EN 16777:2018

SLOVENSKI STANDARD
01-februar-2019
.HPLþQDUD]NXåLODLQDQWLVHSWLNL.YDQWLWDWLYQLSUHVNXVQDQHSRUR]QLKSRYUãLQDK
EUH]PHKDQVNHJDGHORYDQMD]DYUHGQRWHQMHYLUXFLGQHJDGHORYDQMDNHPLþQLK
UD]NXåLOYKXPDQLPHGLFLQL3UHVNXVQDPHWRGDLQ]DKWHYHID]DVWRSQMD
Chemical disinfectants and antiseptics - Quantitative non-porous surface test without
mechanical action for the evaluation of virucidal activity of chemical disinfectants used in
the medical area - Test method and requirements (phase 2/step 2)
Chemische Desinfektionsmittel und Antiseptika-Quantitativer Versuch auf nicht porösen
Oberflächen ohne mechanische Einwirkung zur Bestimmung der viruziden Wirkung im
humanmedizinischen Bereich - Prüfverfahren und Anforderungen (Phase 2, Stufe 2)
Antiseptiques et désinfectants chimiques - Essai quantitatif de surface non-poreuse sans
action mécanique pour l'évaluation de l'activité virucide désinfectants chimiques utilisés
dans le domaine humain médicine - Méthode d'essai et prescriptions (phase 2/étape 2)
Ta slovenski standard je istoveten z: EN 16777:2018
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 16777
EUROPEAN STANDARD
NORME EUROPÉENNE
December 2018
EUROPÄISCHE NORM
ICS 11.080.20
English Version
Chemical disinfectants and antiseptics - Quantitative non-
porous surface test without mechanical action for the
evaluation of virucidal activity of chemical disinfectants
used in the medical area - Test method and requirements
(phase 2/step 2)
Antiseptiques et désinfectants chimiques - Essai Chemische Desinfektionsmittel und Antiseptika -
quantitatif de surface non poreuse sans action Quantitativer Versuch auf nicht porösen Oberflächen
mécanique pour l'évaluation de l'activité virucide des ohne mechanische Einwirkung zur Bestimmung der
désinfectants chimiques utilisés dans le domaine viruziden Wirkung im humanmedizinischen Bereich -
médical - Méthode d'essai et exigences (phase 2/étape Prüfverfahren und Anforderungen (Phase 2, Stufe 2)
2)
This European Standard was approved by CEN on 24 September 2018.

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, Serbia, 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: Rue de la Science 23, B-1040 Brussels
© 2018 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 16777:2018 E
worldwide for CEN national Members.

Contents Page
European foreword . 4
Introduction . 5
1 Scope . 6
2 Normative references . 6
3 Terms and definitions . 7
4 Requirements for virucidal activity on surfaces. 7
5 Test methods . 8
5.1 Principle . 8
5.2 Materials and reagents, including cell cultures . 8
5.2.1 Test organisms . 8
5.2.2 Culture media, reagents and cell cultures . 9
5.3 Apparatus and glassware . 12
5.3.1 General . 12
5.3.2 Usual microbiological laboratory equipment . 13
5.3.3 Test surfaces . 14
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 solution . 14
5.5 Procedure for assessing the virucidal activity of the product . 15
5.5.1 Experimental conditions . 15
5.5.2 Test procedure . 16
5.5.3 Cytotoxicity caused by product solutions . 17
5.5.4 Control of efficiency for suppression of disinfectant virucidal activity . 18
5.5.5 Reference test for virus inactivation . 19
5.5.6 Titration of the virus control . 19
5.6 Experimental data and calculation . 19
5.6.1 Protocol of the results . 19
5.6.2 Calculation of infectivity titre (TCID PFU) . 19
50 –
5.7 Verification of the methodology . 20
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 . 21
Annex A (informative) Examples of viruses sorted according to their presence in the
human body in case of virus infection . 23
Annex B (normative) Detoxification of test mixtures by molecular sieving . 25
B.1 Molecular sieving with Sephadex™ LH 20 . 25
B.1.1 Principle . 25
B.1.2 Sephadex suspension . 25
B.1.3 Procedure. 25
B.2 Molecular sieving using MicroSpin™ S 400 HR . 27
B.3 Determination of the residual virus titre by the large-volume-plating (LVP) method . 27
B.3.1 General . 27
B.3.2 Example for the calculation of titres and the reduction according to the large-
volume-plating Method . 28
Annex C (informative) Calculation of the viral infectivity titre . 30
C.1 Quantal tests - Example of TCID determination by the Spaerman-Kärber method . 30
C.2 Plaque test . 30
C.3 Biometrical evaluation of experimental approaches and assessment of the
disinfecting effect on the virus (reduction [R]): . 31
C.3.1 General . 31
C.3.2 Calculating the virus titre with 95 % confidence interval - Example . 32
C.3.3 Calculating the reduction and its 95 % confidence interval . 32
C.3.4 Calculating the average reduction (R ) and its 95 % confidence interval . 33
(mi)
C.3.5 Practical example . 34
Bibliography . 37

European foreword
This document (EN 16777:2018) 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 June 2019, and conflicting national standards shall be
withdrawn at the latest by June 2019.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
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, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and the United Kingdom.
