FprEN 16777

SLOVENSKI STANDARD
01-oktober-2014
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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 des désinfectants chimiques
utilisés dans le domaine médical - Méthode d'essai et prescriptions (phase 2/étape 2)
Ta slovenski standard je istoveten z: prEN 16777
ICS:
11.080.20 Dezinfektanti in antiseptiki Disinfectants and antiseptics
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EUROPEAN STANDARD
DRAFT
prEN 16777
NORME EUROPÉENNE
EUROPÄISCHE NORM
July 2014
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 quantitatif Chemische Desinfektionsmittel und Antiseptika -
de surface non-poreuse sans action mécanique pour Quantitativer Versuch auf nicht porösen Oberflächen ohne
l'évaluation de l'activité virucide des désinfectants mechanische Einwirkung zur Bestimmung der viruziden
chimiques utilisés dans le domaine médical - Méthode Wirkung im humanmedizinischen Bereich - Prüfverfahren
d'essai et prescriptions (phase 2/étape 2) und Anforderungen (Phase 2, Stufe 2)
This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee CEN/TC 216.

If this draft becomes a European Standard, 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.

This draft European Standard was established by CEN 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.
Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are aware and to
provide supporting documentation.

Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without notice and
shall not be referred to as a European Standard.

EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2014 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 16777:2014 E
worldwide for CEN national Members.

prEN 16777:2014 (E)
Contents
Page
Foreword .3
Introduction .4
1 Scope .5
2 Normative references .5
3 Terms and definitions .5
4 Requirements for virucidal activity on surfaces .6
5 Test methods .6
5.1 Principle .6
5.2 Materials and reagents, including cell cultures .7
5.3 Apparatus and glassware . 10
5.4 Preparation of test organism suspensions and product test solutions . 12
5.5 Procedure for assessing the virucidal activity of the product . 13
5.6 Experimental data and calculation. 17
5.7 Verification of the methodology . 18
5.8 Expression of results . 19
5.9 Test report . 19
Annex A (informative) Examples of viruses sorted according to their presence in the human
body in case of virus infection . 21
Annex B (normative) Detoxification of test mixtures by molecular sieving . 23
B.1 Molecular sieving with SephadexTM LH 20 . 23
Annex C (informative) Calculation of the viral infectivity titre . 25
C.1 Quantal tests . 25
C.2 Plaque test . 26
C.3 Biometrical evaluation of experimental approaches and assessment of the disinfecting
effect on the virus (reduction [R]): . 26
Bibliography . 31

prEN 16777:2014 (E)
Foreword
This document (prEN 16777:2014) has been prepared by Technical Committee CEN/TC 216 “Chemical
disinfectants and antiseptics”, the secretariat of which is held by AFNOR.
This document is currently submitted to the CEN Enquiry.
prEN 16777:2014 (E)
Introduction
This European Standard 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 European Standard.
prEN 16777:2014 (E)
1 Scope
This European Standard 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 European Standard applies to products that are used in the medical area for disinfecting non-porous
surfaces including surfaces of medical devices without mechanical action.
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
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, 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 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.
prEN 16777:2014 (E)
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 Clauses 5, 6, 7, 8, and 9.
Table 1 — Minimum and additional test conditions
Test conditions
Test virus Adenovirus Type 5
Murine Norovirus
Test temperature between 18 °C ± 1 °C and 25 °C ± 1 °C
Contact time according to the manufacturer’s recommendation, but not longer than 5
a
min or 60 min
Interfering substances
a) clean 0,3 g/l bovine serum albumin
and/or
b) dirty 3,0 g/l bovine serum albumin plus 3,0 ml erythrocytes
b Further contact time(s), interfering substance(s) or virus(es)
Additional conditions
a
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.
b
Where appropriate (specific purposes), additional specific virucidal activity shall be determined under other conditions
of time, temperature, and interfering substances (see 5.2.3.3) in accordance with 6.2, in order to take into account
intended specific use conditions. Additional virus(es) can be tested, if relevant.

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 stainless
steel disc and dried. A prepared sample of the product under test is applied in a manner which covers the
dried film.
The disc is maintained at a specified temperature for a defined period of time. The disc is transferred to cell
maintenance medium so that the action of the disinfectant is immediately neutralised. The titre of the virus
recovered from the disc is determined.
The titre of the virus on a disc 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.
prEN 16777:2014 (E)
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
1)
clause 4, table 1
2)
a) Non-enveloped RNA virus
Murine norovirus, strain S99 Berlin
b) Non-enveloped DNA virus
Adenovirus type 5, strain Adenoid 75, ATCC VR-5*
The required incubation temperature for these test organisms is 36 °C ± 1 °C or 37 °C ± 1 °C (5.3.2.3). 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 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.

