SIST EN 17914:2025

SLOVENSKI STANDARD
01-september-2025
Kemična razkužila in antiseptiki - Kvantitativni suspenzijski preskus za
vrednotenje virucidnega delovanja kemičnih razkužil in antiseptikov v živilski in
drugih industrijah, gospodinjstvu in javnih ustanovah - Preskusna metoda in
zahteve (faza 2, stopnja 1)
Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation
of virucidal activity in the food, industrial, domestic and institutional area - Test method
and requirements (Phase 2/Step 1)
Chemische Desinfektionsmittel und Antiseptika - Quantitativer Suspensionsversuch zur
Bestimmung der viruziden Wirkung chemischer Desinfektionsmittel und Antiseptika in
den Bereichen Lebensmittel, Industrie, Haushalt und öffentliche Einrichtungen -
Prüfverfahren und Anforderungen (Phase 2, Stufe 1)
Antiseptiques et désinfectants chimiques - Méthode d’essai quantitatif de suspension
pour l’évaluation de l’activité virucide des antiseptiques et des désinfectants chimiques
dans l’agroalimentaire, l’industrie, la sphère domestique et les collectivités - Méthode
d’essai et exigences (phase 2, étape 1)
Ta slovenski standard je istoveten z: EN 17914:2025
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 17914
EUROPEAN STANDARD
NORME EUROPÉENNE
June 2025
EUROPÄISCHE NORM
ICS 71.100.35
English Version
Chemical disinfectants and antiseptics - Quantitative
suspension test for the evaluation of virucidal activity in
the food, industrial, domestic and institutional area - Test
method and requirements (Phase 2/Step 1)
Antiseptiques et désinfectants chimiques - Essai Chemische Desinfektionsmittel und Antiseptika -
quantitatif de suspension pour l'évaluation de l'activité Quantitativer Suspensionsversuch zur Bestimmung der
virucide dans l'agroalimentaire, l'industrie, la sphère viruziden Wirkung chemischer Desinfektionsmittel
domestique et les collectivités - Méthode d'essai et und Antiseptika in den Bereichen Lebensmittel,
exigences (phase 2/étape 1) Industrie, Haushalt und öffentliche Einrichtungen -
Prüfverfahren und Anforderungen (Phase 2, Stufe 1)
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 17914:2025 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 . 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
5.4 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 solutions .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 Modified method for ready-to-use products .20
5.5.4 Cytotoxicity caused by product test solutions .20
5.5.5 Control of efficiency of suppression of product’s activity .22
5.5.6 Reference test for virus inactivation .22
5.5.7 Titration of the virus control .22
5.5.8 Titration of test samples .23
5.6 Experimental data and calculation .23
5.6.1 Protocol of results .23
5.6.2 Calculation of infectivity titre (TCID50 or PFU) .23
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) Viruses covered by virucidal activity claims described in this
document .27
Annex B (informative) Detoxification of test mixtures .28
TM
B.1 Molecular sieving with Sephadex LH 20 .28
B.1.1 Principle .28
B.1.2 Sephadex suspension .28
B.1.3 Procedure.28
B.2 Molecular sieving using MicroSpin™ S 400 HR .30
B.3 Determination of the residual virus titre by the large-volume-plating (LVP) method
................................................................................................................................................................... 30
B.3.1 General . 30
B.3.2 Example for the calculation of titres and the reduction according to the LVP method
................................................................................................................................................................... 31
Annex C (informative) Calculation of the viral infectivity titre . 33
C.1 Quantal tests — Example of TCID determination by the Spearman-Kärber method
................................................................................................................................................................... 33
C.2 Plaque test . 34
C.3 Biometrical evaluation of experimental approaches and assessment of the
disinfecting effect on the virus (reduction [R]) . 35
C.3.1 General . 35
C.3.2 Calculating the virus titre . 35
C.3.3 Calculating the reduction and its 95 % confidence interval . 36
Annex D (informative) Presentation of test results of one active concentration . 37
Annex E (informative) Preparation of the glutaraldehyde test solutions (v/v) . 39
Bibliography . 40

European foreword
This document (EN 17914:2025) 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 specifies a suspension test for establishing whether a chemical disinfectant or an
antiseptic has a virucidal activity in the area and fields described in the scope.
