EN 14476:2013+A1:2015

SLOVENSKI STANDARD
01-november-2015
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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)
Chemische Desinfektionsmittel und Antiseptika - Quantitativer Suspensionsversuch zur
Bestimmung der viruziden Wirkung im humanmedizinischen Bereich - Prüfverfahren und
Anforderungen (Phase 2, Stufe 1)
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Ta slovenski standard je istoveten z: EN 14476:2013+A1:2015
ICS:
11.080.20 Dezinfektanti in antiseptiki Disinfectants and antiseptics
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN 14476:2013+A1
EUROPEAN STANDARD
NORME EUROPÉENNE
September 2015
EUROPÄISCHE NORM
ICS 11.080.20 Supersedes EN 14476:2013
English 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 Chemische Desinfektionsmittel und Antiseptika -
quantitatif de suspension pour l'évaluation de l'activité Quantitativer Suspensionsversuch zur Bestimmung der
virucide dans le domaine médical - Méthode d'essai et viruziden Wirkung im humanmedizinischen Bereich -
prescriptions (Phase 2/Étape 1) Prüfverfahren und Anforderungen (Phase 2, Stufe 1)
This European Standard was approved by CEN on 5 July 2013 and includes Amendment 1 approved by CEN on 27 July 2015.

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

CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2015 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 14476:2013+A1:2015 E
worldwide for CEN national Members.

Contents Page
European foreword . 4
Introduction . 6
1 Scope . 7
2 Normative references . 7
3 Terms and definitions . 7
4 Requirements . 8
5 Test methods . 10
5.1 Principle . 10
5.2 Materials and reagents, including cell cultures . 10
5.2.1 Test organisms . 10
5.2.2 Culture media, reagents and cell cultures . 11
5.3 Apparatus and glassware . 14
5.4 Preparation of test organism suspensions and product test solutions . 15
5.4.1 Test organisms suspensions (test virus suspension) . 15
5.4.2 Product test solutions . 15
5.5 Procedure for assessing the virucidal activity of the product . 16
5.5.1 General . 16
5.5.2 Test procedure . 17
5.5.3 Modified method for ready-to-use products . 18
5.5.4 Cytotoxicity caused by product test solutions . 18
5.5.5 Control of efficiency of suppression of product’s activity . 20
5.5.6 Reference test for virus inactivation . 20
5.5.7 Titration of the virus control . 20
5.5.8 Titration of test samples . 20
5.6 Experimental data and calculation . 21
5.6.1 Protocol of results . 21
5.6.2 Calculation of infectivity titer (TCID50 or PFU) . 21
5.7 Verification of the methodology . 21
5.8 Expression of results . 22
5.8.1 General . 22
5.8.2 Calculation of the virucidal activity of products . 22
5.9 Test report . 22
Annex A (informative) Examples of viruses sorted according to their presence in the human
body in case of virus infection . 24
Annex B (informative) Detoxification of test mixtures by molecular sieving . 26
TM
B.1 Molecular sieving with Sephadex LH 20 . 26
B.1.1 Principle . 26
B.1.2 Sephadex suspension . 26
B.1.3 Procedure. 26
TM
B.2 Molecular sieving using MicroSpin S 400 HR . 28
Annex C (informative) Calculation of the viral infectivity titre . 29
C.1 Quantal tests — Example of TCID50 determination by the Spearman-Kärber method . 29
C.2 Plaque test . 29
C.3 Biometrical evaluation of experimental approaches and assessment of the
disinfecting effect on the virus (reduction [R]): . 30
C.3.1 General . 30
C.3.2 Calculating the virus titre with 95 % confidence interval . 31
C.3.3 Calculating the reduction and its 95 % confidence interval . 31
C.3.4 Calculating the average reduction (R ) and its 95 % confidence interval. . 32
(mi)
C.3.5 Practical example . 33
Annex D (informative) Presentation of test results of one active concentration . 35
Annex E (informative) Quantitative determination of formaldehyde concentrations . 38
Annex ZA (informative) !Relationship between this European Standard and the Essential
Requirements of EU Directive 93/42/EEC" . 39
Bibliography . 40

