strain NOTE Viral infectivity may also include the expression of at least part of the virus’ genetic information in cells 3.12 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
3.13 virucide product that inactivates viruses under defined conditions NOTE The adjective derived from "virucide" is "virucidal". 3.14 virucidal activity capability of a product to produce a reduction in the number of infectious virus particles of relevant test organisms under defined conditions
3.15 virus titre amount of infectious virus per unit volume present in a cell culture lysate
EN 14476:2005 (E) 6
4
Requirements A product, when tested in accordance with Clause 6 and Clause 7, shall demonstrate at least a decimal log (lg) reduction of 4 in virus titre of the test strains when tested under the test conditions described in Table 1. Table 1 — Required test conditions Test conditions Instrument and surface disinfectantsHygienic handrub and handwash Chemothermal disinfection procedureTest virus Poliovirus and Adenovirus Poliovirus and Adenovirus Parvovirus Test temperature
20 °C ± 1 °C (except for chemothermal disinfection) 20 °C ± 1 °C (except for chemothermal disinfection) according to the recommendation of the manufacturer, but not higher than 60°C Contact time a) obligatory
b) additional
60 min
5 min, 15 min, 30 min 1 min or 30 s, if manufacturer recommends < 1 min 3 min according to the contact time recommended by the manufacturer, but not longer than 60 min - Interfering substances a) clean
and/or b) dirty
0,3 g/l bovine serum albumin and/or 3,0 g/l bovine serum albumin plus 3,0 ml erythrocytes
PBSa)
-
0,3 g/l bovine serum albumin and/or 3,0 g/l bovine serum albumin plus 3,0 ml erythrocytes
a) Phosphate Buffered Saline NOTE The human immunodeficiency virus (HIV) is not considered a virus which requires testing, because it is a highly fragile virus. Therefore testing of the virucidal activity of chemical disinfectants against HIV is not necessary within the framework of this European Standard, if the product is active against poliovirus. In fact, poliovirus is selected as test virus because it has a high resistance to chemicals, is acid-stable and is unaffected by lipid solvents such as ether, and most detergents or quaternary products. 5 Materials and reagents 5.1 Apparatus and glassware 5.1.1 General Sterilise all glassware and parts of the apparatus that will come into contact with the culture media and reagents or the sample, except those that are supplied sterile, by one of the methods described in 5.1.2.2.
EN 14476:2005 (E) 7 5.1.2 Usual microbiological laboratory equipment1) 5.1.2.1 General and, in particular, the following: 5.1.2.2 Apparatus for sterilisation a) for moist heat sterilisation an autoclave capable of being maintained at (12130+) °C for a minimum holding time of 15 min; b) for dry heat sterilisation a hot air oven capable of being maintained at (18050+) °C for a minimum holding time of 30 min, at (17050+) °C for
a minimum holding time of 1 h or at (16050+) °C for
a minimum holding time of 2 h. 5.1.2.3 Water bath capable of being controlled at 20 °C ± 1 °C. 5.1.2.4 Refrigerator, capable of being controlled at 2 °C to 8 °C. 5.1.2.5 pH-meter, having an accuracy of calibration of 0,1 pH units at 25 °C. 5.1.2.6 Inverted microscope for reading cell cultures microscopically 5.1.2.7 Stopwatch 5.1.2.8 Electromechanical agitator , e.g. Vortex ® mixer2). 5.1.2.9 Membrane filtration apparatus for filtration of media (0,22 µm pore size). 5.1.2.10 Microtitre plates or tubes, petri dishes and flasks for cell culture use. 5.1.2.11 Magnetic stirrer for keeping cells in suspension before seeding. 5.1.2.12 CO2 incubator (95% air, 5% CO2), capable of being controlled at 36 °C ± 1 °C, for incubation of cell cultures. An incubator at 37 °C ± 1 °C may be used if an incubator at 36 °C ± 1 °C is not available. 5.1.2.13
Ice producing machine or commercially available ice to cool the cell maintenance medium and the reaction mixtures during the test (see 6.1, 6.6.3, and 6.6.6.1). 5.1.2.14 Basin as ice bath with ice and water. 5.1.2.15 Container: sterile test tubes, culture bottles or flasks of suitable capacity. 5.1.2.16 Graduated sterile pipettes, of nominal capacities 10 ml and 1 ml and 0,1 ml. NOTE Calibrated automatic pipettes may be used. 5.1.2.17 Volumetric flasks calibrated at 20 °C. 5.1.2.18 Mechanical shaker 5.1.2.19 Centrifuge
1) Disposable equipment is an acceptable alternative to reusable glassware. 2) Vortex ® is an example of a suitable product available commercially. This information is given for the convenience of users of this document and does not constitute an endorsement by CEN of this product.
