SIST EN 14476:2013+A2:2019

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)

This European Standard specifies a test method and the minimum requirements for virucidal activity of chemical disinfectant and antiseptic products that form a homogeneous physically stable preparation when diluted with hard water or in the case of ready-to-use products, i. e, products that are not diluted when applied, with water. Products can only be tested at a concentration of 80 % (97 %, with a modified method for special cases) as some dilution is always produced by adding the test organisms and interfering substance.
This European Standard applies to products that are used in the medical area in the fields of hygienic handrub, hygienic handwash, instrument disinfection by immersion, surface disinfection by wiping, spraying, flooding or other means and textile disinfection.
This European Standard applies to areas and situations where disinfection is medically indicated. Such indications occur in patient care, for example:
-   in hospitals, in community medical facilities, and in dental institutions;
-   in clinics of schools, of kindergartens, and of nursing homes;
and may occur in the workplace and in the home. It may also include services such as laundries and kitchens supplying products directly for the patients.
NOTE 1   The method described is intended to determine the activity of commercial formulations or active substances under the conditions in which they are used.
NOTE 2   This method corresponds to a phase 2, step 1 test.
NOTE 3   EN 14885 specifies in detail the relationship of the various tests to one another and to "use recommendations".

Chemische Desinfektionsmittel und Antiseptika - Quantitativer Suspensionsversuch zur Bestimmung der viruziden Wirkung im humanmedizinischen Bereich - Prüfverfahren und Anforderungen (Phase 2, Stufe 1)

Dieses Dokument legt ein Prüfverfahren für und die Mindestanforderungen an die viruzide Wirkung von chemischen Desinfektionsmitteln und Antiseptika fest, die bei Verdünnung mit Wasser standardisierter Härte als homogene, physikalisch stabile Zubereitung vorliegen, bzw. bei gebrauchsfertigen Produkten, z. B. Produkte, die bei der Anwendung nicht mit Wasser verdünnt werden. Die Produkte können nur bei einer Konzentration von 80 % (97 % bei einem modifizierten Verfahren für Sonderfälle) geprüft werden, da durch Zugabe der Prüfkeime und der Belastungssubstanz immer eine gewisse Verdünnung bewirkt wird.
Diese Europäische Norm gilt für Produkte, die im medizinischen Bereich auf den Gebieten der hygienischen Händedesinfektion, hygienischen Händewaschung, Instrumentendesinfektion durch Eintauchen und Oberflächendesinfektion durch Abwischen, Besprühen, Überfluten oder auf sonstige Weise sowie für die Wäschedesinfektion verwendet werden.
Diese Europäische Norm gilt für Bereiche und unter Bedingungen, wo eine Desinfektion aus medizinischen Gründen angezeigt ist. Indikationen dieser Art liegen z. B. vor bei der Patientenbetreuung:
-   in Krankenhäusern, kommunalen medizinischen Einrichtungen und im Dentalbereich;
-   in medizinischen Bereichen in Schulen, Kindergärten und Heimen
und können auch am Arbeitsplatz oder im privaten Bereich gegeben sein. Eingeschlossen sein können auch Einrichtungen wie Wäschereien und Küchen, die der direkten Versorgung von Patienten dienen.
ANMERKUNG 1   Das beschriebene Verfahren dient zur Bestimmung der Wirksamkeit kommerziell erhältlicher Zuberei¬tungen oder Wirkstoffe unter den Bedingungen, unter denen sie angewendet werden.
ANMERKUNG 2   Dieses Verfahren entspricht einer Prüfung der Phase 2, Stufe 1.
ANMERKUNG 3   EN 14885 legt im Einzelnen die Beziehung der verschiedenen Prüfungen untereinander sowie zu den "Anwendungsempfehlungen" fest.

Antiseptiques et désinfectants chimiques - Essai quantitatif de suspension pour l'évaluation de l'activité virucide dans le domaine médical - Méthode d'essai et prescriptions (Phase 2/Étape 1)

La présente Norme européenne spécifie une méthode d’essai et les prescriptions minimales relatives à l’activité virucide des produits antiseptiques et désinfectants chimiques qui forment une préparation homogène, physiquement stable, lorsqu’ils sont dilués dans l’eau dure ou – dans le cas de produits prêts à l’emploi, c’est-à-dire de produits qui ne sont pas dilués lors de leur utilisation – dans l’eau. Les produits ne peuvent être soumis à essai qu’à une concentration de 80 % (97 % avec une méthode modifiée dans certains cas particuliers), car l’ajout des microorganismes d’essai et de la substance interférente s’accompagne forcément d’une dilution.
La présente Norme européenne s’applique aux produits utilisés dans le domaine médical pour le traitement hygiénique des mains par frictions, le lavage hygiénique des mains, la désinfection des instruments par immersion, la désinfection des surfaces par essuyage, pulvérisation, inondation ou d’autres moyens, et la désinfection des textiles.
La présente Norme européenne s’applique aux domaines et situations où la désinfection est indiquée en médecine humaine. De telles indications se produisent pour les soins des patients, par exemple :
   dans les hôpitaux, les centres médicaux communautaires et les institutions dentaires ;
   dans les cliniques d’écoles, de jardins d’enfants et de maisons de soins infirmiers ;
et peuvent se produire sur le lieu de travail et à la maison. Elles peuvent également inclure les services tels que les blanchisseries et les cuisines fournissant directement des produits aux patients.
NOTE 1   La méthode décrite a pour objet de déterminer l’activité de formulations commerciales ou de substances actives dans leurs conditions d’emploi.
NOTE 2   Cette méthode correspond à un essai de phase 2, étape 1.
NOTE 3   L’EN 14885 spécifie de façon détaillée les relations des différents essais entre eux et avec les « recommandations d’emploi ».

