SIST EN 14476:2013

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Chemische Desinfektionsmittel und Antiseptika - Quantitativer Suspensionsversuch zur Bestimmung der viruziden Wirkung im humanmedizinischen Bereich - Prüfverfahren und Anforderungen (Phase 2, Stufe 1)Antiseptiques et désinfectants chimiques - Essai quantitatif de suspension pour l’évaluation de l’activité virucide dans le domaine médical - Méthode d’essai et prescriptions (Phase 2/Étape 1)Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of virucidal activity in the medical area - Test method and requirements (Phase 2/Step 1)11.080.20Dezinfektanti in antiseptikiDisinfectants and antisepticsICS:Ta slovenski standard je istoveten z:EN 14476:2013SIST EN 14476:2013en,fr,de01-november-2013SIST EN 14476:2013SLOVENSKI
STANDARDSIST EN 14476:2005+A1:20071DGRPHãþD



SIST EN 14476:2013



EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 14476
August 2013 ICS 11.080.20 Supersedes EN 14476:2005+A1:2006English Version
Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of virucidal activity in the medical area - Test method and requirements (Phase 2/Step 1)
Antiseptiques et désinfectants chimiques - Essai quantitatif de suspension pour l'évaluation de l'activité virucide dans le domaine médical - Méthode d'essai et prescriptions (Phase 2/Étape 1)
Chemische Desinfektionsmittel und Antiseptika - Quantitativer Suspensionsversuch zur Bestimmung der viruziden Wirkung im humanmedizinischen Bereich - Prüfverfahren und Anforderungen (Phase 2, Stufe 1) This European Standard was approved by CEN on 5 July 2013.
CEN members are bound to comply 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 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 same 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, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre:
Avenue Marnix 17,
B-1000 Brussels © 2013 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 14476:2013: ESIST EN 14476:2013



EN 14476:2013 (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 methods .8 5.1 Principle .8 5.2 Materials and reagents, including cell cultures .9 5.2.1 Test organisms .9 5.2.2 Culture media, reagents and cell cultures .9 5.3 Apparatus and glassware . 12 5.4 Preparation of test organism suspensions and product test solutions . 14 5.4.1 Test organisms suspensions (test virus suspension). 14 5.4.2 Product test solutions . 14 5.5 Procedure for assessing the virucidal activity of the product . 15 5.5.1 General . 15 5.5.2 Test procedure . 16 5.5.3 Modified method for ready-to-use products . 17 5.5.4 Cytotoxicity caused by product test solutions . 17 5.5.5 Control of efficiency of suppression of product’s activity . 18 5.5.6 Reference test for virus inactivation. 18 5.5.7 Titration of the virus control . 19 5.5.8 Titration of test samples . 19 5.6 Experimental data and calculation. 19 5.6.1 Protocol of results . 19 5.6.2 Calculation of infectivity titer (TCID50 or PFU) . 19 5.7 Verification of the methodology . 19 5.8 Expression of results . 20 5.8.1 General . 20 5.8.2 Calculation of the virucidal activity of products . 20 5.9 Test report . 20 Annex A (informative)
Examples of viruses sorted according to their presence in the human body in case of virus infection . 22 Annex B (informative)
Detoxification of test mixtures by molecular sieving. 24 B.1 Molecular sieving with SephadexTM LH 20 . 24 B.1.1 Principle . 24 B.1.2 Sephadex suspension . 24 B.1.3 Procedure . 24 B.2 Molecular sieving using MicroSpinTM S 400 HR . 26 Annex C (informative)
Calculation of the viral infectivity titre . 27 C.1 Quantal tests  Example of TCID50 determination by the Spearman-Kärber method . 27 C.2 Plaque test . 28 C.3 Biometrical evaluation of experimental approaches and assessment of the disinfecting effect on the virus (reduction [R]): . 28 C.3.1 General . 28 C.3.2 Calculating the virus titre with 95 % confidence interval . 29 C.3.3 Calculating the reduction and its 95 % confidence interval . 29 C.3.4 Calculating the average reduction (R(mi)) and its 95 % confidence interval. . 30 C.3.5 Practical example. 31 SIST EN 14476:2013