EN 14476:2005 (E) 8 5.1.2.20 Biological safety cabinet, class II 5.1.2.21 Freezer, -70 °C 5.2 Culture media and reagents 5.2.1 General The reagents shall be of analytical grade and/or appropriate for virological purposes. NOTE To improve reproducibility, it is recommended that commercially available dehydrated material be used for the preparation of culture media. The manufacturer's instructions relating to the preparation of these products should be rigorously followed. 5.2.2 Water 5.2.2.1 General requirements on water The water shall be free from substances that are toxic to cells. It shall be freshly glass double-distilled or demineralised water. Sterilise in the autoclave (5.1.2.2). NOTE 1 If the water is sterilised during the sterilisation of the reagents, this is not necessary. NOTE 2 If distilled water of adequate quality is not available, water for injectable preparations (see bibliographic reference [1]) can be used. 5.2.2.2 Hard water Hard water for dilution of products is prepared as follows: solution A: dissolve 19,84 g anhydrous magnesium chloride (MgCl2) or an equivalent of hydrated magnesium chloride and 46,24 g anhydrous calcium chloride (CaCl2) or an equivalent of hydrated calcium chloride in water (5.2.2.1) and dilute to 1 000 ml; sterilize in the autoclave (5.1.2.2). Store the solution at 2 °C to 8 °C for no longer than one month; solution B: dissolve 35,02 g sodium bicarbonate (NaHCO3) in water (see 5.2.2.1) and dilute to 1 000 ml. Sterilize by membrane filtration. Store the solution at 2 °C to 8 °C for no longer than one week. Hard Water: for the preparation of 1 l, place at least 600 ml water (see 5.2.2.1) in a 1 000 ml volumetric flask (5.1.2.17) and add 6,0 ml of solution A, then 8,0 ml of solution B; mix and dilute to 1 000 ml with water (see 5.2.2.1); the pH of the hard water shall be 7,0 ± 0,2; if necessary adjust the pH by using a solution of approximately 40 g/l (about 1 mol/l) of sodium hydroxide (NaOH) or approximately 36,5 g/l (about 1 mol/l) of hydrochloric acid (HCI); the hard water shall be freshly prepared under aseptic conditions and used within 12 h. NOTE When preparing the product test solutions the addition of the product to this hard water produces a different final water hardness in each test tube. In any case the final hardness is lower than 300 mg/l of calcium carbonate (CaCO3) in the test tube.
EN 14476:2005 (E) 9 5.2.3 Composition and preparation of cell culture media and reagents 5.2.3.1 General Chemicals should be of analytical reagent grade. New or cleaned and dried spatulas should be used between each weighing to prevent cross-contamination of reagents. As each batch of medium is prepared, the lot number of each reagent should be recorded as it is weighed out. 5.2.3.2 Growth and maintenance media Eagle’s minimal essential medium (MEM) or equivalent, supplemented with appropriate concentration of inactivated and mycoplasma-free foetal calf serum (FCS), antibiotics, and other growth factors as needed shall be used. (For reference see bibliographic reference [2]). NOTE Materials and reagents for cell culture may be purchased from biological supply companies. 5.2.3.3 Interfering substances
5.2.3.3.1 General The interfering substance(s) shall be chosen according to the conditions of use laid down for the product (see Table 1). The interfering substance(s) shall be sterile and prepared at 10 times the final concentration in the test (except the fresh defibrinated sheep blood). The method of preparation and sterilisation together with the composition shall be noted in the test report (9.2). 5.2.3.3.2 Clean conditions (bovine serum albumin) Bovine serum albumin should be used as commercially available product or shall be prepared as follows: dissolve 0,3 g of bovine albumin fraction V (suitable for microbiological purposes) in 100 ml of water (see 5.2.2); sterilise by membrane filtration. The final concentration of bovine serum albumin (BSA) in the test is 0,3 g BSA per litre. 5.2.3.3.3 Dirty conditions a) bovine serum albumin: Bovine serum albumin should be used as commercially available product or shall be prepared as follows: dissolve 3 g of bovine albumin fraction V (suitable for microbiological purposes) in 100 ml of water (see 5.2.2); sterilise by membrane filtration. The final concentration of bovine serum albumin (BSA) in the control is 3 g BSA per litre (see 6.6.3). b) sheep erthrocytes: Sterile defibrinated sheep blood should be used as commercially available product or shall be prepared according to EN 14820. Centrifuge erythrocytes from at least 8 ml fresh defibrinated sheep blood at 800 g (5.1.2.19) for 10 min. After discarding the supernatant, resuspend the erythrocytes in sterile phosphate buffered saline (PBS). Repeat this procedure at least three times (the supernatant should be colourless).