Kemična razkužila in antiseptiki - Kvantitativni suspenzijski preskus za vrednotenje virucidnega delovanja v humani medicini - Preskusna metoda in zahteve (faza 2, stopnja 1) (vključno z dopolnilom A2)

Ta evropski standard določa preskusno metodo in minimalne zahteve za virucidno delovanje kemičnih razkužil in antiseptikov, ki tvorijo homogen, fizikalno stabilen pripravek, če so razredčeni s trdo vodo ali, pri proizvodih, ki so pripravljeni za uporabo (tj. proizvodih, ki med uporabo niso razredčeni), z vodo. Proizvode je mogoče preskušati samo pri 80-odstotni koncentraciji (s prilagojeno metodo v posebnih primerih 97-odstotni), ker dodajanje preskusnih organizmov in moteče snovi vedno povzroči razredčenje.
Ta evropski standard se uporablja za proizvode, ki se uporabljajo na področju zdravstva pri higienskem drgnjenju rok, higienskem umivanju rok, dezinfekciji instrumentov s potapljanjem, površinski dezinfekciji z brisanjem, pršenjem, zalivanjem ali pri drugih načinih dezinfekcije in dezinfekciji tekstilij.
Ta evropski standard se uporablja za področja in primere, ko obstajajo zdravniške indikacije za dezinfekcijo. Te indikacije se pojavljajo pri negi bolnikov, na primer:
–   v bolnišnicah, javnih zdravstvenih in zobozdravstvenih ustanovah;
–   v ambulantah šol, vrtcev in domov za starejše;
in lahko se pojavljajo na delovnem mestu ali doma. Vključujejo lahko tudi storitve, kot so pralnice in kuhinje, ki proizvode dostavljajo neposredno bolnikom.
OPOMBA 1:   Opisana metoda je namenjena določevanju dejavnosti komercialnih oblik ali aktivnih snovi pod pogoji, v katerih se uporabljajo.
OPOMBA 2:   Ta metoda ustreza preskusu stopnje 1 faze 2.
OPOMBA 3:   Standard EN 14885 podrobno določa razmerje med različnimi preskusi in »priporočili za uporabo«.

General Information

• Status: Withdrawn
• Publication Date: 11-Aug-2019
• Withdrawal Date: 08-Dec-2025
• ICS: 11.080.20 - Disinfectants and antiseptics
• Technical Committee: KDS - Cosmetics, chemical disinfectants and surface active agents
• Current Stage: 9900 - Withdrawal (Adopted Project)
• Start Date: 21-Nov-2025
• Due Date: 14-Dec-2025
• Completion Date: 09-Dec-2025
• Ref Project: EN 14476:2013+A2:2019 - 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)

Relations

• Replaces: SIST EN 14476:2013+A1:2015 - 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)
• Effective Date: 01-Sep-2019
• Replaced By: SIST EN 14476:2026 - 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)
• Effective Date: 01-Jan-2026
• Consolidates: SIST EN 14476:2013+A1:2015/kFprA2:2018 - 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)
• Effective Date: 01-Sep-2019

Standards Content (Sample)

SLOVENSKI STANDARD
01-september-2019
Nadomešča:
SIST EN 14476:2013+A1:2015
Kemična razkužila in antiseptiki - Kvantitativni suspenzijski preskus za
vrednotenje virucidnega delovanja v humani medicini - Preskusna metoda in
zahteve (faza 2, stopnja 1) (vključno z dopolnilom A2)
Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation
of virucidal activity in the medical area - Test method and requirements (Phase 2/Step 1)
Chemische Desinfektionsmittel und Antiseptika - Quantitativer Suspensionsversuch zur
Bestimmung der viruziden Wirkung im humanmedizinischen Bereich - Prüfverfahren und
Anforderungen (Phase 2, Stufe 1)
Antiseptiques et désinfectants chimiques - Essai quantitatif de suspension pour
l'évaluation de l'activité virucide dans le domaine médical - Méthode d'essai et
prescriptions (Phase 2/Étape 1)
Ta slovenski standard je istoveten z: EN 14476:2013+A2:2019
ICS:
11.080.20 Dezinfektanti in antiseptiki Disinfectants and antiseptics
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN 14476:2013+A2
EUROPEAN STANDARD
NORME EUROPÉENNE
July 2019
EUROPÄISCHE NORM
ICS 11.080.20
English Version
Chemical disinfectants and antiseptics - Quantitative
suspension test for the evaluation of virucidal activity in
the medical area - Test method and requirements (Phase
2/Step 1)
Antiseptiques et désinfectants chimiques - Essai Chemische Desinfektionsmittel und Antiseptika -
quantitatif de suspension pour l¿évaluation de Quantitativer Suspensionsversuch zur Bestimmung der
l¿activité virucide dans le domaine médical - Méthode viruziden Wirkung im humanmedizinischen Bereich -
d¿essai et prescriptions (Phase 2/Étape 1) Prüfverfahren und Anforderungen (Phase 2, Stufe 1)
This European Standard was approved by CEN on 27 July 2015 and includes Amendment 2 approved by CEN on 9 April 2019.

