Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of fungicidal or yeasticidal activity of chemical disinfectants and antiseptics used in food, industrial, domestic and institutional areas - Test method and requirements (phase 2, step 1)

This document specifies a test method and the minimum requirements for fungicidal or yeasticidal 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 - with water. Products can only be tested at a concentration of 80 % or less as some dilution is always produced by adding the test organisms and interfering substance.
This document applies to products that are used in food, industrial, domestic and institutional areas excluding areas and situations where disinfection is medically indicated and excluding products used on living tissues except those for hand hygiene in the above considered areas. The following areas are at least included:
a)   processing, distribution and retailing of:
1)   food of animal origin:   2)   food of vegetable origin:
-   milk and milk products;   -   beverages;
-   meat and meat products;   -   fruits, vegetables and derivatives (including sugar, distillery ...);
-   fish, seafood, and related products;   -   flour, milling and baking;
-   eggs and egg products;   -   animal feeds;
-   animal feeds;   -   etc.
-   etc.   
b)   institutional and domestic areas:
-   catering establishments;
-   public areas;
-   public transports;
-   schools;
-   nurseries;
-   shops;
-   sports rooms;
-   waste containers (bins ...);
-   hotels;
-   dwellings;
-   clinically non-sensitive areas of hospitals;
-   offices;
-   etc.
c)   other industrial areas:
-   packaging material;
-   biotechnology (yeast, proteins, enzymes, ...);
-   pharmaceutical;
-   cosmetics and toiletries;
-   textiles;
-   space industry, computer industry;
-   etc.
EN 14885 specifies in detail the relationship of the various tests to one another and to "use recommendations".

Chemische Desinfektionsmittel und Antiseptika - Quantitativer Suspensionsversuch zur Bestimmung der fungiziden oder levuroziden Wirkung chemischer Desinfektionsmittel und Antiseptika in den Bereichen Lebensmittel, Industrie, Haushalt und öffentliche Einrichtungen - Prüfverfahren und Anforderungen (Phase 2, Stufe 1)

Dieses Dokument legt ein Prüfverfahren und die Mindestanforderungen an die fungizide oder levurozide Wirkung von chemischen Desinfektionsmitteln und Antiseptika fest, die bei Verdünnung in Wasser standardisierter Härte oder – im Falle gebrauchsfertiger Produkte – in Wasser eine homogene, physikalisch stabile Zubereitung bilden. Die Produkte können nur bei einer Konzentration von höchstens 80 % geprüft werden, da durch die Zugabe der Prüforganismen und der Belastungssubstanz immer eine bestimmte Verdünnung auftritt.
Dieses Dokument gilt für Produkte, die im Lebensmittelbereich, in der Industrie, im Haushalt und in öffentlichen Einrichtungen benutzt werden; ausgenommen sind Bereiche und Situationen, in denen die Desinfektion medizinisch angezeigt ist, und Produkte, die auf lebenden Geweben benutzt werden, hier wiederum außer denen, die zur Handhygiene in den vorstehend genannten Bereichen verwendet werden. Es werden mindestens folgende Bereiche abgedeckt:
a) Verarbeitung, Vertrieb und Verkauf von:
1) Lebensmitteln tierischer Herkunft:
— Milch und Milcherzeugnisse;
— Fleisch und Fleischprodukte;
— Fisch, Meerestiere und zugehörige Erzeugnisse;
— Eier und Eierprodukte;
— Tiernahrung;
— usw.
2) Lebensmitteln pflanzlicher Herkunft:
— Getränke;
— Obst, Gemüse und zugehörige Erzeugnisse (einschließlich Zucker, Destillationsprodukte usw.);
— Mehl, gemahlene und gebackene Produkte;
— Tiernahrung;
— usw.
b) öffentliche Einrichtungen und Haushaltsbereiche:
— Verpflegungseinrichtungen;
— öffentliche Bereiche;
— öffentliche Verkehrsmittel;
— Schulen;
— Kindergärten;
— Geschäfte;
— Sportstätten;
— Abfallbehälter (Mülltonnen usw.);
— Hotels;
— Wohngebäude;
— klinisch nicht relevante Bereiche in Krankenhäusern;
— Büroräume;
— usw.
prEN 1650:2018 (D)
7
c) andere industrielle Bereiche:
— Verpackungsmaterial;
— Biotechnologie (Hefe, Proteine, Enzyme usw.);
— Pharmazeutika;
— Kosmetika und Toilettenartikeln;
— Textilien;
— Luft- und Raumfahrtindustrie, Computerindustrie;
— usw.
EN 14885 legt im Einzelnen den Zusammenhang zwischen den verschiedenen Prüfungen sowie zu den „Anwendungsempfehlungen“ fest.
ANMERKUNG 1 Das beschriebene Verfahren dient zur Bestimmung der Wirkung handelsüblicher Zubereitungen oder Wirkstoffe unter den Bedingungen, unter denen sie angewendet werden.
ANMERKUNG 2 Dieses Verfahren entspricht einer Prüfung der Phase 2, Stufe 1.

