SIST EN 17915:2025

SLOVENSKI STANDARD
01-september-2025
Kemična razkužila in antiseptiki - Kvantitativni preskus na neporoznih površinah
brez mehanskega delovanja za vrednotenje virucidnega delovanja kemičnih
razkužil v živilski in drugih industrijah, gospodinjstvu in javnih ustanovah -
Preskusna metoda in zahteve (faza 2, stopnja 2)
Chemical disinfectants and antiseptics - Quantitative non-porous surface test without
mechanical action for the evaluation of virucidal activity of chemical disinfectants used in
the food, industrial, domestic and institutional area - Test method and requirements
(phase 2, step 2)
Chemische Desinfektionsmittel und Antiseptika – Quantitative Prüfung nicht-poröser
Oberflächen ohne mechanische Einwirkung zur Bestimmung der viruziden Wirkung
chemischer Desinfektionsmittel in den Bereichen Lebensmittel, Industrie, Haushalt und
öffentliche Einrichtungen – 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 l’agroalimentaire, l’industrie, la sphère domestique et les collectivités -
Méthode d’essai et exigences (phase 2, étape 2)
Ta slovenski standard je istoveten z: EN 17915:2025
ICS:
71.100.35 Kemikalije za dezinfekcijo v Chemicals for industrial and
industriji in doma domestic disinfection
purposes
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN 17915
EUROPEAN STANDARD
NORME EUROPÉENNE
June 2025
EUROPÄISCHE NORM
ICS 11.080.20; 71.100.35
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 food, industrial, domestic and institutional 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 Quantitative Prüfung nicht-poröser Oberflächen ohne
mécanique pour l'évaluation de l'activité virucide des mechanische Einwirkung zur Bestimmung der
désinfectants chimiques utilisés dans l'agroalimentaire, viruziden Wirkung chemischer Desinfektionsmittel in
l'industrie, la sphère domestique et les collectivités - den Bereichen Lebensmittel, Industrie, Haushalt und
Méthode d'essai et exigences (phase 2, étape 2) öffentliche Einrichtungen - Prüfverfahren und
Anforderungen (Phase 2, Stufe 2)
This European Standard was approved by CEN on 7 March 2025.

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, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye 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
© 2025 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 17915:2025 E
worldwide for CEN national Members.

Contents Page
European foreword . 4
Introduction . 5
1 Scope . 6
2 Normative references . 7
3 Terms and definitions . 7
4 Requirements for virucidal activity on surfaces. 8
5 Test methods . 10
5.1 Principle . 10
5.2 Materials and reagents, including cell cultures . 11
5.2.1 Test organisms . 11
5.2.2 Culture media, reagents and cell cultures . 11
5.3 Apparatus and glassware . 15
5.3.1 General . 15
5.3.2 Standard microbiological laboratory equipment. 15
5.3.3 Test surfaces . 16
5.4 Preparation of test organism suspensions and product test solutions . 17
5.4.1 Test organism suspension (test virus suspension) . 17
5.4.2 Product test solution . 17
5.5 Procedure for assessing the virucidal activity of the product . 18
5.5.1 General . 18
5.5.2 Test procedure . 19
5.5.3 Cytotoxicity caused by product solutions . 20
5.5.4 Control of efficiency for suppression of disinfectant virucidal activity . 22
5.5.5 Reference test for virus inactivation . 22
5.5.6 Titration of the virus control / water control . 22
5.6 Experimental data and calculation . 23
5.6.1 Protocol of the results . 23
5.6.2 Calculation of infectivity titre (TCID PFU) . 23
50 or
5.7 Verification of the methodology . 23
5.8 Expression of results . 24
5.8.1 General . 24
5.8.2 Calculation of the virucidal activity of products . 24
5.8.3 Calculation of the virucidal activity against enveloped viruses . 25
5.9 Test report . 25
Annex A (informative) Detoxification of test mixtures by molecular sieving . 27
A.1 Molecular sieving with Sephadex™ LH 20 . 27
A.2 Molecular sieving using MicroSpin™ S 400 HR . 29
A.3 Determination of the residual virus titre by the large-volume-plating (LVP) method . 29
Annex B (informative) Calculation of the viral infectivity titre . 32
B.1 Quantal tests — Example of TCID determination by the Spearman-Kärber method . 32
B.2 Plaque test . 33
B.3 Biometrical evaluation of experimental approaches and assessment of the
disinfecting effect on the virus (reduction [R]) . 34
Annex C (informative) Presentation of test results of one active concentration. 39
Annex D (informative) Preparation of the glutaraldehyde test solutions (v/v) . 42
Bibliography . 43