This laboratory test takes into account practical conditions of application of the product including contact
time, temperature, test organisms and interfering substances, i.e. conditions which may influence its
action in practical situations. Each utilization concentration of the chemical disinfectant or antiseptic
found by this test corresponds to the chosen experimental conditions.
Prior data generated according to EN 14476 remains valid.
1 Scope
This document specifies a test method and the minimum requirements for virucidal activity of chemical
disinfectant and antiseptic products that form a homogeneous physically stable preparation when diluted
with hard water or, in the case of ready-to-use products (i.e. products that are not diluted when applied),
with water. Products can only be tested at a concentration of 80 % (97 %, with a modified method for
special cases) as some dilution is always produced by adding the test organisms and interfering
substance.
This document is applicable to products that are used in the food, industrial, domestic and institutional
area for surface disinfection by wiping, spraying, flooding or otherwise, and for disinfection of textile and
equipment, excluding areas and situations where disinfection is medically indicated.
This document does not apply to hand disinfection and hygienic hand washing products, as this is
medically indicated in most cases. For hand disinfection and hygienic hand washing products, refer to
EN 14476.
This document does not apply to products used on living tissues and excluding products which fall under
the scope of EN 17272.
This document is applicable at least to the following:
a) processing, distribution and retailing of:
— food of animal origin:
— milk and milk products;
— meat and meat products;
— fish, seafood, and related products;
— eggs and egg products;
— animal feeds;
— etc.;
— 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.
NOTE 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 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 specific product (e.g. glutaraldehyde) in parallel with a product under test for the internal
control of the test
3.4
TCID50
50 % infecting dose of a virus suspension or that dilution of the virus suspension that induce a CPE (3.5)
in 50 % of cell culture units
3.5
viral cytopathic effect
CPE
morphological alteration of cells and/or their destruction as a consequence of virus multiplication
3.6
viral plaque
area of lysis formed in a cell monolayer under semisolid medium due to infection by and multiplication
of a single infectious virus particle
3.7
virus titre
amount of infectious virus per unit volume present in a cell culture lysate or in a solution
3.8
virucidal activity
capability of a product to reduce the number of infectious virus particles
Note 1 to entry: See Table 1.
4 Requirements
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
Test conditions Surface disinfection Surface Surface disinfection Disinfection of textile
a
food area disinfection other industrial
b c
institutional area areas
Minimum Virucidal activity Virucidal activity Virucidal activity Virucidal activity
spectrum of test
adenovirus adenovirus adenovirus adenovirus
organisms
murine norovirus murine norovirus murine norovirus murine norovirus

for
temperatures > 40 °C
c
murine parvovirus murine parvovirus
Virucidal activity Virucidal activity Virucidal activity Virucidal activity
against enveloped against enveloped against enveloped against enveloped
d d d d
viruses viruses viruses viruses
vaccinia virus vaccinia virus vaccinia virus for
temperatures ≤ 40°C
vaccinia virus
c
additional Any relevant test organism
Test temperature according to the manufacturer’s recommendation, but at / between
4 °C and ≤ 40 °C 4 °C and ≤ 40 °C 4 °C and ≤ 40 °C 10 °C and 70 °C for
main wash cycle
10 °C and 20 °C for
rinse cycle
Contact time according to the manufacturer’s recommendation
but no longer than but no longer than but no longer than but no longer than
60 min 60 min 60 min 60 min for wash cycle
20 min for rinse cycle
Interfering substance
clean conditions 0,3 g/l bovine 0,3 g/l bovine 0,3 g/l bovine 0,3 g/l bovine albumin
albumin solution albumin solution albumin solution solution (rinse cycle)
and/or and/or and/or
dirty conditions 3,0 g/l bovine 3,0 g/l bovine 3,0 g/l bovine 3,0 g/l bovine albumin
albumin solution albumin solution albumin solution solution (wash cycle)
in dairy industry 1,0 % reconstituted
e
milk
(= 1,0 g/l milk
powder)
Additional any relevant any relevant any relevant any relevant substance
conditions substance substance substance
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 (Annex A).
e
Additional interfering substances may be tested according to specific fields of application such as milk (5.2.2.8.5)
for dairies.