Antiseptiques et désinfectants chimiques - Essai quantitatif de suspension pour l'évaluation de l'activité fongicide ou levuricide des antiseptiques et des désinfectants chimiques utilisés dans le domaine de l'agro-alimentaire, dans l'industrie, dans les domaines domestiques et en collectivité - Méthode d'essai et prescriptions (phase 2, étape 1)

Le présent document spécifie une méthode d’essai et les exigences minimales relatives à l’activité fongicide ou levuricide des produits antiseptiques et désinfectants chimiques qui forment une préparation homogène, physiquement stable, lorsqu’ils sont dilués dans de l’eau dure ou - dans le cas de produits prêts à l’emploi - dans l’eau. Les produits ne peuvent être soumis à l’essai qu’à une concentration inférieure ou égale à 80 % car l’addition des microorganismes d’essai et de la substance interférente s’accompagne nécessairement d’une dilution.
Le présent document s’applique aux produits utilisés dans le domaine de l’agroalimentaire, dans l’industrie, dans les domaines domestiques et les collectivités, à l’exclusion des domaines et des situations où la désinfection est médicalement prescrite et à l’exclusion des produits utilisés sur les tissus vivants (excepté ceux destinés à l’hygiène des mains dans les domaines indiqués ci-dessus). Les domaines concernés sont notamment :
a)   la transformation, la distribution et le commerce de détail des :
1)   aliments d’origine animale :   2)   aliments d’origine végétale :
—   lait et produits laitiers ;   —   boissons ;
—   viande et produits carnés ;   —   fruits, légumes et leurs dérivés (y compris le sucre, les produits de distillation, etc.) ;
—   poisson, fruits de mer et leurs dérivés ;   —   farine, minoterie et boulangerie ;
—   œufs et produits dérivés ;   —   alimentation animale ;
—   alimentation animale ;   —   etc. ;
—   etc. ;   
b)   les domaines domestiques et les collectivités :
—   établissements de restauration ;
—   lieux publics ;
—   transports publics ;
—   écoles ;
—   crèches ;
—   magasins ;
—   salles de sport ;
—   conteneurs pour déchets (bennes, etc.) ;
—   hôtels ;
—   locaux d’habitation ;
—   zones cliniquement non sensibles des hôpitaux ;
—   bureaux ;
—   etc. ;
c)   les autres secteurs industriels :
—   matériaux d’emballage ;
—   biotechnologie (levure, protéines, enzymes, etc.) ;
—   industrie pharmaceutique ;
—   cosmétiques et produits d’hygiène corporelle ;
—   textiles ;
—   industrie spatiale, secteur informatique ;
—   etc.
L’EN 14885 spécifie de façon détaillée la relation entre les différents essais et les « recommandations d’utilisation ».

Kemična razkužila in antiseptiki - Kvantitativni suspenzijski preskus za vrednotenje fungicidnega delovanja ali delovanja kemičnih razkužil in antiseptikov na kvasovke v živilski in drugih industrijah, gospodinjstvu in javnih ustanovah - Preskusna metoda in zahteve (faza 2, stopnja 1)

