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
Published
Publication Date
16-Jul-2019
Current Stage
6060 - Definitive text made available (DAV) - Publishing
Due Date
17-Jul-2019
Completion Date
17-Jul-2019

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SLOVENSKI STANDARD
SIST EN 14476:2013+A2:2019
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
SIST EN 14476:2013+A2:2019 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

---------------------- Page: 1 ----------------------
SIST EN 14476:2013+A2:2019
---------------------- Page: 2 ----------------------
SIST EN 14476:2013+A2:2019
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.
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SIST EN 14476:2013+A2:2019
EN 14476:2013+A2:2019 (E)
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

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

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

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SIST EN 14476:2013+A2:2019
EN 14476:2013+A2:2019 (E)

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

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SIST EN 14476:2013+A2:2019
EN 14476:2013+A2:2019 (E)
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)."
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SIST EN 14476:2013+A2:2019
EN 14476:2013+A2:2019 (E)

#• 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.
---------------------- Page: 7 ----------------------
SIST EN 14476:2013+A2:2019
EN 14476:2013+A2:2019 (E)
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.
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SIST EN 14476:2013+A2:2019
EN 14476:2013+A2:2019 (E)
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
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SIST EN 14476:2013+A2:2019
EN 14476:2013+A2:2019 (E)
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.
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SIST EN 14476:2013+A2:2019
EN 14476:2013+A2:2019 (E)
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
virucidal activity 40 °C or higher: only
spectrum virucidal
Adenovirus Parvovirus
activity adenovirus
Murine Norovirus
murine norovirus
!Virucidal activity
Virucidal activity
against enveloped
against enveloped
viruses
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
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
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
handwash)
erythrocytes erythrocytes
d, e

Additional clean or dirty ; any relevant substance any relevant substance any relevant

conditions any relevant substance substance
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SIST EN 14476:2013+A2:2019
EN 14476:2013+A2:2019 (E)

#The test for “limited spectrum virucidal activity” will cover all enveloped viruses (Annex A) and norovirus,

rotavirus and adenovirus.$

!The test for #"virucidal activity against enveloped viruses"$ will cover all enveloped viruses only (Annex

A)."

The contact times for surface disinfectants stated in this table are chosen on the basis of the practical conditions of

the product. The recommended contact time for the use of the product is within the responsibility of the

manufacturer. Products intended to disinfect surfaces that are likely to come into contact with the patient and / or

the medical staff and surfaces, which are frequently touched by different people, leading to the transmission of

microorganisms to the patient, shall be tested with a contact time of maximum 5 min. The same applies where the

contact time of the product shall be limited for practical reasons. Products for other surfaces than stated above may

be tested with a contact time of maximum 60 min.
Hygienic handrub shall be tested as a minimum under clean conditions.
Hygienic handwash shall be tested as a minimum under dirty conditions.

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

to Clause 4, Table 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.

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SIST EN 14476:2013+A2:2019
EN 14476:2013+A2:2019 (E)
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: +49 38351 7-0, fax: +49 038351 7-121. http://www.fli.bund.de.

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SIST EN 14476:2013+A2:2019
EN 14476:2013+A2:2019 (E)

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
...

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