Table 2 — Minimum and additional test conditions for non-professional users, non-medical
Test conditions Surface disinfection domestic area Disinfection of textile domestic area
a
Minimum spectrum Virucidal activity Virucidal activity
of test organisms
adenovirus
murine norovirus
for temperatures > 40 °C
murine parvovirus
Virucidal activity against enveloped Virucidal activity against enveloped
b b
viruses viruses
vaccinia virus for temperatures ≤ 40 °C
vaccinia virus
additional Any relevant test organism
Test temperature according to the manufacturer’s recommendation, but at / between
4 °C and ≤ 40 °C 10 °C and 70 °C for main wash cycle
10 °C and 20 °C for rinse cycle
Contact time according to the manufacturer’s recommendation
but no longer than but no longer than
60 min 60 min for wash cycle
20 min for rinse cycle
Interfering substance
clean conditions 0,3 g/l bovine albumin solution 0,3 g/l bovine albumin solution (rinse cycle)
and/or
dirty conditions 3,0 g/l bovine albumin solution 3,0 g/l bovine albumin solution (wash cycle)
Additional conditions any relevant substance any relevant substance
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 (Annex A).
5 Test methods
5.1 Principle
5.1.1 A sample of the product as delivered and/or diluted with hard water (or water for ready to use
products) is added to a test suspension of viruses in a solution of an interfering substance. The mixture
is maintained at one of the temperatures and the contact times specified in Clause 4, Tables 1 and 2 and
5.5.1.1. At the end of this contact time, an aliquot is taken; the virucidal action in this portion is
immediately suppressed by a validated method (dilution of the sample in ice-cold cell maintenance
medium). The dilutions are transferred into cell culture units (petri dishes, tubes or wells of microtitre
plates) either using monolayer or cell suspension. Infectivity tests are done either by plaque test or
quantal tests. After incubation, the titres of infectivity are calculated according to Spearman and Kärber
(quantal tests, Clause C.1, Table C.2), by plaque counting (plaque test, C.2) or large-volume-plating (LVP)
(Clause B.3) and evaluated. Reduction of virus infectivity is calculated from differences of lg virus titres
before (virus control – 0 min contact time) (5.5.7) and after treatment with the product test solution
(5.5.2).
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, 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, Tables 1 and 2 :
a) Non-enveloped RNA virus
1) Murine norovirus, strain S99 Berlin
b) Non-enveloped DNA virus
1) Adenovirus type 5, strain Adenoid 75, ATCC VR-5
2) Murine parvovirus, minute virus of mice, strain Crawford, ATCC VR-1346
c) Enveloped DNA virus
1) Modified vaccinia virus, strain Ankara (MVA), ATCC VR-1508.
The required incubation temperature for these test organisms is 36 °C ± 1 °C or 37 °C ± 1 °C (5.3.1.3). The
same temperature (either 36 °C or 37 °C) shall be used for all incubations performed during a test and its
control and validation.
If additional test organisms are used, they shall be kept and used under optimum growth conditions
(temperature, time, atmosphere, media) noted in the test report. If these additional test organisms are
not classified at a reference centre, their identification characteristics shall be stated. In addition, they
shall be held by the testing laboratory or national culture collection under a reference for five years.
5.2.2 Culture media, reagents and cell cultures
5.2.2.1 General
All weights of chemical substances given in this 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.

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.
5.2.2.2 Water
The water shall be freshly glass-distilled water or demineralized water which meets the requirements
described in Table 3. If distilled water of adequate quality is not available, water for injections (see
bibliographic reference [1]) may be used.
Sterilize in the autoclave [5.3.1.1 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 × 12 H 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
0,40 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 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), 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 (5.3.1.7) or in the
autoclave [5.3.1.1 a)]. Autoclaving - if used - may cause a loss of liquid. In this case, make up to
1 000 ml with water (5.2.2.2) under aseptic conditions. Store the solution in the refrigerator (5.3.1.8)
for no longer than one month;
— prepare solution B: dissolve 35,02 g sodium bicarbonate (NaHCO ) in water (5.2.2.2) and dilute to
1 000 ml. Sterilize by membrane filtration (5.3.1.7). Store the solution in the refrigerator (5.3.1.8) for
no longer than one week;
— place 600 ml to 700 ml of water (5.2.2.2) in a 1 000 ml volumetric flask (5.3.1.12) and add 6,0 ml
(5.3.1.9) of solution A, then 8,0 ml of solution B. Mix and dilute to 1 000 ml with water (5.2.2.2). The
pH (5.3.1.4) of the hard water shall be 7,0 ± 0,2. 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 (CaCO3) is lower than 375 mg/l.