EN 14476:2005 (E) 10 c) bovine albumin and erythrocyte solution: Resuspend 3 ml of packed erythrocytes with 97 ml of 3 % w/v of bovine albumin solution. The final concentration of sheep erythrocytes and albumin in the test procedure is 3 ml/l and 3 g/l respectively. To avoid contamination this mixture should be split in portions probably needed per day and stored in separate containers for a maximum of 7 days at 2 °C to 8 °C. 5.3 Viruses 5.3.1 Test viruses The virucidal activity shall be evaluated using the test viruses listed in Table 2 depending on the intended use of the product. Table 2 — Viruses for virucidal testing Intended use of disinfectant Test viruses (group, virustype, strain) Instruments and surface disinfectants Hygienic handrub and handwash
Non-enveloped RNA virus Picornavirus group - poliovirus type 1, LSc-2aba Non-enveloped DNA virus Adenovirus group - adenovirus type 5, strain Adenoid 75, ATCC VR-5 Chemothermal disinfection procedure Bovine Parvovirus, strain Haden, ATCC VR-767 a Only virus material that passed the requirements for the production of oral polio vaccine of the World Health Organisation (WHO) must be used. (Other stocks derived from LSc-2ab cannot be used any longer). 5.3.2 Reference virus suspension Virus suspension of a defined virus strain is maintained in reference centres. Virus suspension of a defined strain may be obtained from a national or international collections (e.g. American Type Culture Collection (ATCC)). In the case of polio virus LSc-2ab only virus material that passed the requirements for the production of oral polio vaccine of the World Health Organisation (WHO) must be used. (Other laboratory strains with the name “LSc-2ab” but that do not fulfil these requirements cannot be used any longer). The virus suspension is kept in small volumes at temperatures below –70 °C or preferably at -196 °C under nitrogen. NOTE Stock virus suspensions are prepared from reference virus suspensions. 5.3.3 Stock virus suspension The virus has to be multiplied on a large scale to obtain a virus suspension of the same characteristics as the reference virus suspension. Due to safety reasons, only 10 passages from the original seed virus are allowed in case of the polio virus vaccine strain. For multiplication and preparation of the virus suspension see Clause 6 and Clause 7. The virus suspension is kept in small volumes below -70 °C or preferably at -196 °C under nitrogen.
EN 14476:2005 (E) 11 NOTE It is envisaged that a European standard on the preservation of viruses will supplement this method. 5.3.4 Test virus suspension The virus suspension that is used in the virucidal testing of the disinfectant. 6 Test method (general) 6.1 Principle A suspension of a test virus is added to the product test solution (6.6.2) under clean or dirty conditions (5.2.3.3). This test mixture is maintained at the temperature(s) given in Table 1. An aliquot is taken at the specified contact times (see Table 1). The virucidal activity is immediately suppressed by transfer of the sample into 9 volumes of ice-cold cell maintenance medium (MEM + 2 % FCS). A dilution series with a factor of ten or preferably less is prepared in an ice-cold medium as soon as possible, at least within 30 min. Dilutions shall be prepared in sterile tubes or microtitre plates. Pipettes shall be changed after each dilution to avoid carry-over of viruses. The dilutions are transferred into cell culture units (petri dishes, tubes or wells of microtitre plates) either using monolayer or cell suspension. Infectivity tests are done either by plaque test or quantal tests. For the quantal test at least 6 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 positive results lying between 100 % and 0 % (see Table C.1). Titre values are calculated according to Spaerman and Kärber (see Annex C and bibliographic references [3] and [4]). Reduction of virus infectivity is calculated from differences of lg virus titres before and after treatment with the product.
6.2 Choice of experimental conditions The selection of contact time, temperature, test virus strains and interfering substance(s) shall be carried out according to the practical use considered for the product: a) contact time: t in min: contact times shall be chosen according to Table 1; b) temperature: θ in °C: temperatures shall be chosen according to Table 1; c) test virus strains: test virus strains shall be chosen according to Table 2; d) interfering substance(s): interfering substances shall be chosen according to 5.2.3.3. 6.3 Preparation of the test virus suspension The stock virus suspension (5.3.3) is multiplied in an appropriate cell line which produces high titres of infectious viruses. The cell debris shall then be separated by low speed centrifugation. This preparation is called "test virus suspension". The test virus suspension is used undiluted for the test procedure (see Clause 6 and Clause 7).