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, Turkey and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2019 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 14476:2013+A2:2019 E
worldwide for CEN national Members.

Contents Page
European foreword . 4
Introduction . 6
1 Scope . 7
2 Normative references . 7
3 Terms and definitions . 7
4 Requirements . 8
5 Test methods . 10
5.1 Principle . 10
5.2 Materials and reagents, including cell cultures . 10
5.2.1 Test organisms . 10
5.2.2 Culture media, reagents and cell cultures . 11
5.3 Apparatus and glassware . 14
5.4 Preparation of test organism suspensions and product test solutions . 15
5.4.1 Test organisms suspensions (test virus suspension) . 15
5.4.2 Product test solutions . 16
5.5 Procedure for assessing the virucidal activity of the product . 16
5.5.1 General . 16
5.5.2 Test procedure . 17
5.5.3 Modified method for ready-to-use products . 18
5.5.4 Cytotoxicity caused by product test solutions . 19
5.5.5 Control of efficiency of suppression of product’s activity . 20
5.5.6 Reference test for virus inactivation . 20
5.5.7 Titration of the virus control . 21
5.5.8 Titration of test samples . 21
5.6 Experimental data and calculation . 21
5.6.1 Protocol of results . 21
5.6.2 Calculation of infectivity titer (TCID50 or PFU) . 21
5.7 Verification of the methodology . 21
5.8 Expression of results . 22
5.8.1 General . 22
5.8.2 Calculation of the virucidal activity of products . 22
5.9 Test report . 22
Annex A (informative) Examples of viruses sorted according to their presence in the human
body in case of virus infection . 25
Annex B (informative) Detoxification of test mixtures by molecular sieving . 27
TM
B.1 Molecular sieving with Sephadex LH 20 . 27
B.1.1 Principle . 27
B.1.2 Sephadex suspension . 27
B.1.3 Procedure. 27
TM
B.2 Molecular sieving using MicroSpin S 400 HR . 29
Annex C (informative) Calculation of the viral infectivity titre . 32
C.1 Quantal tests — Example of TCID50 determination by the Spearman-Kärber method . 32
C.2 Plaque test . 33
C.3 Biometrical evaluation of experimental approaches and assessment of the
disinfecting effect on the virus (reduction [R]): . 33
C.3.1 General . 33
C.3.2 Calculating the virus titre with 95 % confidence interval . 34
C.3.3 Calculating the reduction and its 95 % confidence interval . 34
C.3.4 Calculating the average reduction (R ) and its 95 % confidence interval. . 35
(mi)
C.3.5 Practical example . 36
Annex D (informative) Presentation of test results of one active concentration . 38
Annex E (informative) Quantitative determination of formaldehyde concentrations . 41
Annex ZA (informative) !Relationship between this European Standard and the Essential
Requirements of EU Directive 93/42/EEC . 42
Bibliography . 43