EN 14476:2013 (E) 3 Annex D (informative)
Presentation of test results of one active concentration . 33 Annex E (informative)
Quantitative determination of formaldehyde concentrations . 36 Bibliography . 37
SIST EN 14476:2013



EN 14476:2013 (E) 4 Foreword This document (EN 14476:2013) 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 February 2014 and conflicting national standards shall be withdrawn at the latest by February 2014. 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 14476:2005+A1:2006. The document was revised to adapt it to the latest state of science, to correct errors and ambiguities, to harmonise the structure and wording with other existing tests of CEN/TC 216 or in preparation and to improve the readability of the standard and thereby make it more understandable. The following list is a list of significant technical changes since the last edition: • The scope was expanded for the following fields of application within the medical area, i.e. products for textile disinfection. • “Obligatory test conditions” were replaced by “minimum test conditions” (test temperatures and contact times can be chosen within limits) that have to be performed to pass the test. • An additional modified method is described to test ready-to-use products in a higher concentration than
80 %, i.e. 9 7%. Data obtained using the former version of EN 14476 may still be used. Other methods to evaluate the efficacy of chemical disinfectants and antiseptics for different applications in the medical area are in preparation. According to the CEN/CENELEC Internal Regulations, the national standards organisations of the following countries are bound to implement this European Standard: 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, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. SIST EN 14476:2013



EN 14476:2013 (E) 5 Introduction This European Standard specifies a suspension test for establishing whether a chemical disinfectant or an antiseptic has a virucidal activity in the area and fields described in the scope. This laboratory test takes into account practical conditions of application of the product including contact time, temperature, test organisms and interfering substances, i.e. conditions which may influence its action in practical situations. Each utilisation concentration of the chemical disinfectant or antiseptic found by this test corresponds to the chosen experimental conditions. SIST EN 14476:2013



EN 14476:2013 (E) 6 1 Scope This European 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, i. e, products that are not diluted when applied,– with water. Products can only be tested at a concentration of 80 % (97 %, with a modified method for special cases) 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 medical area in the fields of hygienic handrub, hygienic handwash, instrument disinfection by immersion, surface disinfection by wiping, spraying, flooding or other means and textile disinfection. This European Standard applies 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. 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. NOTE 3 EN 14885 specifies in detail the relationship of the various tests to one another and to “use recommendations”. 2 Normative 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 amendments) applies. EN 12353, Chemical disinfectants and antiseptics — Preservation of test organisms used for the determination of bactericidal (including Legionella), mycobactericidal, sporicidal, fungicidal and virucidal (including bacteriophages) activity 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 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) SIST EN 14476:2013



EN 14476:2013 (E) 7 3.3 reference test for virus inactivation
test with a defined product (e.g. formaldehyde) in parallel with a product under test for the internal control of the test 3.4 TCID50 50 % infecting dose of a virus suspension or that dilution of the virus suspension that induce a CPE (3.5) in 50 % of cell culture units 3.5 viral cytopathic effect
CPE
morphological alteration of cells and/or their destruction as a consequence of virus multiplication 3.6 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
3.7 virus titre amount of infectious virus per unit volume present in a cell culture lysate or in a solution 4 Requirements The product shall demonstrate at least a decimal log (lg) reduction of 4 in virus titre when tested in accordance with Table 1 and Clause 5. SIST EN 14476:2013