EN 14476:2005 (E) 12 It is suggested that the minimum titre of the virus suspension is at least 108 TCID50/ml. In any case, it shall be sufficiently high to at least enable a titre reduction of 4 lg to verify the method (see 8.3).
In exceptional cases the virus suspension may be concentrated by appropriate methods (e.g. ultracentrifugation). 6.4 Cell culture preparation for virucidal testing Cell monolayers shall be > 90 % confluent before inoculation. Cell lines and appropriate culture media are selected in accordance with their sensitivity to the test viruses (see 5.3.1 and Clause 7). Cells for virus titration, if used as suspensions in quantal tests, shall be added to the dilutions of the test mixture (6.6.3) in such a density as to enable the formation of a monolayer in at least two days in the cell control. 6.5 Infectivity assay 6.5.1 Quantal tests (endpoint titration) 6.5.1.1 Virus titration on cells in suspension on microtiter plates Transfer 0,1 ml of each dilution (6.1) into six or eight wells of a microtitre plate, beginning with the highest dilution. Add 0,1 ml of cell culture suspension in such a density as to enable the formation of a monolayer (> 90 %) in at least 2 days in the cell control. Six or eight wells will serve as the cell control and does not receive any viral suspension. The viral cytopathic effect is read by using an inverted microscope after the appropriate incubation time (according to the virus type). 6.5.1.2 Virus titration on monolayers of cells on microtiter plates Transfer 0,1 ml of each dilution (6.1) into six or eight wells of a microtiter plate containing a confluent (> 90 %) cell monolayer without any medium. The last row of six or eight wells will receive 0,1 ml of culture medium and will serve as the cell control. After 1 h of incubation at 37 °C, 0,1 ml of cell maintenance medium is added to each well. Change pipettes for tubes or wells when adding medium. 6.5.1.3 Virus titration on monolayers of cells in cell culture tubes 6.5.1.2 applies with the modification that the volumes are multiplied by 5 to 10. 6.5.2 Plaque assay (for poliovirus) Plastic tray wells (surface diameter 30 mm to 35 mm) with confluent cell monolayers are washed once with PBS and inoculated with 0,2 ml of serial dilutions of virus in MEM + 2 % FCS. Three wells are generally used per dilution. After absorption period of 1 h at 37 °C, during which the cell monolayers are kept moist by tilting the dishes every 8 min to 10 min, the inoculum is removed and the cell monolayers are washed once with PBS.
Subsequently, the wells are overlaid with 3 ml of a mixture consisting of 1 % melted agarose or another appropriate semisolid medium and 2 times concentrated MEM with 4 % FCS. The cultures are incubated for 2 to 3 days at 37 °C in a CO2 incubator (see 5.1.2.12). Plaques can be counted (see C.2) after addition of 2 ml of a second overlay with the same composition of the first and also containing 5 % of a 1:1000 solution of neutral red and further incubation (in the dark) at 37 °C for 24 h to 48 h in a CO2 incubator (see 5.1.2.12). Counting can be performed also after addition of crystal violet. The cell monolayers are fixed by adding 2 ml of 10 % trichloroacetic acid (TCA) over the agar overlay for 10 min to 15 min at room temperature. The agar overlay is then removed and 2 ml of 0,1 % crystal violet in 20 % ethanol are added. After 10 min to 15 min at room temperature, the wells are extensively washed with water and the plaques (white spots) are counted (see C.2).
EN 14476:2005 (E) 13 6.6 Virucidal testing 6.6.1 General Before testing, equilibrate all reagents (except the test virus suspension (6.3)), i.e. interfering substance, water (see 5.2.2), trypsin, PBS, cell media, diluent, and product test solution (6.6.2) to 20 °C ± 1 °C using the water bath (5.1.2.3) and in case of chemothermal process (see Table 1) to the additional test temperature. Check that the temperature of the reagents is stabilised at the test temperature of 20 °C ± 1 °C or the additional test temperature. Tubes are filled with ice-cold MEM + 2 % FCS for the various titrations that have to be performed after the different contact times and placed in an ice bath. Microtiter plates are labelled for identification. 6.6.2 Preparation of the product test solution Details of samples of the product as received from the producer or from any other source shall be recorded. The concentration of a product test solution shall be 1,25 times the desired test concentration because it is diluted to 80% during the test and the method validation.
Product test solutions shall be prepared in hard water (see 5.2.2.2) at minimum three different concentrations to include one concentr
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