5.2.2.8 Interfering substance
5.2.2.8.1 General
The interfering substance shall be chosen according to the conditions of use laid down for the product.
The interfering substance shall be sterile and prepared at 10 times its final concentration in the test
(50 times in the case of the modified method, 5.2.2.8.4).
The ionic composition (e.g. pH, calcium and/or magnesium hardness) and chemical composition (e.g.
mineral substances, protein, carbohydrates, lipids and detergents) shall be specified.
NOTE The term “interfering substance” is used even if it contains more than one substance.
5.2.2.8.2 Clean conditions (bovine albumin solution – low concentration)
Dissolve 0,30 g of bovine albumin fraction V (suitable for microbiological purposes) in a final volume of
100 ml of water (5.2.2.2).
Sterilize by membrane filtration (5.3.1.7), keep in a refrigerator (5.3.1.8) and use within one month.
The final concentration of the bovine albumin in the test procedure (5.5) shall be 0,3 g/l.
5.2.2.8.3 Dirty conditions (bovine albumin solution – high concentration)
Dissolve 3,00 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 (5.3.1.7), keep in a refrigerator (5.3.1.8) and use within one month.
The final concentration of bovine albumin in the test procedure (5.5) shall be 3 g/l.
5.2.2.8.4 Clean and dirty conditions for the modified method for ready-to-use products
Follow the procedures for preparation according to 5.2.2.8.2 and 5.2.2.8.3, but prepare the interfering
substance in fivefold higher concentrations, for the dirty conditions maximum 50 ml to avoid problems
with the filtration.
a) Clean conditions (5.2.2.8.2) – dissolve 1,50 g bovine albumin (instead of 0,30 g) in a final volume of
100 ml of water (5.2.2.2);
b) Dirty conditions (5.2.2.8.3) – dissolve 7,50 g bovine albumin (instead of 1,50 g) in a final volume of
50 ml of water (5.2.2.2).
5.2.2.8.5 Milk (for dairies)
Skimmed milk, guaranteed free of antibiotics and additives, shall be prepared as follows:
— prepare a solution of 100 g milk-powder in 1 000 ml water (5.2.2.2).
— 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. Heat for 30 min at (105 ± 3) °C [or 5 min at (121 ± 3 °C)].
— for the modified method (5.5.3) prepare a solution of 50,0 % (v/v) in water (5.2.2.2) by adding 50
parts of reconstituted milk to 50 parts of water. Heat for 30 min at (105 ± 3) °C [or 5 min at
(121 ± 3 °C)].
The final concentration of reconstituted milk in the test procedure (5.5) is 1,0 g/l of milk-powder.
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
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 [5.3.1.1 a)];
b) by dry heat, in the hot air oven [5.3.1.1 b)].
5.3.1 Usual microbiological laboratory equipment and in particular the following:
5.3.1.1 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.
5.3.1.2 Water baths, capable of being controlled at 20 °C ± 1 °C, and at additional test
temperatures ± 1 °C (5.5.1).
5.3.1.3 CO Incubator (95 % air, 5 % C0 ), capable of being controlled either at 36 °C ± 1 °C or
2 2
37 °C ± 1 °C (5.2.1).
5.3.1.4 pH-meter, having a maximum permissible error of no more than ± 0,1 pH units at
20 °C ± 1 °C.
Disposable sterile equipment is an acceptable alternative to reusable glassware.
5.3.1.5 Stopwatch.
5.3.1.6 Shakers. ®
a) Electromechanical agitator, e.g. Vortex mixer ;
b) Mechanical shaker.
5.3.1.7 Membrane filtration apparatus, constructed of a material compatible with the substances
to be filtered, with a filter holder of at least 50 ml volume, and suitable for use of filters of diameter 47 mm
to 50 mm and 0,22 µm pore size for sterilization of hard water (5.2.2.7) and bovine albumin (5.2.2.8.2,
5.2.2.8.3 and 5.2.2.8.4).