European foreword
This document (EN 17915:2025) 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 December 2025 and conflicting national standards shall
be withdrawn at the latest by December 2025.
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.
Any feedback and questions on this document should be directed to the users’ national standards body.
A complete listing of these bodies can be found on the CEN website.
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, France, Germany, Greece, Hungary, Iceland, Ireland,
Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of North
Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye 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 does 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 food, industrial, domestic and institutional area
for disinfecting non-porous surfaces without mechanical action, excluding areas and situations where
disinfection is medically indicated and excluding products used on living tissues.
This document applies at least to the following:
a) processing, distribution and retailing of:
1) food of animal origin:
— milk and milk products;
— meat and meat products;
— fish, seafood, and related products;
— eggs and egg products;
— animal feeds;
— etc.;
2) food of vegetable origin:
— beverages;
— fruits, vegetables and derivatives (including sugar, distillery, etc.);
— flour, milling and baking;
— animal feeds;
— etc.;
b) institutional and domestic areas:
— catering establishments;
— public areas;
— public transports;
— schools;
— nurseries;
— shops;
— sports rooms;
— waste containers (bins, etc.);
— hotels;
— dwellings;
— clinically non sensitive areas of hospitals;
— offices;
— etc.;
c) industries other than food:
— packaging material;
— biotechnology (yeast, proteins, enzymes, etc.);
— pharmaceutical;
— cosmetics and toiletries;
— textiles;
— space industry, computer industry;
— etc.
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 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
resistant steels for general purposes
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.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https://www.iso.org/obp
— IEC Electropedia: available at https://www.electropedia.org/
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. glutaraldehyde) 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
3.8
virucidal activity
capability of a product to reduce the number of infectious virus particles
4 Requirements for virucidal activity on surfaces
The product shall demonstrate at least a decimal log (lg) reduction of 4 in virus titre, when tested in
accordance with Tables 1 and 2 and Clause 5.
Table 1 — Minimum and additional test conditions for professional users, non-medical
Minimum Surface disinfection food Surface disinfection Surface disinfection other
a b c
conditions
area institutional area industrial areas
Minimum
f
Virucidal activity Virucidal activity
Virucidal activity
spectrum of
adenovirus adenovirus
murine norovirus
test organisms
murine norovirus murine norovirus

c
murine parvovirus
Virucidal activity against Virucidal activity against Virucidal activity against
d d d
enveloped viruses enveloped viruses enveloped viruses
vaccinia virus vaccinia virus vaccinia virus
c
additional
any relevant test organism
Test In a range from 4 °C to 40 °C In a range from 4 °C to 40 °C In a range from 4 °C to 40 °C
temperature
For tests performed at room For tests performed at room For tests performed at room
temperature, the range shall temperature, the range shall temperature, the range shall
be between 18 °C and 25 °C be between 18 °C and 25 °C be between 18 °C and 25 °C
Contact time according to the according to the according to the
manufacturer’s manufacturer’s manufacturer’s
recommendation, but not recommendation, but not recommendation, but not
e e e
longer than 60 min longer than 60 min longer than 60 min
Interfering
substances
clean conditions 0,3 g/l bovine serum 0,3 g/l bovine serum 0,3 g/l bovine serum
albumin albumin albumin
and/or and/or and/or
dirty conditions 3,0 g/l bovine serum 3,0 g/l bovine serum 3,0 g/l bovine serum
albumin albumin albumin
g
in dairy industry
1,0 % reconstituted milk
(= 1,0 g/l milk powder)
Additional any relevant substance any relevant substance any relevant substance
conditions
a
Processing, distribution and retailing of food of animal origin, food of vegetable origin.
b
Excluding nursing homes, any other medical area or medical indicated outbreak management in any area, e.g.
an outbreak in a kindergarten with enteroviruses.
c
Murine parvovirus may be relevant for special areas, e.g. in pharmaceutical manufacturing of vaccines with
parvovirus as vector.
d
The test for “virucidal activity against enveloped viruses” will cover all enveloped viruses only.
e
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.
f
Adenovirus does not need to be tested for surfaces in food processing, distribution and retailing.
g
Additional interfering substances may be tested according to specific fields of application such as milk