European foreword
This document (EN 14476:2013+A2:2019) 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 #January 2020$ and conflicting national standards
shall be withdrawn at the latest by #January 2020$.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent
rights.
This document supersedes #EN 14476:2013+A1:2015$.
This document includes Amendment 1 approved by CEN on 2015-07-27 and Amendment 2 approved
by CEN on 2019-04-09.
The start and finish of text introduced or altered by amendment 1 is indicated in the text by tags !".
The start and finish of text introduced or altered by amendment 2 is indicated in the text by tags #$.
!This document has been prepared under a mandate given to CEN by the European Commission and
the European Free Trade Association, and supports essential requirements of EU Directive(s).
For relationship with EU Directive(s), see informative Annex ZA, which is an integral part of this
document."
The document was revised to adapt it to the latest state of science, to correct errors and ambiguities, to
harmonise the structure and wording with other existing tests of CEN/TC 216 or in preparation and to
improve the readability of the standard and thereby make it more understandable. The following list is
a list of significant technical changes since the last edition:
• The scope was expanded for the following fields of application within the medical area, i.e. products
for textile disinfection.
• “Obligatory test conditions” were replaced by “minimum test conditions” (test temperatures and
contact times can be chosen within limits) that have to be performed to pass the test.
• An additional modified method is described to test ready-to-use products in a higher concentration
than 80 %, i.e. 9 7%;
!
• For the hygienic handrub and handwash method a test for virucidal activity against enveloped
viruses with Vacciniavirus was added.
• The relationship between this European Standard and the MDD was added (Foreword and
Annex ZA).
• The value of v in C.1 was corrected (0,001 instead of 0,0001)."
n
#• For the surface disinfection a test for virucidal activity against enveloped viruses with vaccinia
virus was added and a test for limited spectrum virucidal activity with adenovirus and murine
norovirus was added;
• The spelling of Vaccinavirus is corrected to vaccinia virus (Table 1);
• The limited spectrum virucidal activity will cover norovirus, rotavirus and adenovirus;
• The vaccinia virus strain Elstree was added as alternative strain [5.2.1c)1)], [5.5.1.1.e)];
• For dirty conditions (5.2.2.8.3) the resuspension shall be done in PBS and not in water (editorial
change reflecting the actual practice);
• the dilution in ice-cold medium for the control of efficiency of suppression of products activity
(5.5.5.1) was clarified;
• addition of the large-volume-platting method (5.5.4.3, B.3)$
#The changes mentioned above have no impact on the test results obtained with reference to the
previous version. Those results are still valid.$
Other methods to evaluate the efficacy of chemical disinfectants and antiseptics for different
applications in the medical area are in preparation.
According to the CEN-CENELEC Internal Regulations, the national standards organizations of the
following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,
Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland,
Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of
Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey 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 utilisation concentration of the chemical disinfectant or
antiseptic found by this test corresponds to the chosen experimental conditions.
1 Scope
This European Standard specifies a test method and the minimum requirements for virucidal activity of
chemical disinfectant and antiseptic products that form a homogeneous physically stable preparation
when diluted with hard water – or in the case of ready-to-use products, i. e, products that are not
diluted when applied,– with water. Products can only be tested at a concentration of 80 % (97 %, with a
modified method for special cases) as some dilution is always produced by adding the test organisms
and interfering substance.
This European Standard applies to products that are used in the medical area in the fields of hygienic
handrub, hygienic handwash, instrument disinfection by immersion, surface disinfection by wiping,
spraying, flooding or other means and textile disinfection.
This European Standard applies to areas and situations where disinfection is medically indicated. Such
indications occur in patient care, for example:
— in hospitals, in community medical facilities, and in dental institutions;
— in clinics of schools, of kindergartens, and of nursing homes;
and may occur in the workplace and in the home. It may also include services such as laundries and
kitchens supplying products directly for the patients.
NOTE 1 The method described is intended to determine the activity of commercial formulations or active
substances under the conditions in which they are used.
NOTE 2 This method corresponds to a phase 2, step 1 test.
NOTE 3 EN 14885 specifies in detail the relationship of the various tests to one another and to “use
recommendations”.
2 Normative references
#The following documents 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 terminological databases for use in standardization at the following addresses:
• IEC Electropedia: available at http://www.electropedia.org/
• ISO Online browsing platform: available at http://www.iso.org/obp$
3.1
cytotoxicity
morphological alteration of cells and/or their destruction or their reduced sensitivity to virus
multiplication caused by the product
3.2
plaque forming units
PFU
number of infectious virus particles per unit volume (ml)
3.3
reference test for virus inactivation
test with a defined product (e.g. formaldehyde) in parallel with a product under test for the internal
control of the test
3.4
TCID
50 % infecting dose of a virus suspension or that dilution of the virus suspension that induce a CPE (3.5)
in 50 % of cell culture units
3.5
viral cytopathic effect
CPE
morphological alteration of cells and/or their destruction as a consequence of virus multiplication
3.6
viral plaque
area of lysis formed in a cell monolayer under semisolid medium due to infection by and multiplication
of a single infectious virus particle
3.7
virus titre
amount of infectious virus per unit volume present in a cell culture lysate or in a solution
4 Requirements
The product shall demonstrate at least a decimal log (lg) reduction of 4 in virus titre when tested in
accordance with Table 1 and Clause 5.
Table 1 — Minimum and additional test conditions
Test Conditions Hygienic handrub Instrument Surface disinfection Textile
and handwash disinfection
disinfection
Minimum #Virucidal activity #Virucidal activity #Virucidal activity Parvovirus
spectrum of
Poliovirus Poliovirus Poliovirus
test organisms
Adenovirus Adenovirus Adenovirus

Murine Norovirus Murine Norovirus Murine Norovirus
Limited spectrum when temperature is #Limited
a
virucidal activity 40 °C or higher: only
spectrum virucidal
Adenovirus Parvovirus
a
activity adenovirus
Murine Norovirus
murine norovirus
!Virucidal activity
Virucidal activity
against enveloped
against enveloped
b
viruses
b
viruses
Vacciniavirus"
vaccinia virus$
additional Any relevant test organism
Test
according to the manufacturer’s recommendation, but at / between
temperature
20 °C 20 °C and 70 °C 4 °C and 30 °C 30 °C and 70 °C
Contact time according to the manufacturer’s recommendation
but between but no longer than
30 s and 120 s 60 min 5 min or 20 min
c
60 min
Interfering substance
clean conditions 0,3 g/l bovine albumin 0,3 g/l bovine albumin 0,3 g/l bovine albumin
solution (hygienic solution solution