European foreword
This document (EN 14476:2013+A1:2015) 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 March 2016 and conflicting national standards shall be
withdrawn at the latest by March 2016.
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 14476:2013".
This document includes Amendment 1 approved by CEN on 2015-07-27.
The start and finish of text introduced or altered by amendment is indicated in the text by tags !".
!This document has been prepared under a mandate given to CEN by the European Commission and
the European Free Trade Association, and supports essential requirements of EU Directive(s).
For relationship with EU Directive(s), see informative Annex ZA, which is an integral part of this
document."
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%;
!
• For the hygienic handrub and handwash method a test for virucidal activity against enveloped
viruses with Vacciniavirus was added.
• The relationship between this European Standard and the MDD was added (Foreword and
Annex ZA).
• The value of v in C.1 was corrected (0,001 instead of 0,0001)."
n
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 organizations 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.
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.
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)
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
TCID
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.
Table 1 — Minimum and additional test conditions
Test Conditions Hygienic handrub and Instrument disinfection Surface disinfection Textile
handwash
disinfection
Minimum Poliovirus Poliovirus Poliovirus Parvovirus
spectrum of
Adenovirus Adenovirus Adenovirus
test organisms
Murine Norovirus Murine Norovirus Murine Norovirus

Limited spectrum when temperature is
a
virucidal activity 40 °C or higher: only
Adenovirus Parvovirus
Murine Norovirus
!Virucidal activity
against enveloped
b
viruses
Vacciniavirus"
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 20 min
c
60 min
Interfering substance
clean conditions 0,3 g/l bovine albumin 0,3 g/l bovine albumin 0,3 g/l bovine albumin
solution (hygienic solution solution

d
handrub)
and/or and/or
dirty conditions 3,0 g/l bovine albumin  3,0 g/l bovine albumin
solution plus 3,0 ml/l solution plus 3,0 ml/l
3,0 g/l bovine albumin 3,0 g/l bovine albumin
erythrocytes (hygienic erythrocytes
solution plus 3,0 ml/l solution plus 3,0 ml/l
e
handwash)
erythrocytes erythrocytes
d, e
Additional clean or dirty ; any relevant substance any relevant substance any relevant substance
f
conditions 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 test for virucidal activity against enveloped virus will cover all enveloped viruses only (Annex A)."
c
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.
d
Hygienic handrub shall be tested as a minimum under clean conditions.
e
Hygienic handwash shall be tested as a minimum under dirty conditions.
f
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.
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
1)
to Clause 4, Table 1
2)
a) Non-enveloped RNA virus
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
!
c) Enveloped DNA virus
1) Vacciniavirus, strain Ankara (MVA), ATCC VR-1508."