The vacuum source used shall give an even filtration flow rate. In order to obtain a uniform distribution
of the microorganisms over the membrane and to prevent overlong filtration, the device shall be set so
as to obtain the filtration of 100 ml in 20 s to 40 s. The filtration of the interfering substance (5.2.2.8) for
the modified method (5.5.3) may take longer.
5.3.1.8 Refrigerator, capable of being controlled at 2 °C to 8 °C.
5.3.1.9 Graduated pipettes, of nominal capacities 10 ml, 1 ml, 100 µl, 10 µl or calibrated automatic
pipettes.
5.3.1.10 Ice producing machine or commercially available ice to cool the cell maintenance medium
and the reaction mixtures during the test.
5.3.1.11 Basin as ice bath with ice and water.
5.3.1.12 Volumetric flasks.
5.3.1.13 Centrifuge (400 g to 1 000 g ).
N N
5.3.1.14 Microtitre plates or tubes, petri dishes and flasks for cell culture use.
5.3.1.15 Magnetic stirrer for keeping cells in suspension before seeding.
5.3.1.16 Inverted microscope for reading cell cultures microscopically.
5.3.1.17 Container: sterile test tubes, culture bottles or flasks of suitable capacity.
5.3.1.18 Biological safety cabinet, class II.
5.3.1.19 Freezer, −70 °C or less.
5.3.1.20 Equipment for cell storage in liquid nitrogen
®
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.
5.4 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 suspension is used undiluted for the test procedure (5.5.2 or 5.5.3).
5.4.2 Product test solutions
The concentration of a product test solution shall be 1,25 times the desired test concentration (= real test
concentration) because it is diluted to 80 % during the test (5.5.2).
Product test solutions shall be prepared in hard water (5.2.2.7) at a minimum of three different
concentrations to include one concentration in the active range and one concentration in the non-active
range (5.7). The product as received may be used as one of the product test solutions; in this case, the
highest tested concentration is 80 %. Ready-to-use products may be tested at 97 % [modified method
(5.5.3)]. In this case, the “real test concentration” is 97 %.
Dilutions of ready-to-use products shall be prepared in water (5.2.2.2) instead of hard water.
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 (5.3.1.12) on a volume/volume basis in hard water (5.2.2.7).
For liquid products, dilutions of the product shall be prepared with hard water in volumetric flasks
(5.3.1.12) on a volume/volume basis.
The product test solutions shall be prepared freshly and used in the test within 2 h. They shall give a
physically homogenous preparation, stable during the whole procedure. If during the procedure a visible
inhomogeneity appears due to the formation of a precipitate or flocculate (for example, through the
addition of the interfering substance), it shall be recorded in the test report.
The concentration of the product stated in the test report shall be the desired test concentration. Record
the test concentration in terms of mass per volume or volume per volume and details of the product
sample as received.
5.5 Procedure for assessing the virucidal activity of the product
5.5.1 General
5.5.1.1 Experimental conditions
The experimental conditions may be selected according to the practical use considered for the product
(Clause 4):
a) temperature θ (in °C):
The temperatures to be tested are specified in Clause 4, Tables 1 and 2.
The allowed deviation for each chosen temperature is ± 1 °C.
b) contact time t (in min):
The contact times to be tested are specified in Clause 4, 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 substance:
The interfering substance to be tested is either:
0,30 g/l bovine albumin (5.2.2.8.2) representing clean conditions
or
3,0 g/l bovine albumin (5.2.2.8.3) representing dirty conditions
according to Clause 4, Tables 1 and 2 and practical applications. Additional interfering substances
may be tested according to specific fields of application such as Milk (5.2.2.8.5) for dairies.
d) test organisms
The test organisms to be tested are specified in Clause 4, Tables 1 and 2 and 5.2.1. Additional test
organisms may be tested.
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.
Murine parvovirus is multiplied in A9 cells (e.g. ATCC CCL-1.4) or other cell lines of appropriate
sensitivity.
Modified vaccinia virus Ankara is multiplied in BHK-21cells (e.g. ATCC CCL-10) or other cell lines of
appropriate sensitivity.
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