d
handrub)
and/or and/or
dirty conditions 3,0 g/l bovine albumin  3,0 g/l bovine
solution plus 3,0 ml/l albumin solution plus
3,0 g/l bovine albumin 3,0 g/l bovine albumin
erythrocytes (hygienic 3,0 ml/l erythrocytes
solution plus 3,0 ml/l solution plus 3,0 ml/l
e
handwash)
erythrocytes erythrocytes
d, e
Additional clean or dirty ; any relevant substance any relevant substance any relevant
f
conditions any relevant substance substance
a
#The test for “limited spectrum virucidal activity” will cover all enveloped viruses (Annex A) and norovirus,
rotavirus and adenovirus.$
b
!The test for #"virucidal activity against enveloped viruses"$ will cover all enveloped viruses only (Annex
A)."
c
The contact times for surface disinfectants stated in this table are chosen on the basis of the practical conditions of
the product. The recommended contact time for the use of the product is within the responsibility of the
manufacturer. Products intended to disinfect surfaces that are likely to come into contact with the patient and / or
the medical staff and surfaces, which are frequently touched by different people, leading to the transmission of
microorganisms to the patient, shall be tested with a contact time of maximum 5 min. The same applies where the
contact time of the product shall be limited for practical reasons. Products for other surfaces than stated above may
be tested with a contact time of maximum 60 min.
d
Hygienic handrub shall be tested as a minimum under clean conditions.
e
Hygienic handwash shall be tested as a minimum under dirty conditions.
f
For the additional conditions, the concentration defined as a result can be lower than the one obtained under the
minimum test conditions.
5 Test methods
5.1 Principle
5.1.1 A sample of the product as delivered and/or diluted with hard water (or water for ready to use
products) is added to a test suspension of viruses in a solution of an interfering substance. The mixture
is maintained at one of the temperatures and the contact times specified in Clause 4 and 5.5.1.1. At the
end of this contact time, an aliquot is taken; the virucidal action in this portion is immediately
suppressed by a validated method (dilution of the sample in ice-cold cell maintenance medium). The
dilutions are transferred into cell culture units (petri dishes, tubes or wells of microtitre plates) either
using monolayer or cell suspension. Infectivity tests are done either by plaque test or quantal tests.
After incubation, the titres of infectivity are calculated according to Spearman and Kärber (quantal
tests, C.1) or by plaque counting (plaque test, C.2) and evaluated. Reduction of virus infectivity is
calculated from differences of lg virus titres before (virus control) and after treatment with the product.
NOTE Handwash products are always prediluted with hard water (5.2.2.7). The resulting solution is regarded
as a ready-to-use product (5.4.2).
5.1.2 The test is performed using the test organisms as specified in Clause 4, Table 1.
5.1.3 Other contact times and temperatures within the limits specified in Clause 4, Table 1 may be
used. Additional interfering substances and test organisms may be used.
5.2 Materials and reagents, including cell cultures
5.2.1 Test organisms
The virucidal activity shall be evaluated using the following strains as test organisms selected according
1)
to Clause 4, Table 1
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)
a) Non-enveloped RNA virus
1) Poliovirus type 1, LSc 2ab (Picornavirus)
2) Murine norovirus, strain S99 Berlin
b) Non-enveloped DNA virus
1) Adenovirus type 5, strain Adenoid 75, ATCC VR-5*
2) Murine Parvovirus, minute virus of mice, strain Crawford, ATCC VR-1346
!
c) Enveloped DNA virus
1) #Modified vaccinia virus Ankara (MVA), ATCC VR-1508, or vaccinia virus strain Elstree, ATCC
VR-1549.$"
The required incubation temperature for these test organisms is 36 °C ± 1 °C or 37 °C ± 1 °C (5.3.1.3).
The same temperature (either 36 °C or 37 °C) shall be used for all incubations performed during a test
and its control and validation.
If additional test organisms are used, they shall be kept and used under optimum growth conditions
(temperature, time, atmosphere, media) noted in the test report. If these additional test organisms are
not classified at a reference centre, their identification characteristics shall be stated. In addition, they
shall be held by the testing laboratory or national culture collection under a reference for five years.
5.2.2 Culture media, reagents and cell cultures
5.2.2.1 General
All weights of chemical substances given in this European Standard refer to the anhydrous salts.
Hydrated forms may be used as an alternative, but the weights required shall be adjusted to allow for
consequent molecular weight differences.
The reagents shall be of analytical grade and/or appropriate for microbiological purposes. They shall be
free from substances that are toxic or inhibitory to the test organisms.
To improve reproducibility, it is recommended that commercially available – if appropriate the material
is used for the preparation of culture media. The manufacturer's instructions relating to the preparation
of these products should be rigorously followed.
For each culture medium and reagent, a time limitation for use should be fixed.
All specified pH values are measured at 20 °C ± 1 °C.
5.2.2.2 Water
The water shall be freshly glass-distilled water and not demineralised water. If distilled water of
adequate quality is not available, water for injections (see bibliographic reference [1]) may be used.