(5.2.2.8.4) for dairies.
Table 2 — Minimum and additional test conditions for non-professional users, non-medical
Minimum conditions Surfaces disinfection domestic area
a
Minimum spectrum of
Virucidal activity
test organisms
b
Virucidal activity against enveloped viruses
vaccinia virus
additional any relevant test organism
Test temperature at room temperature between 18 °C and 25 °C
Additional temperature between 4 °C and and ≤ 40 °C
c
Contact time
according to the manufacturer’s recommendation, but not longer than 60 min
Interfering substances
clean conditions 0,3 g/l bovine serum albumin
and/or
dirty conditions 3,0 g/l bovine serum albumin
Additional conditions Further contact time(s), interfering substance(s) or virus(es)
a
Non-professional area, higher virucidal activity to be claimed via norms of the human medicine area, e.g.
EN 14476, EN 16777 as in these situations the application is very likely medically indicated.
b
The test for “virucidal activity against enveloped viruses” will cover all enveloped viruses only.
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.
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 (or water for ready to use products)
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, Tables 1 and 2.
5.1.3 Other contact times and temperatures within the limits specified in Clause 4, Tables 1 and 2 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 strain as test organism selected according to
Clause 4, Tables 1 and 2 :
a) Non-enveloped RNA virus
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
Modified vaccinia virus, strain Ankara (MVA), ATCC VR-1508
The required incubation temperature for the test organism is 36 °C ± 1 °C or 37 °C ± 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 document 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 material – if appropriate- 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 or demineralized water, which meets the requirements of
Table 3. If distilled water of adequate quality is not available, water for injections (see bibliographic
reference [1]) may be used.
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 document and does not constitute an endorsement by CEN of the product
named.
Sterilize in the autoclave [5.3.2.1 a)]. Sterilization is not necessary if the water is used e.g. for preparation
of culture media and subsequently sterilized. Alternatively, cell culture grade water is suitable for
preparation of cell culture media and other solutions used in cell culture.
Table 3 — Consolidated classification of water qualities
Unit Type I Type II
(ultrapure (purified
water) water)
Resistance (M/cm)Ω > 18 > 1
Conductivity µS/cm < 0,056 < 1
Silicates(SiO ) ppb or µg/L 3 3
TOC ppb or µg/l 50 50
Bacteria CFU/ml < 1 < 100
Endotoxin EU/ml < 0,001 0,03
Type I Required for critical laboratory applications (e.g. for
HPLC, GC; MS etc.). Also for the production of buffers for
cell culture, as well as for applications in molecular
biology.
Type II Used for buffers and microbiological culture media. Used
as system water for laboratory automats and for
dissolving controls and reagents
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 1 000 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.
5.2.2.5 Foetal calf serum (FCS)
FCS shall be certified free of viruses and mycoplasma. Extraneous viruses and mycoplasma can 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.

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.
5.2.2.6 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 - can 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
1 000 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 1 000 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 1 000 ml with water (5.2.2.2). The pH (5.3.2.4)
of the hard water shall be 7,0 ± 0,2. If necessary, adjust the pH by using a solution of approximately
40 g/l (approximately 1 mol/l) of sodium hydroxide (NaOH) or approximately 36,5 g/l
(approximately 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 specified.
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 – low concentration)
Dissolve 0,3 g of bovine albumin fraction V (suitable for microbiological purposes) in a final volume of
100 ml of water (5.2.2.2).
Sterilize by membrane filtration (5.3.2.11), keep in a refrigerator (5.3.2.6) and use within one month.
The final concentration of bovine albumin in the test procedure (5.5.2) is 0,3 g/l.
5.2.2.8.3 Dirty conditions (bovine albumin solution – high concentration)
Dissolve 3,0 g of bovine albumin fraction V (suitable for microbiological purposes) in a final volume of
100 ml of water (see 5.2.2.2). Sterilize by membrane filtration (5.3.2.11), keep in a refrigerator (5.3.2.6)
and use within one month.
The final concentration of bovine albumin in the test procedure (5.5.2) is 3,0 g/l.
5.2.2.8.4 Milk (for dairies)
Skimmed milk, guaranteed free of antibiotics and additives, shall be reconstituted at a rate of 100 g
powder per litre of water (see 5.2.2.2). The working solution shall be prepared as follows:
— prepare a solution of 10,0 % (v/v) in water (5.2.2.2) by adding 10 parts of reconstituted milk to 90
parts of water.;
— sterilize for 30 min at 105 °C ± 3 °C [or 5 min at 121 °C ± 3 °C].
The final concentration of reconstituted milk should be 1,0 % (v/v) in the test procedure (5.5.2) or 1,0 g/l
of milk-powder during the test.
5.2.2.9 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.
5.2.2.10 Cell cultures
Before virus inoculation, cell monolayers shall be at appropriate level of confluence based on the specific
virus (see also EN 12353). 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 no longer than 2 days in the
cell control.
Cell lines are selected in accordance with their sensitivity to the test organisms (5.2.1). For details of cell
lines, see 5.5.1.1 e).
5.2.2.11 Reference glutaraldehyde (Glutaral, 1,5-Pentanedial) CAS Number 111-30-8
Required chemical and physical parameters for use as reference standard for testing disinfectant
preparations are specified in Table 4.
Table 4 — Required chemical and physical parameters
Parameters Specifications
Solution 50 % Solution 25 %
a
Appearance clear liquid clear liquid
pH-value 3,1 to 4,5 3,1 to 4,5
Concentration of glutaraldehyde (by titration) 50,0 % to 52,0 % 25,0 % to 26,0 %
Stability (ambient conditions) 12 months 12 months
a
Yellowish colour indicates a beginning polymerization
The specification above (see Table 4) should be checked on the certificate of analyses. The pH values shall
be tested and confirmed regularly by the laboratory. If the pH values and / or appearance are out of
specification the glutaraldehyde shall not be used.
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 Standard 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 ( 121 ) °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 °C ± 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 dishes, 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.