Introduction
This document describes a surface test method for establishing whether a product proposed as a
disinfectant in the fields described in Clause 1 has or does not have virucidal activity on non-porous
surfaces.
The laboratory test closely simulates practical conditions of application. Chosen conditions (contact
time, temperature, organisms on surfaces etc.) reflect parameters which are found in practical
situations including conditions which may influence the action of disinfectants. Each use concentration
found from this test corresponds to defined experimental conditions.
The conditions are intended to cover general purposes and to allow reference between laboratories and
product types. Each utilization concentration of the chemical disinfectant or antiseptic found by this test
corresponds to defined experimental conditions.
However for special applications the recommendations of use of a product can differ and therefore
additional test conditions might be needed, which cannot be covered by this document.
1 Scope
This document specifies a test method and the minimum requirements for virucidal activity of chemical
disinfectants 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 applies to products that are used in the medical area for disinfecting non-porous
surfaces including surfaces of medical devices without mechanical action.
This document 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 on viruses in the conditions in which they are used.
NOTE 2 This method corresponds to a phase 2, step 2 test.
EN 14885 specifies in detail the relationship of the various tests to one another and to “use
recommendations”.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements 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 14476, 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)
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
EN 10088-1, Stainless steels — Part 1: List of stainless steels
EN 10088-2, Stainless steels — Part 2: Technical delivery conditions for sheet/plate and strip of corrosion
resisting steels for general purposes
3 Terms and definitions
For the purposes of this document, the terms and definitions given in EN 14885 and EN 14476 apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
• IEC Electropedia: available at http://www.electropedia.org/
• ISO Online browsing platform: available at http://www.iso.org/obp
4 Requirements for virucidal activity on surfaces
The product shall demonstrate at least a decimal log (lg) reduction of 4 in virus titre of the adenovirus
and murine norovirus test strains when tested in accordance with Table 1 and Clause 5. As described in
EN 14885, to claim virucidal activity against enveloped virus the product shall pass both EN 14476 and
this standard with vaccinia virus and to claim limited spectrum virucidal activity the product shall pass
both EN 14476 and this standard with adenovirus and murine norovirus. However, to claim the
virucidal activity the product shall pass standards EN 14476 with poliovirus, adenovirus and murine
norovirus and this standard with adenovirus and murine norovirus, because poliovirus is not resistant
to drying.
Table 1 — Minimum and additional test conditions
Minimum spectrum of test a
Virucidal activity
organisms
adenovirus
murine norovirus
b
Limited spectrum virucidal activity
adenovirus
murine norovirus
c
Virucidal activity against enveloped viruses
vaccinia virus
Test temperature between 18 °C and 25 °C

Additional temperature between 4 °C and 30 °C
Contact time according to the manufacturer’s recommendation, but not longer than
d
5 min or 60 min
Interfering substances 0,3 g/l bovine serum albumin
a) clean and/or
b) dirty 3,0 g/l bovine serum albumin plus 3,0 ml erythrocytes
e Further contact time(s), interfering substance(s) or virus(es)
Additional conditions
a
Poliovirus (as used in the corresponding suspension test) cannot be used for surfaces, because of drying
problems.
b
The test for “limited spectrum virucidal activity” will cover all enveloped viruses (Annex A) and norovirus,
rotavirus and adenovirus.
c
The test for “virucidal activity against enveloped viruses” will cover all enveloped viruses only (Annex A).
d
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.
e
Where appropriate (specific purposes), additional specific virucidal activity shall be determined under other
conditions of time, temperature, and interfering substances (see 5.2.2.8) in accordance with 5.5, in order to take
into account intended specific use conditions. Additional virus(es) can be tested, if relevant. For the additional
conditions, the concentration defined as a result can be lower than the one obtained under the minimum test
conditions.