2) Virus strains may be obtained from a national or international culture collection. Regarding Poliovirus only virus material that passed the
requirements for the production of oral polio vaccine of the World Health Organisation (WHO) shall be used (Other stocks derived from LSc-
2ab cannot be used any longer). LSc-2ab can be obtained from NIBSC (www.nibsc.ac.uk: contact Dr. Javier Martin) or from Eurovir Hygiene
Institut (www.eurovir.de: contact Dr. Jursch).
Murine Norovirus may be obtained from Friedrich-Loeffler-Insitut !Bundesforschungsinstitut" für Tiergesundheit, Hauptsitz Insel Riems
Südufer 10, 17493, Greifswald-Insel Riems; phone: [phone], fax: [phone]. http://www.fli.bund.de.
Sterilise in the autoclave [5.3.1.1 a)]. Sterilisation is not necessary if the water is used e.g. for
preparation of culture media and subsequently sterilised.
See 5.2.2.7 for the procedure to prepare hard water.
5.2.2.3 Phosphate buffered saline (PBS)
Sodium chloride (NaCl) 8,00 g
Potassium chloride (KCl) 0,20 g
Disodium hydrogen phosphate, 12-hydrate (Na2HPO4 x 2,89 g
12H2O)
Potassium phosphate, monobasic (KH2PO4) 0,20 g
Water (5.2.2.2) to 1 000,0 ml
5.2.2.4 Neutral Red (1:1000 solution)
Prepare neutral red (Sigma N7005) stock solution at 0,1 mg/ml in water (5.2.2.2). Filter through a
0,40 µm pore size filter and store 4 °C in the dark.
5.2.2.5 Foetal calf serum (FCS)
FCS has to be certified free of viruses and mycoplasma. Extraneous viruses and mycoplasma may
interfere with cell and virus growth resulting in false results.
For RAW 264.7 cells, special FCS has to be used due to the cells’ high sensitivity to endotoxins.
5.2.2.6 Trichloroacetic acid (10 % solution) (TCA)
Dissolve 10 g of TCA crystals in 80 ml of water (5.2.2.2), then adjust the volume to 100 ml with water.
Stir to complete solution.
5.2.2.7 Hard water for dilution of products
For the preparation of 1 l of hard water, the procedure is as follows:
— prepare solution A: dissolve 19,84 g magnesium chloride (MgCl ) and 46,24 g calcium chloride
(CaCl ) in water (5.2.2.2) and dilute to 1 000 ml. Sterilise by membrane filtration (5.3.1.7) or in the
autoclave [5.3.1.1 a)]. Autoclaving – if used - may cause a loss of liquid. In this case make up to
1 000 ml with water (5.2.2.2) under aseptic conditions. Store the solution in the refrigerator
(5.3.1.8) for no longer than one month;
— prepare solution B: dissolve 35,02 g sodium bicarbonate (NaHCO ) in water (5.2.2.2) and dilute to
1000 ml. Sterilise by membrane filtration (5.3.1.7). Store the solution in the refrigerator (5.3.1.8)
for no longer than one week;
— place 600 ml to 700 ml of water (5.2.2.2) in a 1 000 ml volumetric flask (5.3.1.12) and add 6,0 ml
(5.3.1.9) of solution A, then 8,0 ml of solution B. Mix and dilute to 1 000 ml with water (5.2.2.2). The
pH (5.3.1.4) of the hard water shall be 7,0 ± 0,2. (5.3.1.4). If necessary, adjust the pH by using a
solution of approximately 40 g/l (about 1 mol/l) of sodium hydroxide (NaOH) or approximately
36,5 g/l (about 1 mol/l) of hydrochloric acid (HCl).
The hard water shall be freshly prepared under aseptic conditions and used within 12 h.
NOTE When preparing the product test solutions (5.4.2), the addition of the product to the hard water
produces different final water hardness in each test tube. In any case, the final hardness in the test tube expressed
as calcium carbonate (CaCO ) is lower than 375 mg/l.
5.2.2.8 Interfering substance
5.2.2.8.1 General
The interfering substance shall be chosen according to the conditions of use laid down for the product.
The interfering substance shall be sterile and prepared at 10 times its final concentration in the test (50
times in the case of the modified method, 5.2.2.8.4).
The ionic composition (e.g. pH, calcium and/or magnesium hardness) and chemical composition (e.g.
mineral substances, protein, carbohydrates, lipids and detergents) shall be defined.
“Diluent” is generally used in the other European Standards in the medical area to prepare the
interfering substance. Since there is no experience in virucidal testing with diluent, water (5.2.2.2) is
used instead.
NOTE The term “interfering substance” is used even if it contains more than one substance.
5.2.2.8.2 Clean conditions (bovine albumin solution – low concentration)
Dissolve 0,30 g of bovine albumin fraction V (suitable for microbiological purposes) in 100 ml of water
(5.2.2.2).
Sterilise by membrane filtration (5.3.1.7), keep in a refrigerator (5.3.1.8) and use within one month.
The final concentration of the bovine albumin in the test procedure (5.5) shall be 0,3 g/l ;
5.2.2.8.3 Dirty conditions (Mixture of bovine albumin solution – high concentration with sheep
erythrocytes)
Dissolve 3,00 g of bovine albumin fraction V (suitable for microbiological purposes) in 97 ml of water
(5.2.2.2).
Sterilise by membrane filtration (5.3.1.7).
Prepare at least 8,0 ml fresh defibrinated sheep blood (5.2.2.9). Centrifuge the erythrocytes at 800 g
N
for 10 min (5.3.1.13). After discarding the supernatant, resuspend erythrocytes in #PBS (5.2.2.3)$.
Repeat this procedure at least 3 times, until the supernatant is colourless.
Resuspend 3 ml of the packed sheep erythrocytes in the 97 ml of sterilised bovine albumin solution (see
above). To avoid later contamination this mixture should be split in portions probably needed per day.
Keep the mixture in separate containers for a maximum of 7 d in a refrigerator (5.3.1.8).
The final concentration of bovine albumin and sheep erythrocytes in the test procedure (5.5) shall be
3 g/l and 3 ml/l respectively.
5.2.2.8.4 Clean and dirty conditions for the modified method for ready-to-use products (5.5.4)
Follow the procedures for preparation according to 5.2.2.8.2 and 5.2.2.8.3, but prepare the interfering
substance in fivefold higher concentrations, for the dirty conditions maximum 50 ml to avoid problems
with the filtration.
a) Clean conditions (5.2.2.8.2) – dissolve 1,50 g bovine albumin (instead of 0,3 g) in 100 ml of water
(5.2.2.2);
b) dirty conditions (5.2.2.8.3) – dissolve 7,5 g bovine albumin (instead of 1,5 g) in 42,5 ml (instead of
48,5 ml) of water (5.2.2.2). Prepare at least 20 ml (instead of 4,0 ml) sheep blood. Resuspend 7,5 ml
(instead of 1,5 ml) of the packed sheep erythrocytes in 42,5 ml of sterilised bovine albumin solution
to obtain 50 ml.
5.2.2.9 Defibrinated sheep blood
The defibrinated sheep blood shall be sterile (aseptic blood-letting and preparation). The defibrinated
sheep blood can be pooled from more than one sheep and can be acquired from a commercial supplier.
5.2.2.10 Medium for cell cultures
Eagle’s minimal essential medium (MEM) or equivalent, supplemented with FCS (5.2.2.5), antibiotics,
and other growth factors as needed shall be used.
a) A growth medium for cell multiplication is supplemented with 10 % FCS. Add 10 parts of FCS
(5.2.2.5) to 90 parts of MEM.
b) A maintenance medium to maintain the cell culture metabolism without stimulation of cell
proliferation is supplemented with 2 % FCS. Add 2 parts of FCS (5.2.2.5) to 98 parts of MEM.
Other media may be used if appropriate for certain cell lines.
See also bibliographic reference [2]. See EN 12353 for a detailed description.
5.2.2.11 Cell cultures
Cell monolayers shall be >90 % confluent before inoculation. Cell lines are selected in accordance with
their sensitivity to the test organisms (5.2.1). Cells for virus titration, if used as suspensions in quantal
tests, shall be added to the dilutions of the test mixture (5.5.2) in such a density as to enable the
formation of a monolayer in at least two days in the cell control. Cell cultures can be used as cell
monolayers or in suspensions for quantal tests. For details of cell lines see 5.5.1.1 e).
5.3 Apparatus and glassware
Sterilise all glassware and parts of the apparatus that will come into contact with the culture media and
reagents or the sample, except those which are supplied sterile, by one of the following methods:
a) by moist heat, in the autoclave [5.3.1.1 a)];
b) by dry heat, in the hot air oven [5.3.1.1 b)].
3)
5.3.1 Usual microbiological laboratory equipment and, in particular, the following:
5.3.1.1 Apparatus for sterilisation (moist and dry heat):
+3
a) For moist heat sterilisation, an autoclave capable of being maintained at (121 ) °C for a minimum
holding time of 15 min;
+5
b) for dry heat sterilisation, a hot air oven capable of being maintained at ( 180 ) °C for a minimum
+5 +5
holding time of 30 min, at (170 ) °C for a minimum holding time of 1 h or at (160 ) °C for a
0 0
minimum holding time of 2 h.
5.3.1.2 Water baths, capable of being controlled at 20 °C ± 1 °C, and at additional test temperatures
± 1 °C (5.5.1).
5.3.1.3 C0 Incubator (95 % air, 5 % C0 ), capable of being controlled either at 36 °C ± 1 °C or
2 2
37 °C ± 1 °C (5.2.1).
5.3.1.4 pH-meter, having an inaccuracy of calibration of no more than ± 0,1 pH units at 20 °C ± 1 °C.