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. 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.
prEN 16777:2014 (E)
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 0,40 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), 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 (5.3.2.7) 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 (5.3.2.8) 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. Sterilize by membrane filtration (5.3.2.7). Store the solution in the refrigerator (5.3.2.8) for no
longer than one week;
 place 600 ml to 700 ml of water (5.2.2.2) in a 1000 ml volumetric flask (5.3.2.12) and add 6,0 ml (5.3.2.9)
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.
prEN 16777:2014 (E)
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 100 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 100 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 (see 6.6.3).
b) sheep erthrocytes:
Prepare at least 8,0 ml fresh defibrinated sheep blood (5.2.2.9). Centrifuge the erythrocytes at 800 g for
N
10 min (5.3.2.13). After discarding the supernatant, resuspend erythrocytes in water (5.2.2.2). Repeat this
procedure at least 3 times, until the supernatant is colourless.
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.
prEN 16777:2014 (E)
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 5.5.1.1e).
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:
c) by moist heat, in the autoclave [5.3.2.1 a)];
d) by dry heat, in the hot air oven [5.3.2.1 b)].
3)
5.3.2 Usual microbiological laboratory equipment
And, in particular, the following:
5.3.2.1 Apparatus for sterilization (moist and dry heat)
+3
e) For moist heat sterilization, an autoclave capable of being maintained at (121 ) °C for a minimum
holding time of 15 min;
+5
f) for dry heat sterilization, a hot air oven capable of being maintained at (180 ) °C for a minimum holding
+5 +5
time of 30 min, at (170 ) °C for a minimum holding time of 1 h or at (160 ) °C for a minimum holding
0 0
time of 2 h.
5.3.2.2 Water baths, capable of being controlled at 20 °C ± 1 °C, and at additional test temperatures
± 1 °C (5.5.1).
3) Disposable sterile equipment is an acceptable alternative to reusable glassware.
prEN 16777:2014 (E)
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.
4)
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 Sterile microtitre plates, six well plates for cell culture, and flasks for cell culture use.
5.3.2.11 Membrane filtration apparatus for filtration of media, 0,2 μm pore size
5.3.2.12 CO incubator (95% air, 5% CO ), capable of being controlled at 36 °C ± 1 °C, for incubation of
2 2
cell cultures. An incubator at 37 °C ± 1 °C may be used if an incubator at 36 °C ± 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 6.1, 6.5.1, and 6.6.3)
5.3.2.16 Basin as ice bath with ice and water
5.3.2.17 Mechanical shaker
5.3.2.18 Centrifuge
5.3.2.19 Biological safety cabinet, class II
5.3.2.20 Freezer, -70 °C
5.3.2.21 Desiccator with vacuum manometer
5.3.2.22 Vacuum Diaphragm Pump (throughput max. 3,8 m /h final vacuum < 75mbar)
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 % (V/V) Decon 90 or of
5)
1% Blanisol-Pur for 60 min, in a manner that they don’t stick together and the surface is not damaged.

4) 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) 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.
prEN 16777:2014 (E)
Rinse the discs with running freshly distilled water (5.2.2.2) or demineralised 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 70 % (V/V) ethanol for 15 min. Remove the discs and rinse them with double distilled water for
at least 10 seconds. 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 is called "test
N
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 solution
Details of samples 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.
Dilutions of ready-to-use products, i.e. products which are not diluted when applied, shall be prepared in water
(5.2.2.6).
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 (5.3.2.12) 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 on a volume/volume basis using
volumetric flasks (5.3.2.12).
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.
prEN 16777:2014 (E)
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 of between 18 °C ± 1 °C and 25 °C ± 1 °C; additional
temperatures may be chosen and shall be reported in the test report (8.2).
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 (8.2).
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 (8.2).
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.
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
5.5.2 Test procedure
The test shall be performed at a temperature of between 18 °C ± 1 °C and 25 °C ± 1 °C. Place each test disc
(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 virus suspension (5.4.1 and 6.3) are mixed with one volume of interfering substance solution
(5.2.3.3).
For each test virus prepare 8 test discs (2 discs/concentration of the product plus 2 discs for the control) by
inoculating 50 μl of the virus suspension plus interfering substance onto the disc in 2 aliquots of 25 µl each,
paying attention to deliver the suspension it in the centre of the disc (5.2.5) (avoiding to touch the disc edges).
Remove the lid of the Petri plate and place it in upright position into a desiccator (5.1.2.21). Close the lid of the
6)
desiccator and the desiccator valve . Connect the desiccator and the diaphragm pump (5.1.2.22) with a tube.
Open the valve and switch on the pump. Check that after maximum 5 min the vacuum is between 80 and 130
mbar. During drying time the pump shall not be switched off, otherwise the pressure increases.