EN 14476:2013 (E) 8 Table 1 — Minimum and additional test conditions Test Conditions Hygienic handrub and handwash Instrument disinfection Surface disinfection Textile
disinfection
Minimum spectrum of test organisms
Poliovirus Adenovirus Murine Norovirus Limited spectrum virucidal activity a Adenovirus
Murine Norovirus Poliovirus Adenovirus Murine Norovirus
when temperature is
40 °C or higher: only
Parvovirus Poliovirus Adenovirus Murine Norovirus
Parvovirus additional Any relevant test organism Test temperature according to the manufacturer’s recommendation, but at / between
20 °C
20 °C and 70 °C
4 °C and 30 °C
30 °C and 70 °C
Contact time according to the manufacturer’s recommendation
but between
but no longer than
but no longer than
but no longer than
30 s and 120 s 60 min
5 min or
60 min b 20 min Interfering substance
clean conditions
0,3 g/l bovine albumin solution (hygienic handrub) c 0,3 g/l bovine albumin solution
and/or
0,3 g/l bovine albumin solution
and/or
dirty conditions 3,0 g/l bovine albumin solution plus 3,0 ml/l erythrocytes (hygienic handwash) d
3,0 g/l bovine albumin solution plus 3,0 ml/l erythrocytes
3,0 g/l bovine albumin solution plus 3,0 ml/l erythrocytes
3,0 g/l bovine albumin solution plus 3,0 ml/l erythrocytes Additional conditions e clean or dirty c, d;
any relevant substance any relevant substance any relevant substance any relevant substance
a The test for limited spectrum virucidal activity will cover all enveloped viruses (Annex A) and the specified test organisms . b The contact times for surface disinfectants stated in this table are chosen on the basis of the practical conditions of the product. The recommended contact time for the use of the product is within the responsibility of the manufacturer. Products intended to disinfect surfaces that are likely to come into contact with the patient and / or the medical staff and surfaces, which are frequently touched by different people, leading to the transmission of microorganisms to the patient, shall be tested with a contact time of maximum 5 min. The same applies where the contact time of the product shall be limited for practical reasons. Products for other surfaces than stated above may be tested with a contact time of maximum 60 min.
c Hygienic handrub shall be tested as a minimum under clean conditions.
d Hygienic handwash shall be tested as a minimum under dirty conditions.
e For the additional conditions, the concentration defined as a result can be lower than the one obtained under the minimum test conditions.
5 Test methods 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 viruses in a solution of an interfering substance. The mixture is maintained at one of the temperatures and the contact times specified in Clause 4 and 5.5.1.1. At the end of this contact time, an aliquot is taken; the virucidal action in this portion is immediately suppressed by a validated method (dilution of the sample in ice-cold cell maintenance medium). 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. After incubation, the titres of infectivity are calculated according to Spearman and Kärber (quantal tests, C.1) or by plaque counting (plaque test, C.2) and evaluated. Reduction of virus infectivity is calculated from differences of lg virus titres before (virus control) and after treatment with the product. SIST EN 14476:2013