Ta dokument določa preskusno metodo in minimalne zahteve za fungicidno delovanje ali delovanje kemičnih razkužil in antiseptikov na kvasovke, ki tvorijo homogen, fizikalno stabilen pripravek, če so razredčeni s trdo vodo oziroma, pri izdelkih, ki so pripravljeni za uporabo, z vodo. Izdelke je mogoče preskušati samo pri 80-odstotni ali nižji koncentraciji, ker dodajanje preskusnih organizmov in moteče snovi vedno povzroči razredčenje.
Ta dokument se uporablja za izdelke, ki se uporabljajo v živilski in drugih industrijah, gospodinjstvu in javnih ustanovah, izključeni pa so področja in primeri, ko obstajajo zdravstvene indikacije za dezinfekcijo ter izdelki, ki se uporabljajo na živih tkivih, razen tistih, ki se uporabljajo za higieno rok v zgoraj navedenih področjih. Naslednja področja so najmanj vključena:
a) predelava, distribucija in prodaja:
1) hrane živalskega izvora:           2) hrane rastlinskega izvora:
– mleko in mlečni izdelki;   – napitki;
– meso in mesni izdelki;   – sadje, zelenjava in derivati (vključno s sladkorjem, destilati ...);
– ribe, morski sadeži in podobno;   – moka, mletje in peka;
– jajca in jajčni izdelki;      – živalska krma;
– živalska krma;                      – itn.
– itn.   
b) javne ustanove in gospodinjstva:
– priprava in dostava hrane;
– javne ustanove;
– javni prevozi;
– šole;
– vrtci;
– trgovine;
– športni objekti;
– zbiralniki odpadkov (koši ...);
– hoteli;
– bivališča;
– klinično neobčutljivi prostori bolnišnic;
– pisarne;
– itn.
c) druga industrijska področja:
– embalaža;
– biotehnologija (kvasovke, proteini, encimi ...);
– farmacevtski izdelki;
– kozmetični in toaletni izdelki;
– tkanine;
– vesoljska in računalniška industrija;
– itn.
Standard EN 14885 podrobno določa razmerje med različnimi preskusi in priporočili za uporabo.
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.

General Information

Status
Published
Publication Date
13-Aug-2019
Current Stage
6060 - Definitive text made available (DAV) - Publishing
Due Date
14-Aug-2019
Completion Date
14-Aug-2019

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SLOVENSKI STANDARD
SIST EN 1650:2019
01-november-2019
Nadomešča:
SIST EN 1650:2008+A1:2013
Kemična razkužila in antiseptiki - Kvantitativni suspenzijski preskus za

vrednotenje fungicidnega delovanja ali delovanja kemičnih razkužil in antiseptikov

na kvasovke 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 fungicidal or yeasticidal activity of chemical disinfectants and antiseptics used in food,

industrial, domestic and institutional areas - Test method and requirements (phase 2,

step 1)

Chemische Desinfektionsmittel und Antiseptika - Quantitativer Suspensionsversuch zur

Bestimmung der fungiziden oder levuroziden 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 - Essai quantitatif de suspension pour

l'évaluation de l'activité fongicide ou levuricide des antiseptiques et des désinfectants

chimiques utilisés dans le domaine de l'agro-alimentaire, dans l'industrie, dans les

domaines domestiques et en collectivité - Méthode d'essai et prescriptions (phase 2,

étape 1)
Ta slovenski standard je istoveten z: EN 1650:2019
ICS:
71.100.35 Kemikalije za dezinfekcijo v Chemicals for industrial and
industriji in doma domestic disinfection
purposes
SIST EN 1650:2019 en,fr,de

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

---------------------- Page: 1 ----------------------
SIST EN 1650:2019
---------------------- Page: 2 ----------------------
SIST EN 1650:2019
EN 1650
EUROPEAN STANDARD
NORME EUROPÉENNE
August 2019
EUROPÄISCHE NORM
ICS 71.100.35 Supersedes EN 1650:2008+A1:2013
English Version
Chemical disinfectants and antiseptics - Quantitative
suspension test for the evaluation of fungicidal or
yeasticidal activity of chemical disinfectants and
antiseptics used in food, industrial, domestic and
institutional areas - 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

fongicide ou levuricide des antiseptiques et des fungiziden oder levuroziden Wirkung chemischer

désinfectants chimiques utilisés dans le domaine de Desinfektionsmittel und Antiseptika in den Bereichen

l'agro-alimentaire, dans l'industrie, dans les domaines Lebensmittel, Industrie, Haushalt und öffentliche

domestiques et en collectivité - Méthode d'essai et Einrichtungen - Prüfverfahren und Anforderungen

prescriptions (phase 2, étape 1) (Phase 2, Stufe 1)
This European Standard was approved by CEN on 10 June 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 1650:2019 E

worldwide for CEN national Members.
---------------------- Page: 3 ----------------------
SIST EN 1650:2019
EN 1650:2019 (E)
Contents Page