The determined virucidal concentration of the test product is suggested as being suitable for practical
situations of use.
5 Test methods
5.1 Principle
5.1.1 A test suspension of viruses in a solution of interfering substances is inoculated onto a test
surface and dried. A prepared sample of the product under test is applied in a manner which covers the
dried film.
The test surface is maintained at a specified temperature for a defined period of time. The test surface is
transferred to cell maintenance medium so that the action of the disinfectant is immediately
neutralized. The titre of the virus recovered from the test surface is determined.
The titre of the inoculum on a test surface treated with hard water in place of the disinfectant is also
determined and the reduction in virus titre attributed to the product is calculated by difference.
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 1
a) Non-enveloped RNA virus
Murine norovirus, strain S99 Berlin
NOTE Virus strains can be obtained from a national or international culture collection. Murine norovirus can
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.
b) Non-enveloped DNA virus
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.
Adenovirus type 5, strain Adenoid 75, ATCC VR-5
c) Enveloped DNA virus
Vaccinia virus, strain modified vaccinia virus Ankara (MVA), ATCC VR-1508 or vaccinia virus strain
Elstree, ATCC VR-1549
The required incubation temperature for these test organisms is (36 ± 1) °C or (37 ± 1) °C (5.3.2.12).
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 ± 1) °C.
5.2.2.2 Water
The water shall be freshly glass-distilled water and not demineralized water. If distilled water of
adequate quality is not available, water for injections (see bibliographic reference [1]) may be used.
Sterilize in the autoclave [5.3.2.1a)]. Sterilization is not necessary if the water is used e.g. for
preparation of culture media and subsequently sterilized.
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 (Na HPO x 12H O) 2,89 g
2 4 2
Potassium phosphate, monobasic (KH PO ) 0,20 g
2 4
Water (5.2.2.2) to 1000,0 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
0,44 µm pore size filter and store at 4 °C in the dark.

Sigma N 7005 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.
5.2.2.5 Foetal calf serum (FCS)
FCS shall 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 shall 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), and 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. Sterilize by membrane filtration (5.3.2.11) or in
the autoclave [5.3.2.1 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
(5.3.2.6) 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. Sterilize by membrane filtration (5.3.2.11). Store the solution in the refrigerator (5.3.2.6)
for no longer than one week;
— place 600 ml to 700 ml of water (5.2.2.2) in a 1000 ml volumetric flask (5.3.2.9) and add 6,0 ml of
solution A, then 8,0 ml of solution B. Mix and dilute to 1000 ml with water (5.2.2.2). The pH
(5.3.2.4) of the hard water shall be 7,0 ± 0,2. (5.3.2.4). 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.
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 serum albumin)
Bovine serum albumin shall 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 97 ml of water
(5.2.2.2);
— sterilize by membrane filtration;
— keep in a refrigerator and use within one month.
The final concentration of bovine serum albumin (BSA) in the test is 0,3 g BSA per litre.
5.2.2.8.3 Dirty conditions
a) bovine serum albumin:
Bovine serum albumin shall 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 97 ml of
water (see 5.2.2.2);
— sterilize by membrane filtration;
— keep in a refrigerator and use within one month.
The final concentration of bovine serum albumin (BSA) in the control is 3 g BSA per litre.
b) sheep erthrocytes:
Prepare at least 8,0 ml fresh defibrinated sheep blood (5.2.2.9). Centrifuge the erythrocytes at
800 g for 10 min (5.3.2.18). After discarding the supernatant, resuspend erythrocytes in PBS
N
(5.2.2.3). Repeat this procedure at least 3 times, until the supernatant is colourless.
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 shall 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.
5.2.2.9 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 no longer than 2 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 5.5.1e).
5.2.2.12 Reference glutardialdehyde (Glutaral, 1,5-Pentanedial) CAS Number 111-30-8
Required chemical and physical parameters for use as reference standard for testing disinfectant
preparations are defined in Table 2.
Table 2 — Required chemical and physical parameters of glutardialdehyde
Parameters Specifications
Solution 50 % Solution 25 %
a
clear liquid clear liquid
Appearance
pH-value 3,1 to 4,5 3,1 to 4,5
Concentration of glutardialdehyde (by
50,0 % to 52,0 % 25,0 % to 26,0 %
titration)
Stability (ambient conditions) 12 months 12 months
a
Note: Yellowish colour indicates a beginning polymerization.