EN 14476:2013 (E) 9 NOTE Handwash products are always prediluted with hard water (5.2.2.7). The resulting solution is regarded as a ready-to-use product (5.4.2). 5.1.2 The test is performed using the test organisms as specified in Clause 4, Table 1. 5.1.3 Other contact times and temperatures within the limits specified in Clause 4, Table 1 may be used. Additional interfering substances and test organisms may be used. 5.2 Materials and reagents, including cell cultures 5.2.1 Test organisms The virucidal activity shall be evaluated using the following strains as test organisms selected according to Clause 4, Table 11)
a) Non-enveloped RNA virus2)
1) Poliovirus type 1, LSc 2ab (Picornavirus) 2) Murine norovirus, strain S99 Berlin b) Non-enveloped DNA virus 1) Adenovirus type 5, strain Adenoid 75, ATCC VR-5* 2) Murine Parvovirus, minute virus of mice, strain Crawford, ATCC VR-1346 The required incubation temperature for these test organisms is 36 °C ± 1 °C or 37 °C ± 1 °C (5.3.1.3). The same temperature (either 36 °C or 37 °C) shall be used for all incubations performed during a test and its control and validation. If additional test organisms are used, they shall be kept and used under optimum growth conditions (temperature, time, atmosphere, media) noted in the test report. If these additional test organisms are not classified at a reference centre, their identification characteristics shall be stated. In addition, they shall be held by the testing laboratory or national culture collection under a reference for five years. 5.2.2 Culture media, reagents and cell cultures 5.2.2.1 General All weights of chemical substances given in this European Standard refer to the anhydrous salts. Hydrated forms may be used as an alternative, but the weights required shall be adjusted to allow for consequent molecular weight differences. The reagents shall be of analytical grade and/or appropriate for microbiological purposes. They shall be free from substances that are toxic or inhibitory to the test organisms.
1) The ATCC numbers are the collection numbers of strains supplied by these culture collections. This information is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of the product named. 2) Virus strains may be obtained from a national or international culture collection. Regarding Poliovirus only virus material that passed the requirements for the production of oral polio vaccine of the World Health Organisation (WHO) shall be used (Other stocks derived from LSc-2ab cannot be used any longer). LSc-2ab can be obtained from NIBSC (www.nibsc.ac.uk: contact Dr. Javier Martin) or from Eurovir Hygiene Institut (www.eurovir.de: contact Dr. Jursch). Murine Norovirus may be obtained from Friedrich-Loeffler-Insitut Bundesforschunsinstitut für Tiergesundheit, Hauptsitz Insel Riems Südufer 10, 17493, Greifswald-Insel Riems; phone: +49 38351 7-0, fax: +49 038351 7-121. http://www.fli.bund.de. SIST EN 14476:2013



EN 14476:2013 (E) 10 To improve reproducibility, it is recommended that commercially available – if appropriate the material is used for the preparation of culture media. The manufacturer's instructions relating to the preparation of these products should be rigorously followed.
For each culture medium and reagent, a time limitation for use should be fixed.
All specified pH values are measured at 20 °C ± 1 °C. 5.2.2.2 Water The water shall be freshly glass-distilled water and not demineralised water. If distilled water of adequate quality is not available, water for injections (see bibliographic reference [1]) may be used. Sterilise in the autoclave [5.3.1.1 a)]. Sterilisation is not necessary if the water is used e.g. for preparation of culture media and subsequently sterilised.
See 5.2.2.7 for the procedure to prepare hard water. 5.2.2.3 Phosphate buffered saline (PBS) Sodium chloride (NaCl) 8,00 g Potassium chloride (KCl) 0,20 g Disodium hydrogen phosphate, 12-hydrate (Na2HPO4 x 12H2O) 2,89 g Potassium phosphate, monobasic (KH2PO4) 0,20 g Water (5.2.2.2) to 1 000,0 ml 5.2.2.4 Neutral Red (1:1000 solution) Prepare neutral red (Sigma N7005) stock solution at 0,1 mg/ml in water (5.2.2.2). Filter through a 0,40 µm pore size filter and store 4 °C in the dark. 5.2.2.5 Foetal calf serum (FCS) FCS has to be certified free of viruses and mycoplasma. Extraneous viruses and mycoplasma may interfere with cell and virus growth resulting in false results. For RAW 264.7 cells, special FCS has to be used due to the cells’ high sensitivity to endotoxins. 5.2.2.6 Trichloroacetic acid (10 % solution) (TCA) Dissolve 10 g of TCA crystals in 80 ml of water (5.2.2.2), then adjust the volume to 100 ml with water. Stir to complete solution.
5.2.2.7 Hard water for dilution of products For the preparation of 1 l of hard water, the procedure is as follows:  prepare solution A: dissolve 19,84 g magnesium chloride (MgCl2) and 46,24 g calcium chloride (CaCl2) in water (5.2.2.2) and dilute to 1 000 ml. Sterilise by membrane filtration (5.3.1.7) or in the autoclave [5.3.1.1 a)]. Autoclaving – if used - may cause a loss of liquid. In this case make up to 1 000 ml with water (5.2.2.2) under aseptic conditions. Store the solution in the refrigerator (5.3.1.8) for no longer than one month;  prepare solution B: dissolve 35,02 g sodium bicarbonate (NaHCO3) in water (5.2.2.2) and dilute to 1000 ml. Sterilise by membrane filtration (5.3.1.7). Store the solution in the refrigerator (5.3.1.8) for no longer than one week;  place 600 ml to 700 ml of water (5.2.2.2) in a 1 000 ml volumetric flask (5.3.1.12) and add 6,0 ml (5.3.1.9) of solution A, then 8,0 ml of solution B. Mix and dilute to 1 000 ml with water (5.2.2.2). The pH (5.3.1.4) of the hard water shall be 7,0 ± 0,2. (5.3.1.4). If necessary, adjust the pH by using a solution of SIST EN 14476:2013