European foreword ....................................................................................................................................................... 4

Introduction .................................................................................................................................................................... 5

1 Scope .................................................................................................................................................................... 6

2 Normative references .................................................................................................................................... 7

3 Terms and definitions ................................................................................................................................... 7

4 Requirements ................................................................................................................................................... 7

5 Test method ...................................................................................................................................................... 9

5.1 Principle ............................................................................................................................................................. 9

5.2 Materials and reagents ............................................................................................................................... 10

5.2.1 Test organisms .............................................................................................................................................. 10

5.2.2 Culture media and reagents ..................................................................................................................... 10

5.3 Apparatus and glassware .......................................................................................................................... 13

5.4 Preparation of test organism suspensions and product test solutions .................................... 14

5.4.1 Test organism suspensions (test and validation suspension) ..................................................... 14

5.4.2 Product test solutions ................................................................................................................................. 18

5.5 Procedure for assessing the fungicidal or yeasticidal activity of the product ....................... 19

5.5.1 General ............................................................................................................................................................. 19

5.5.2 Dilution-neutralization method ............................................................................................................ 20

5.5.3 Membrane filtration method .................................................................................................................. 22

5.6 Experimental data and calculation ........................................................................................................ 24

5.6.1 Explanation of terms and abbreviations ............................................................................................. 24

5.6.2 Calculation ...................................................................................................................................................... 25

5.7 Verification of methodology ..................................................................................................................... 28

5.7.1 General ............................................................................................................................................................. 28

5.7.2 Control of weighted mean counts ........................................................................................................... 28

5.7.3 Basic limits ..................................................................................................................................................... 28

5.7.4 Microscopic observation ........................................................................................................................... 28

5.8 Expression of results and precision ...................................................................................................... 29

5.8.1 Reduction ........................................................................................................................................................ 29

5.8.2 Control of active and non-active product test solution (5.4.2) .................................................... 29

5.8.3 Limiting test organism and fungicidal/yeasticidal concentration ............................................. 29

5.8.4 Precision, replicates .................................................................................................................................... 29

5.9 Interpretation of results - conclusion ................................................................................................... 30

5.9.1 General ............................................................................................................................................................. 30

5.9.2 Fungicidal activity for general purposes ............................................................................................. 30

5.9.3 Yeasticidal activity for general purposes ............................................................................................ 30

5.9.4 Yeasticidal activity for hand hygiene .................................................................................................... 30

5.10 Test report ...................................................................................................................................................... 30

Annex A (informative) Referenced strains in national collections ......................................................... 32

Annex B (informative) Examples of neutralizers of the residual antimicrobial activity of

chemical disinfectants and antiseptics and rinsing liquids .......................................................... 33

Annex C (informative) Graphical representations of dilution-neutralization method and

membrane filtration method ................................................................................................................... 35

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SIST EN 1650:2019
EN 1650:2019 (E)

Annex D (informative) Example of a typical test report .............................................................................. 39

Annex E (informative) Precision of the test result ........................................................................................ 44

Bibliography ................................................................................................................................................................. 47

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SIST EN 1650:2019
EN 1650:2019 (E)
European foreword

This document (EN 1650:2019) 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 February 2020 and conflicting national standards shall

be withdrawn at the latest by February 2020.

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.

This document supersedes EN 1650:2008+A1:2013.

Data obtained by using the latest version of EN 1650:2008+A1:2013 are still valid.

The main changes in relation to EN 1650:2008+A1:2013 are:
— inclusion of hand hygiene;

— handrub and handwash test conditions and test requirements have been harmonized with

EN 13624;

— interfering substance for breweries, soft drinks, cosmetics and cleaning in place have been deleted.

A sentence to allow additional interfering substance for specific applications has been added;

— the obligatory conditions (temperature and contact time) have been deleted. The text has been

harmonized with EN 13624 keeping specified time intervals and temperature steps.

According to the CEN-CENELEC Internal Regulations, the national standards organisations 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, Turkey and the

United Kingdom.
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SIST EN 1650:2019
EN 1650:2019 (E)
Introduction

This European Standard describes a suspension test for establishing whether a chemical disinfectant or

antiseptic has or does not have a fungicidal or yeasticidal activity in the 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 substance, i.e. conditions which may influence

its action in practical situations.