3)
Disposable sterile equipment is an acceptable alternative to reusable glassware.
5.3.1.5 Stopwatch.
5.3.1.6 Shakers.
® 4)
a) Electromechanical agitator, e.g. Vortex mixer ;
b) Mechanical shaker.
5.3.1.7 Membrane filtration apparatus, constructed of a material compatible with the substances
to be filtered, with a filter holder of at least 50 ml volume, and suitable for use of filters of diameter
47 mm to 50 mm and 0,22 µm pore size for sterilisation of hard water (5.2.2.7) and bovine albumin
(5.2.2.8.2, 5.2.2.8.3 and 5.2.2.8.4).
The vacuum source used shall give an even filtration flow rate. In order to obtain a uniform distribution
of the micro-organisms over the membrane and to prevent overlong filtration, the device shall be set so
as to obtain the filtration of 100 ml in 20 s to 40 s. The filtration of the interfering substance (5.2.2.8) for
the modified method (5.5.3) may take a longer time.
5.3.1.8 Refrigerator, capable of being controlled at 2 °C to 8 °C.
5.3.1.9 Graduated pipettes, of nominal capacities 10 ml, 1 ml, 100 µl, 10 µl or calibrated automatic
pipettes.
5.3.1.10 Ice producing machine or commercially available ice to cool the cell maintenance
medium and the reaction mixtures during the test.
5.3.1.11 Basin as ice bath with ice and water.
5.3.1.12 Volumetric flasks.
5.3.1.13 Centrifuge (400 g to 1000 g ).
N N
5.3.1.14 Microtitre plates or tubes, petri dishes and flasks for cell culture use.
5.3.1.15 Magnetic stirrer for keeping cells in suspension before seeding.
5.3.1.16 Inverted microscope for reading cell cultures microscopically.
5.3.1.17 Container: sterile test tubes, culture bottles or flasks of suitable capacity.
5.3.1.18 Biological safety cabinet, class II.
5.3.1.19 Freezer, -70 °C or less.
5.4 Preparation of test organism suspensions and product test solutions
5.4.1 Test organisms suspensions (test virus suspension)
The test organisms and their stock cultures shall be prepared and kept in accordance with EN 12353.
The stock virus suspension is multiplied in an appropriate cell line that produces high titres of
infectious viruses. The cell debris is separated by centrifugation (400 g for 15 min). This preparation
N
is called "test virus suspension".