EN 14476:2013 (E) 11 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 (HCl).
The hard water shall be freshly prepared under aseptic conditions and used within 12 h. NOTE When preparing the product test solutions (5.4.2), the addition of the product to the hard water produces different final water hardness in each test tube. In any case, the final hardness in the test tube expressed as calcium carbonate (CaCO3) is lower than 375 mg/l. 5.2.2.8 Interfering substance 5.2.2.8.1 General The interfering substance shall be chosen according to the conditions of use laid down for the product.
The interfering substance shall be sterile and prepared at 10 times its final concentration in the test (50 times in the case of the modified method, 5.2.2.8.4). The ionic composition (e.g. pH, calcium and/or magnesium hardness) and chemical composition (e.g. mineral substances, protein, carbohydrates, lipids and detergents) shall be defined. “Diluent” is generally used in the other European Standards in the medical area to prepare the interfering substance. Since there is no experience in virucidal testing with diluent, water (5.2.2.2) is used instead.
NOTE The term “interfering substance” is used even if it contains more than one substance. 5.2.2.8.2 Clean conditions (bovine albumin solution – low concentration) Dissolve 0,30 g of bovine albumin fraction V (suitable for microbiological purposes) in 100 ml of water (5.2.2.2). Sterilise by membrane filtration (5.3.1.7), keep in a refrigerator (5.3.1.8) and use within one month. The final concentration of the bovine albumin in the test procedure (5.5) shall be 0,3 g/l ; 5.2.2.8.3 Dirty conditions (Mixture of bovine albumin solution – high concentration with sheep erythrocytes) Dissolve 3,00 g of bovine albumin fraction V (suitable for microbiological purposes) in 97 ml of water (5.2.2.2). Sterilise by membrane filtration (5.3.1.7). Prepare at least 8,0 ml fresh defibrinated sheep blood (5.2.2.9). Centrifuge the erythrocytes at 800 gN for 10 min (5.3.1.13). After discarding the supernatant, resuspend erythrocytes in water (5.2.2.2). Repeat this procedure at least 3 times, until the supernatant is colourless.
Resuspend 3 ml of the packed sheep erythrocytes in the 97 ml of sterilised bovine albumin solution (see above). To avoid later contamination this mixture should be split in portions probably needed per day. Keep the mixture in separate containers for a maximum of 7 d in a refrigerator (5.3.1.8). The final concentration of bovine albumin and sheep erythrocytes in the test procedure (5.5) shall be 3 g/l and 3 ml/l respectively. 5.2.2.8.4 Clean and dirty conditions for the modified method for ready-to-use products (5.5.4) Follow the procedures for preparation according to 5.2.2.8.2 and 5.2.2.8.3, but prepare the interfering substance in fivefold higher concentrations, for the dirty conditions maximum 50 ml to avoid problems with the filtration. SIST EN 14476:2013



EN 14476:2013 (E) 12 a) Clean conditions (5.2.2.8.2) – dissolve 1,50 g bovine albumin (instead of 0,3 g) in 100 ml of water (5.2.2.2); b) dirty conditions (5.2.2.8.3) – dissolve 7,5 g bovine albumin (instead of 1,5 g) in 42,5 ml (instead o
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