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.
5.3.2.12 CO incubator (95 % air, 5 % CO ), capable of being controlled at 36 °C ± 1 °C, for incubation
2 2
of cell cultures.
An incubator at 37 °C ± 1 °C may be used if an incubator at 36 °C ± 1 °C is not available.
5.3.2.13 Graduated sterile pipettes of nominal capacities 10 ml and 1 ml and 0,1 ml.
NOTE Calibrated automatic pipettes can 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.
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.
5.3.2.24 Equipment for cell storage in liquid nitrogen.
5.3.3 Test surfaces
These shall be 1.4301 (EN 10088-1) stainless steel discs (2 cm diameter discs) with Grade 2b 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 Decon
®, 5
90 or of 1 % Blanisol-Pur for 60 min, ensuring that the carriers are separated and the surface is not
damaged. Rinse the discs with running freshly distilled water (5.2.2.2) or demineralized water for 10 s.
Do not allow them to dry.
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 ISO-Propanol (IPA) (for example 70 % v/v) 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.

Decon 90® and Blanisol-Pur are examples of suitable products available commercially. This information is given
for the convenience of users of this document and does not constitute an endorsement by CEN of those products.
5.4 Preparation of test organism suspensions and product test solutions
5.4.1 Test organism suspension (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”.
The test virus 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.
It is suggested that the minimum titre of the test 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 virus suspension may be concentrated by appropriate methods (e.g.
ultracentrifugation).
The test virus suspension is used undiluted for the test procedure (5.5.2 or 5.5.3).
5.4.2 Product test solution
Detailed 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 a minimum of 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 (5.2.2.2).
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 fill 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 General
5.5.1.1 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:
test temperature shall be chosen between 4 °C ± 1 °C and 40 °C ± 1 °C. A temperature controlled
chamber should be usted for temperature other than ambient. For thest performed at room
temperature, the range shall be between 18 °C and 25 °C);
b) Contact time, t (min):
The contact times to be tested are specified in Clause 4, Tables 1 and 2.
The allowed deviation for each chosen contact time is ± 10 s, except for 1 min or less where it is ± 5 s.
c) Interfering substances:
The interfering substance to be tested is the bovine albumin solution under clean conditions
(5.2.2.8.2) or under dirty conditions (5.2.2.8.3), according to practical applications.
Additional interfering substance can be selected according to specific fields of application (Tables 1
and 2), such as Milk (5.2.2.8.4), for dairies and shall be recorded in the test report (5.9)
d) Test organisms
The test organisms to be tested are specified in Clause 4, Tables 1 and 2.
Additional strains may be chosen and shall be reported in the test report (5.9).
e) Cell line(s):
Adenovirus is multiplied in HeLa cells (e.g. ATCC CCL-2) or other cell lines of appropriate sensitivity
such as A549 cells (e.g. ATCC CCL-185).
Murine norovirus is multiplied in RAW 264.7 cells (e.g. ATCC TIB-71) or other cell lines of appropriate
sensitivity.
Modified vaccinia virus Ankara is multiplied in BHK-21 cells (e.g. ATCC CCL-10) or other cell lines of
appropriate sensitivity.
Murine parvovirus is multiplied in A9 cells (e.g. ATCC CCL-1.4) or other cell lines of appropriate
sensitivity.
5.5.1.2 Preparation
Tubes are filled with ice-cold maintenance medium (5.2.2.9 b)) for the various titrations that shall be
performed after the different contact times and placed in an ice bath. Microtitre plates are labelled for
identification.
The procedures described under 5.5.3 to 5.5.6 are always carried out in parallel with the test procedure
(5.5.2).
5.5.1.3 Equilibration of temperature
Prior to testing, equilibrate all reagents [product test solutions (5.4.2), PBS (5.2.2.3), cell culture (5.2.2.9),
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 wate
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