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 !Bundesforschungsinstitut" 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.
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.
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 (MgCl ) and 46,24 g calcium chloride
(CaCl ) 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 (NaHCO ) 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 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 (CaCO ) 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 g
N
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.
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 of
48,5 ml) of water (5.2.2.2). Prepare at least 20 ml (instead of 4,0 ml) sheep blood. Resuspend 7,5 ml
(instead of 1,5 ml) of the packed sheep erythrocytes in 42,5 ml of sterilised bovine albumin solution
to obtain 50 ml.
5.2.2.9 Defibrinated sheep blood
The defibrinated sheep blood shall be sterile (aseptic blood-letting and preparation). The defibrinated
sheep blood can be pooled from more than one sheep and can be acquired from a commercial supplier.
5.2.2.10 Medium for cell cultures
Eagle’s minimal essential medium (MEM) or equivalent, supplemented with FCS (5.2.2.5), antibiotics,
and other growth factors as needed shall be used.
a) A growth medium for cell multiplication is supplemented with 10 % FCS. Add 10 parts of FCS
(5.2.2.5) to 90 parts of MEM.
b) A maintenance medium to maintain the cell culture metabolism without stimulation of cell
proliferation is supplemented with 2 % FCS. Add 2 parts of FCS (5.2.2.5) to 98 parts of MEM.
Other media may be used if appropriate for certain cell lines.
See also bibliographic reference [2]. See EN 12353 for a detailed description.
5.2.2.11 Cell cultures
Cell monolayers shall be >90 % confluent before inoculation. Cell lines are selected in accordance with
their sensitivity to the test organisms (5.2.1). Cells for virus titration, if used as suspensions in quantal
tests, shall be added to the dilutions of the test mixture (5.5.2) in such a density as to enable the
formation of a monolayer in at least two days in the cell control. Cell cultures can be used as cell
monolayers or in suspensions for quantal tests. For details of cell lines see 5.5.1.1 e).
5.3 Apparatus and glassware
Sterilise all glassware and parts of the apparatus that will come into contact with the culture media and
reagents or the sample, except those which are supplied sterile, by one of the following methods:
a) by moist heat, in the autoclave [5.3.1.1 a)];
b) by dry heat, in the hot air oven [5.3.1.1 b)].
3)
5.3.1 Usual microbiological laboratory equipment and, in particular, the following:
5.3.1.1 Apparatus for sterilisation (moist and dry heat):
+3
a) For moist heat sterilisation, an autoclave capable of being maintained at (121 ) °C for a minimum
holding time of 15 min;
+5
b) for dry heat sterilisation, a hot air oven capable of being maintained at (180 ) °C for a minimum
+5 +5
holding time of 30 min, at (170 ) °C for a minimum holding time of 1 h or at (160 ) °C for a
0 0
minimum holding time of 2 h.
5.3.1.2 Water baths, capable of being controlled at 20 °C ± 1 °C, and at additional test temperatures
± 1 °C (5.5.1).
5.3.1.3 C0 Incubator (95 % air, 5 % C0 ), capable of being controlled either at 36 °C ± 1 °C or
2 2
37 °C ± 1 °C (5.2.1).
5.3.1.4 pH-meter, having an inaccuracy of calibration of no more than ± 0,1 pH units at 20 °C ± 1 °C.
5.3.1.5 Stopwatch.
5.3.1.6 Shakers.
® 4)
a) Electromechanical agitator, e.g. Vortex mixer ;
b) Mechanical shaker.
5.3.1.7 Membrane filtration apparatus, constructed of a material compatible with the substances
to be filtered, with a filter holder of at least 50 ml volume, and suitable for use of filters of diameter
47 mm to 50 mm and 0,22 µm pore size for sterilisation of hard water (5.2.2.7) and bovine albumin
(5.2.2.8.2, 5.2.2.8.3 and 5.2.2.8.4).
The vacuum source used shall give an even filtration flow rate. In order to obtain a uniform distribution
of the micro-organisms over the membrane and to prevent overlong filtration, the device shall be set so
as to obtain the filtration of 100 ml in 20 s to 40 s. The filtration of the interfering substance (5.2.2.8) for
the modified method (5.5.3) may take a longer time.
5.3.1.8 Refrigerator, capable of being controlled at 2 °C to 8 °C.
5.3.1.9 Graduated pipettes, of nominal capacities 10 ml, 1 ml, 100 µl, 10 µl or calibrated automatic
pipettes.
3)
Disposable sterile equipment is an acceptable alternative to reusable glassware.
4) ®
Vortex is an example of a suitable product available commercially. This information is given for the convenience of users
of this European Standard and does not constitute an endorsement by CEN of this product.
5.3.1.10 Ice producing machine or commercially available ice to cool the cell maintenance
medium and the reaction mixtures during the test.
5.3.1.11 Basin as ice bath with ice and water.
5.3.1.12 Volumetric flasks.
5.3.1.13 Centrifuge (400 g to 1000 g ).
N N
5.3.1.14 Microtitre plates or tubes, petri dishes and flasks for cell culture use.
5.3.1.15 Magnetic stirrer for keeping cells in suspension before seeding.
5.3.1.16 Inverted microscope for reading cell cultures microscopically.
5.3.1.17 Container: sterile test tubes, culture bottles or flasks of suitable capacity.
5.3.1.18 Biological safety cabinet, class II.
5.3.1.19 Freezer, -70 °C or less.
5.4 Preparation of test organism suspensions and product test solutions
5.4.1 Test organisms suspensions (test virus suspension)
The test organisms and their stock cultures shall be prepared and kept in accordance with EN 12353.
The stock virus suspension is multiplied in an appropriate cell line that produces high titres of
infectious viruses. The cell debris is separated by centrifugation (400 g for 15 min). This preparation is
N
called "test virus suspension".
It is suggested that the minimum titre of the virus suspension - determined by a quantal test [5.5.2 a)]
or by plaque test [5.5.2 b)] - is at least 10 TCID /ml. In any case, it shall be sufficiently high to at least
enable a titre reduction of 4 lg to verify the method.
In exceptional cases the test suspension may be concentrated by appropriate methods (e.g.
ultracentrifugation).
The test suspension is kept in small volumes below -70 °C or preferably at -196 °C under nitrogen. Due
to safety reasons, and – in some cases – to limit the possibility of genetic mutations, only 10 passages
from the original seed (e.g. virus from culture collection) are allowed.
The test suspension is used undiluted for the test procedure (5.5.2 or 5.5.3).
5.4.2 Product test solutions
The concentration of a product test solution shall be 1,25 times the desired test concentration (= real
test concentration) because it is diluted to 80 % during the test (5.5.2).
Product test solutions shall be prepared in hard water (5.2.2.7) at minimum three different
concentrations to include one concentration in the active range and one concentration in the non-active
range (5.7). The product as received may be used as one of the product test solutions, in this case the
highest tested concentration is 80 %. Ready-to-use products may be tested at 97 % [modified method
(5.5.3)]. In this case, the “real test concentration” is 97 %.
Dilutions of ready-to-use products shall be prepared in water (5.2.2.2) instead of hard water. Handwash
products are always prediluted with hard water (5.2.2.7) to achieve a 62,5 % solution. This solution
simulates the addition of tap water in practice (1:1). Such a product is nevertheless regarded as a
“ready-to-use product”. The modified method (5.5.3) cannot be used, since 62,5 % represents the
highest accepted concentration (50 %), multiplied by 1,25.
For solid products, dissolve the product as received by weighing at least 1,0 g ± 10 mg of the product in
a volumetric flask and filling up with hard water (5.2.2.7). Subsequent dilutions (= lower
concentrations) shall be prepared in volumetric flasks (5.3.1.12) on a volume/volume basis in hard
water (5.2.2.7).
For liquid products, dilutions of the product shall be prepared with hard water in volumetric flasks
(5.3.1.12) on a volume/volume basis.
The product test solutions shall be prepared freshly and used in the test within 2 h. They shall give a
physically homogenous preparation, stable during the whole procedure. If during the procedure a
visible inhomogeneity appears due to the formation of a precipitate or flocculate (for example, through
the addition of the interfering substance), it shall be recorded in the test report.
The concentration of the product stated in the test report shall be the desired test concentration.
Record the test concentration in terms of mass per volume or volume per volume and details of the
product sample as received.
5.5 Procedure for assessing the virucidal activity of the product
5.5.1 General
5.5.1.1 Experimental conditions
The experimental conditions may be selected according to the practical use considered for the product
(Clause 4):
a) temperature θ (in °C): The temperatures to be tested are specified in Clause 4, Table 1.
The allowed deviation for each chosen temperature is ± 1 °C.
b) contact time t (in min):
The contact times to be tested are specified in Clause 4, Table 1.
The allowed deviation for each chosen contact time is ± 10 s, except for 1 min or less where it is
± 5 s.
c) interfering substance:
The interfering substance to be tested is either 0,30 g/l bovine albumin (5.2.2.8.2) representing
clean conditions or a mixture of 3 ml/l sheep erythrocytes and 3,0 g/l bovine albumin (5.2.2.8.3)
representing dirty conditions – according to Clause 4, Table 1 and practical applications. Additional
interfering substances may be tested according to specific fields of application.
d) test organisms:
The test organisms to be tested are specified in Clause 4, Table 1 and 5.2.1. Additional test
organisms may be tested.
e) cell line(s):
Adenovirus is multiplied in HeLa cells or other cell lines of appropriate sensitivity