6) The desiccator does not need to contain chemical substances to reduce the humidity. In the vacuum tube there must
be a syringe prefilter to avoid the escape of microorganisms into the ambient air. The filter can be autoclaved 5 times;
then, the filter must be replaced by a new one.
prEN 16777:2014 (E)
Alternatively, 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 0,05 ml of the virus suspension plus
interfering substance on to each test surface. 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 incubator (e.g. with or without a fan). 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 dried carriers within 60 minutes, to avoid virus inactivation with time.
After drying, carefully pick up each disc and place it, inoculated side up, in a sterile Petri plate and ensure that
the disc is in a horizontal position. Cover the dried inoculum of 6 discs with 100 µl of the test solution. Care
has to 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 3 test discs ensuring that the dried
inoculum is totally covered.
After the appropriate contact time transfer each of the discs 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 (EMEM) or Dulbecco’s Modified Eagle Medium (DMEM) according to the cell line
used. Mix (5.3.2.7) each container for 30 s to re-suspend the virus. Visually examine each carrier and, in case
of incomplete elution, mix further 30 s (5.3.2.7).
Immediately after elution, prepare a series of ten-fold dilutions of the virus suspension in ice-cold maintenance
medium (EMEM + 2% FCS). Pipette tips shall be changed after each dilution to avoid carry-over of viruses.
The dilutions are inoculated on cell culture. After incubation the titre of virus is calculated.
Reduction of virus titre is calculated from differences of dried virus titres before and after treatment with the
product.
After treatment with the product, the infectivity is tested with one of the following procedures:
a) Quantal tests (endpoint titration) – [the procedures 1) to 2) are alternatives]
1) Virus titration on cells in suspension on microtitre plates:
Transfer 0,1 ml of each dilution into six or eight wells of a microtitre plate (5.3.1.14), beginning with
the highest dilution. Add 0,1 ml of cell culture suspension (5.2.2.11) in such a density as to enable
the formation of a monolayer (> 90 %) in at least 2 d in the cell control. In parallel six or eight wells do
not receive any viral suspension and will serve as the cell control.
The viral cytopathic effect (CPE) is read by using an inverted microscope after the appropriate
incubation time (according to the virus type).
2) Virus titration on monolayers of cells on microtitre plates:
Transfer 0,1 ml of each dilution into six or eight wells of a microtitre plate (5.3.1.14) containing a
confluent (> 90 %) cell monolayer (5.2.2.11) without any medium. The last row of six or eight wells
will receive 0,1 ml of culture medium (5.2.2.10) and will serve as the cell control. After 1 h of
incubation at 37 °C, 0,1 ml of cell culture medium (5.2.2.10) is added to each well. Change pipettes
for tubes or wells when adding medium.
Dilute the viral suspension by tenfold dilution steps with a volume of 0,1 ml of viral suspension plus an
appropriate volume of culture maintenance medium (EMEM + 2% FCS). Pipettes shall be changed after each
dilution step.
prEN 16777:2014 (E)
Transfer 0,1 ml of each dilution into six or eight wells of a microtitre plate, beginning with the highest dilution.
Add 0,1 ml of cell culture in such a density to enable the formation of a monolayer (> 90 %) in at least 2 days
in the cell control. The last row of six or eight wells shall not receive any virus suspension and serve as the
cell control.
After the appropriate incubation time, according the virus type, the viral cytopathic effect is read using an
inverted microscope.
b) Plaque assay
Plastic tray wells (surface diameter 30 mm to 35 mm) with confluent cell monolayers are washed once with
phosphate buffered saline (PBS) (5.2.2.3) and inoculated with 0,2 ml of serial dilutions of virus in EMEM + 2%
FCS. Six to eight wells are generally used per dilution. After absorption period of 1 h at 37°C, during which the
cell monolayers are kept moist by tilting the dishes every 8 min to 10 min, the inoculum is removed and the
cell monolayers are washed once with PBS. Subsequently, the wells are overlaid with 3 ml of a mixture
consisting of 2% melted agarose or another appropriate semisolid medium and 2 times concentrated MEM
with 4% FCS. The cultures are incubated for 5 to 6 days at 37°C in a CO incubator (5.1.2.12). Plaques can
be counted after addition of 2 ml of a second overlay with the same composition of the first and also
containing 5% of a 1:1000 solution of neutral red and further incubation (in the dark) at 37°C for 24 h to 48 h in
a CO incubator (5.1.2.12). Counting can be performed also after addition of crystal violet. The cell
monolayers are fixed by adding 2 ml of 10% trichloroacetic acid (TCA) (5.2.2.6) over the agar overlay for 10
min to 15 min at room temperature. The agar overlay is then removed and 2 ml of 0,1% crystal violet in 20%
ethanol are added. After 10 min to 15 min at room temperature, the wells are extensively washed with water
and the plaques (white spots) are counted.
NOTE Determination of CPE can be used as an alternative to plaque assay.
5.5.3 Cytotoxicity caused by product solutions
5.5.3.1 Cytotoxic effect
To check for possible morphological alteration of cells by the disinfectant, mix 45 µl MEM 2 % FCS + 5 µl of
interfering substance, inoculate on a carrier and dry under desiccator (6.6.3). Add 100 µl of product (at the
dilutions used in the test) and leave for 5 min at test temperature. Elute with 0,9 ml MEM + 0 % FCS vortex 30
s. Serial dilutions (dilution step: 1:10) are prepared in the culture medium and are inoculated into cell cultures
(monolayers or suspended cells). This has to be done in parallel with the test. Any microscopic changes in the
cells are recorded when reading the tests for CPE (3 discs).
5.5.3.2 Interference control – control of cell susceptibility
The aim of the interference control is to verify that the susceptibility of the cells for the virus infection is not
influenced negatively by the treatment with the disinfectant.
Comparative virus titrations are performed on cells that have or have not been treated with disinfectants to
check the reduction of the sensitivity to viruses as follows:
Treatment of cells:
a) monolayers: 0,1 ml of the lowest apparently non-cytotoxic dilution (no microscopic alteration) of the
product test solution (5.4.2) or PBS (5.2.2.3) and 0,1 ml of culture medium are distributed onto each of
6 established cell cultures in microtitre plates with 96 wells (5.3.2.14). After 1 h at 37 °C the
supernatant is discarded;
b) suspension cells: a volume of the lowest apparently non-cytotoxic dilution of the product or PBS
(5.2.2.3) is added to a volume of double concentrated cell suspension. After 1 h at 37 °C the cells are
centrifuged and resuspended in culture medium.
prEN 16777:2014 (E)
Comparative titration of viruses:
-1 -10
The virus is diluted from 10 to 10 and titrated on the treated or untreated cells in parallel as in 5.5.2 a) or
b).
Only those dilutions of the product solution can be used for the determination of the residual infectivity which:
a) show a low degree of cell destruction (< 25 % of monolayer);