4) ®
Vortex is an example of a suitable product available commercially. This information is given for the convenience of users
of this European Standard and does not constitute an endorsement by CEN of this product.
It is suggested that the minimum titre of the virus suspension - determined by a quantal test [5.5.2 a)]
or by plaque test [5.5.2 b)] - is at least 10 TCID /ml. In any case, it shall be sufficiently high to at least
enable a titre reduction of 4 lg to verify the method.
In exceptional cases the test suspension may be concentrated by appropriate methods (e.g.
ultracentrifugation).
The test suspension is kept in small volumes below -70 °C or preferably at -196 °C under nitrogen. Due
to safety reasons, and – in some cases – to limit the possibility of genetic mutations, only 10 passages
from the original seed (e.g. virus from culture collection) are allowed.
The test suspension is used undiluted for the test procedure (5.5.2 or 5.5.3).
5.4.2 Product test solutions
The concentration of a product test solution shall be 1,25 times the desired test concentration (= real
test concentration) because it is diluted to 80 % during the test (5.5.2).
Product test solutions shall be prepared in hard water (5.2.2.7) at minimum three different
concentrations to include one concentration in the active range and one concentration in the non-active
range (5.7). The product as received may be used as one of the product test solutions, in this case the
highest tested concentration is 80 %. Ready-to-use products may be tested at 97 % [modified method
(5.5.3)]. In this case, the “real test concentration” is 97 %.
Dilutions of ready-to-use products shall be prepared in water (5.2.2.2) instead of hard water. Handwash
products are always prediluted with hard water (5.2.2.7) to achieve a 62,5 % solution. This solution
simulates the addition of tap water in practice (1:1). Such a product is nevertheless regarded as a
“ready-to-use product”. The modified method (5.5.3) cannot be used, since 62,5 % represents the
highest accepted concentration (50 %), multiplied by 1,25.
For solid products, dissolve the product as received by weighing at least 1,0 g ± 10 mg of the product in
a volumetric flask and filling up with hard water (5.2.2.7). Subsequent dilutions (= lower
concentrations) shall be prepared in volumetric flasks (5.3.1.12) on a volume/volume basis in hard
water (5.2.2.7).
For liquid products, dilutions of the product shall be prepared with hard water in volumetric flasks
(5.3.1.12) on a volume/volume basis.
The product test solutions shall be prepared freshly and used in the test within 2 h. They shall give a
physically homogenous preparation, stable during the whole procedure. If during the procedure a
visible inhomogeneity appears due to the formation of a precipitate or flocculate (for example, through
the addition of the interfering substance), it shall be recorded in the test report.
The concentration of the product stated in the test report shall be the desired test concentration.
Record the test concentration in terms of mass per volume or volume per volume and details of the
product sample as received.
5.5 Procedure for assessing the virucidal activity of the product
5.5.1 General
5.5.1.1 Experimental conditions
The experimental conditions may be selected according to the practical use considered for the product
(Clause 4):
a) temperature θ (in °C): The temperatures to be tested are specified in Clause 4, Table 1. The allowed
deviation for each chosen temperature is ± 1 °C.
b) contact time t (in min): The contact times to be tested are specified in Clause 4, Table 1. The
allowed deviation for each chosen contact time is ± 10 s, except for 1 min or less where it is ± 5 s.
c) interfering substance: The interfering substance to be tested is either 0,30 g/l bovine albumin
(5.2.2.8.2) representing clean conditions or a mixture of 3 ml/l sheep erythrocytes and 3,0 g/l
bovine albumin (5.2.2.8.3) representing dirty conditions – according to Clause 4, Table 1 and
practical applications. Additional interfering substances may be tested according to specific fields
of application.
d) test organisms: The test organisms to be tested are specified in Clause 4, Table 1 and 5.2.1.
Additional tes
...