The specification above (see Table 2) should be checked on the certificate of analyses. The pH values
shall be tested and confirmed regularly by the laboratory. In case pH values and/or appearance are out
of specification the glutardialdehyde cannot be used anymore.
5.3 Apparatus and glassware
5.3.1 General
Sterilize 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 [5.3.2.1 a)];
b) by dry heat, in the hot air oven [5.3.2.1 b)].
5.3.2 Usual microbiological laboratory equipment
And, in particular, the following:
5.3.2.1 Apparatus for sterilization (moist and dry heat)
+3
a) For moist heat sterilization, an autoclave capable of being maintained at ( ) °C for a minimum
holding time of 15 min;
+5
b) for dry heat sterilization, 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.
5.3.2.2 Water baths, capable of being controlled at (20 ± 1) °C, and at additional test
temperatures ± 1 °C (5.5.1).
5.3.2.3 Inverted microscope for reading cell cultures microscopically
5.3.2.4 pH meter, having an accuracy of calibration of 0,1 pH units at 20 °C.
5.3.2.5 Stopwatch
5.3.2.6 Refrigerator, capable of being controlled at 2 °C to 8 °C.
5.3.2.7 Electromechanical agitator e.g. Vortex ® mixer
5.3.2.8 Containers: Petri plates, sterile test tubes, culture bottles or flasks of suitable capacity.
5.3.2.9 Volumetric flasks, calibrated at 20°C.
5.3.2.10 Microtitre plates or tubes, petri dishes and flasks for cell culture use.
5.3.2.11 Membrane filtration apparatus for filtration of media, 0,22 μm pore size
5.3.2.12 CO incubator (95 % air, 5 % CO ), capable of being controlled at (36 ± 1) °C, for
2 2
incubation of cell cultures. An incubator at (37 ± 1) °C may be used if an incubator at (36 ± 1) °C is not
available.
5.3.2.13 Graduated sterile pipettes of nominal capacities 10 ml and 1 ml and 0,1 ml.
NOTE Calibrated automatic pipettes may be used.
5.3.2.14 Magnetic stirrer for keeping cells in suspension before seeding
5.3.2.15 Ice producing machine or commercially available ice to cool the cell maintenance
medium and the reaction mixtures during the test (see 5.5.2 and 5.5.4)
5.3.2.16 Basin as ice bath with ice and water
5.3.2.17 Mechanical shaker
5.3.2.18 Centrifuge
Disposable sterile equipment is an acceptable alternative to reusable glassware.
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.
5.3.2.19 Biological safety cabinet, class II
5.3.2.20 Freezer, −70 °C or less
5.3.2.21 Desiccator with vacuum manometer
5.3.2.22 Vacuum Diaphragm Pump (e.g. throughput max. 3,8 m /h final vacuum < 75mbar)
5.3.2.23 Cryotubes
5.3.3 Test surfaces
These shall be 1.4301 (EN 10088-1) stainless steel discs (2 cm diameter discs) with Grade 2 B finish on
both sides, in accordance with the requirements of EN 10088-2. The discs shall be as flat as possible and
this is best achieved by using stainless steel of a gauge of 1,2 mm or 1,5 mm. The discs shall be handled
only with forceps and used only once.
Prior to use the discs shall be placed in a container with an appropriate quantity of 5 % per volume
®, 5
Decon 90 or of 1 % Blanisol-Pur for 60 min, in a manner that they don’t stick together and the
surface is not damaged. Rinse the discs with running freshly distilled water (5.2.2.2) or demineralized
water for 10 s, avoiding them to dry at any extent.
Rinse the discs with water (5.2.2.2) for a further 10 s to ensure complete removal of the surfactant. To
supply a satisfactory flow of water, a sterilized fluid dispensing pressure vessel with suitable hose and
connectors or other suitable method can be used and regulated to supply approximately 2000 ml per
min. Dip the disc in a bath containing isopropanol (IPA) for 15 min. Remove the discs and rinse them
with water (5.2.2.2) for at least 10 s. Sterilize by autoclaving.
NOTE Suitable stainless steel discs can usually be purchased from local engineering companies.
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
N
is 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.
If necessary 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 between −135°C and −196°C in
liquid nitrogen vapor phase or liquid nitrogen (with suitable tight cryotubes, 5.3.2.23).
Due to safety reasons, and – in some cases – to avoid 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 solution
Detail sample information of the product as received from the producer or from any other source shall
be recorded.