b) produce a titre reduction of the virus of < 1 lg.
5.5.3.3 Elimination of cytotoxicity
If the cytotoxicity is so great that the reduction of the residual infectivity titre cannot be followed over the range
of 4 lg TCID , special techniques have to be used, such as:
TM
7)
a) molecular sieving with a molecular sieve filter, i.e. Sephadex LH 20 (annex B);
7)
b) ultrafiltration with Minicon® (Millipore) or with ready to use i.e. MicroSpinTM S 400 HR columns (GE
Healthcare) (annex B).
Appropriate controls are required to demonstrate that the procedure does not reduce the infectivity of the
virus. The virucidal activity is measured by comparing the titres of the treated and untreated viral suspensions.
Use according to the instructions of the manufacturer.
5.5.4 Control of efficiency for suppression of disinfectant activity
5.5.4.1 Dilution in ice-cold medium
Immediately after preparation of the test mixture (5.5.2) mix [5.3.2.6a)] and pipette 0,5 ml of the test mixture
into 4,5 ml of ice-cold MEM + 2 % FCS (5.2.2.5). Mix again and start the clock. Incubate the mixture in the ice
-8
bath (5.3.2.11) for 30 min ± 10 s. Immediately prepare dilutions up to 10 , and titrate the virus [5.5.2 a) or b)
applies]. This control is performed in
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