Poliovirus is multiplied in HeLa cells or other cell lines of appropriate sensitivity

Norovirus is multiplied in RAW 264.7 cells (ATCC TIB-71) or other cell lines of appropriate
sensitivity
Parvovirus is multiplied in A9 cells (ATCC CCL-1.4) or other cell lines of appropriate sensitivity.

!Vacciniavirus is multiplied in BHK-21cells (ATCC CCL-10) or other cell lines of appropriate
sensitivity."
5.5.1.2 Preparation
Tubes are filled with ice-cold maintenance medium (5.2.2.10 b)) for the various titrations that have to
be performed after the different contact times and placed in an ice bath. Microtitre plates are labelled
for identification.
The procedures described under 5.5.4 to 5.5.8 are always carried out in parallel with the test procedure
(5.5.2).
5.5.1.3 Special instruction for ready-to-use products
In the case of ready-to-use products use water (5.2.2.2) instead of hard water, but observe the
exception with handwash products (5.1.1, NOTE).
5.5.1.4 Equilibration of temperature
Prior to testing, equilibrate all reagents [product test solutions (5.4.2), PBS (5.2.2.3), cell culture
(5.2.2.11), water (5.2.2.2), hard water (5.2.2.7) and interfering substance (5.2.2.8)] to the test
temperature of θ (5.5.1.1 a)) using the water bath (5.3.1.2) controlled at θ. Check that the temperature
of the reagents is stabilised at θ. The test suspension (5.4.1) is kept in the ice bath (5.3.1.11) to avoid
titre loss.
Water (5.2.2.2) shall be equilibrated at a temperature of 20 °C ± 1 °C.
In the case of ready-to-use-products, water (5.2.2.2
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