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. The product as received may be used as one of the product test solutions.

5 ®
Decon 90 and Blanisol-Pur are examples of suitable products available commercially. This information is given
for the convenience of users of this standard and does not constitute an endorsement by CEN of those products.
Dilutions of ready-to-use products, i.e. products which are not diluted when applied, shall be prepared
in water (5.2.2.2).
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 (5.3.2.9) 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 on a volume/volume
basis using volumetric flasks (5.3.2.9).
The product test solutions shall be prepared freshly and used in the test within 2 h. They shall give a
physically homogeneous preparation, stable during the whole procedure. If during the procedure a
visible inhomogeneity appears due to the formation of a precipitate or flocculant 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 Experimental conditions
The selection of contact temperature, contact time and interfering substances shall be carried out
according to the practical use considered for the product (Clause 4) as follows:
a) Test temperature, θ in °C:
The test temperature shall be room temperature [between (18 ± 1) °C and (25 ± 1) °C]; additional
temperatures may be chosen between (4 ± 1) °C and (30 ± 1) °C and shall be reported in the test
report (5.9).
b) Contact time, t (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; additional times may be chosen and
shall be reported in the test report (5.9).
c) Virus strains:
Strains shall be as specified in Clause 4, Table 1.
Additional strains may be chosen and shall be reported in the test report (5.9).
d) Interfering substances:
The interfering substance to be tested is the bovine albumin solution (5.2.2.8.2) under clean
conditions or albumin and sheep erythrocytes under dirty conditions (5.2.2.8.3), according to
practical applications.
Additional interfering substance can be selected and shall be recorded in the test report (5.9)
e) Cell line(s):
Adenovirus is multiplied in HeLa cells or other cell lines of appropriate sensitivity.
Murine norovirus is multiplied in RAW 264.7 cells (ATCC TIB-71) or other cell lines of appropriate
sensitivity.
Modified vaccinia virus Ankara is multiplied in BHK-21 cells (ATCC CCL-10) or other cell lines of
appropriate sensitivity. Vaccinia virus strain Elstree is multiplied in Vero cells (ATCC CCL-81), CV-1
cells (ATCC CCL-70) or other cell lines of appropriate susceptibility.
5.5.2 Test procedure
The test shall be performed at a temperature of between (18 ± 1) °C and (25 ± 1) °C. Place each test
surface (5.3.3) aseptically in a Petri plate (5.3.2.8) and ensure that the plate is in a horizontal position.
Nine volumes of test virus suspension (5.4.1) are mixed with one volume of interfering substance
solution (5.2.2.8).
For each test organism, for each concentration of the product and contact time prepare 2 test surfaces
plus 2 test surfaces for the water control (5.5.6). The 2 test surfaces shall be used in the same working
session, in parallel. Place the test surfaces in a sterile Petri dish under the laminar air flow and ensure
that the dish is in a horizontal position. Prepare the test surfaces by inoculating 50 μl of the virus
suspension plus interfering substance on to each test surface, paying attention to deliver the suspension
it in the centre of the test surface (5.3.3) (avoiding to touch the test surface edges). Dry the surfaces
until they are visibly dry.
It is understood that drying of the test surfaces will occur at different rates due to the ambient
conditions of the laboratory and the design of the laminar air flow cabinet. For this reason no time
duration is given and the minimum required time for the surfaces to become visibly dry should be
established for each laboratory. The drying time should not exceed 60 min and if it does, alternative
drying conditions shall be used. Allow the test surfaces to equilibrate with the chosen test temperature
(θ ± 1)°C.
Use the test surfaces within 60 min, to avoid virus inactivation with time.
Immediately after drying, carefully pick up each test surface and place it, inoculated side up, in a sterile
Petri plate and ensure that the test surface is in a horizontal position. Cover the dried inoculum on the
test surfaces with 100 µl of the test solution. Care shall be taken, not to touch pipette tip to carrier. For
the water control, place 100 µl of hard water (5.2.2.7), or water (5.2.2.2), if the product has been diluted
in water, onto the other test surfaces ensuring that the dried inoculum is totally covered.
After the chosen contact time transfer immediately each of the test surfaces to a separate container (flat
bottom between 4 cm and 5 cm at the base, with cap) and add 0,9 ml of ice-cold cell culture medium
without FCS e.g. Eagle's minimal essential medium (MEM) or Dulbecco’s Modified Eagle Medium
(DMEM) according to the cell
...