The conditions are intended to cover general purposes and to allow reference between laboratories and

product types. Each utilization concentration of the chemical disinfectant or antiseptic found by this test

corresponds to defined experimental conditions. However, for some applications the recommendations

of use of a product may differ and therefore additional test conditions should be used.

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SIST EN 1650:2019
EN 1650:2019 (E)
1 Scope

This document specifies a test method and the minimum requirements for fungicidal or yeasticidal

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 - with water.

Products can only be tested at a concentration of 80 % or less as some dilution is always produced by

adding the test organisms and interfering substance.

This document applies to products that are used in food, industrial, domestic and institutional areas

excluding areas and situations where disinfection is medically indicated and excluding products used

on living tissues except those for hand hygiene in the above considered areas. The following areas are at

least included:
a) processing, distribution and retailing of:
1) food of animal origin: 2) food of vegetable origin:
— milk and milk products; — beverages;
— meat and meat products; — fruits, vegetables and derivatives
(including sugar, distillery ...);
— fish, seafood, and related products; — flour, milling and baking;
— eggs and egg products; — animal feeds;
— animal feeds; — etc.
— etc.
b) institutional and domestic areas:
— catering establishments;
— public areas;
— public transports;
— schools;
— nurseries;
— shops;
— sports rooms;
— waste containers (bins ...);
— hotels;
— dwellings;
— clinically non-sensitive areas of hospitals;
— offices;
— etc.
---------------------- Page: 8 ----------------------
SIST EN 1650:2019
EN 1650:2019 (E)
c) other industrial areas:
— packaging material;
— biotechnology (yeast, proteins, enzymes, ...);
— pharmaceutical;
— cosmetics and toiletries;
— textiles;
— space industry, computer industry;
— etc.

EN 14885 specifies in detail the relationship of the various tests to one another and to “use

recommendations”.
2 Normative references

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements of this document. For dated references, only the edition cited applies. For

undated references, the latest edition of the referenced document (including any amendments) applies.

EN 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

ISO 4793, Laboratory sintered (fritted) filters — Porosity grading, classification and designation

3 Terms and definitions

For the purposes of this document, the terms and definitions given in EN 14885 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 https://www.iso.org/obp
4 Requirements

The product shall demonstrate a reduction of at least a 4 decimal logarithm (lg) when diluted with hard

water (5.2.2.7) or - in the case of ready-to-use products - with water (5.2.2.2) and tested in accordance

with Clause 5 under simulated clean conditions (0,3 g/l bovine albumin solution - 5.2.2.8.2) or

simulated dirty conditions (3 g/l bovine albumin solution - 5.2.2.8.3) according to its practical

applications and under the other adopted test conditions as described in 5.5.1.1, Tables 1 and 2 here

below.
---------------------- Page: 9 ----------------------
SIST EN 1650:2019
EN 1650:2019 (E)
Table 1 — Test conditions for general purpose disinfection
Test Conditions Yeasticidal activity Fungicidal activity
Test organism
Candida albicans
(see 5.2.1)
Candida albicans
Aspergillus brasiliensis
obligatory
Example of
Saccharomyces cerevisiae
any relevant test
additional test
organism
Saccharomyces cerevisiae var. diastaticus
microorganisms
Test temperature
in a range from 4 °C to
in a range from 4 °C to 40 °C
40 °C
in a range from 1 min
to 60 min (from 1 min
in a range from 1 min to 60 min (from 1 min to 5 min at
Contact time
to 5 min at intervals of
intervals of 1 min and from 5 min to 60 min at intervals of
1 min and from 5 min
5 min)
to 60 min at intervals
of 5 min)
0,3 g/l Bovine Albumin
Clean conditions 0,3 g/l Bovine Albumin for C. albicans for C. albicans and A.
brasiliensis
3,0 g/l Bovine Albumin
Dirty conditions 3,0 g/l Bovine Albumin for C. albicans for C. albicans and A.
brasiliensis
additional any relevant substance any relevant substance
Lg reduction
≥ 4 lg ≥ 4 lg
(decimal lg)

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

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SIST EN 1650:2019
EN 1650:2019 (E)
Table 2 — Test conditions for hand hygiene
Test Conditions Yeasticidal activity
Test organism
(see 5.2.1)
Candida albicans
obligatory
Test temperature
20 °C
Contact time
30 s or 60 s
Clean conditions
0,3 g/l Bovine Albumin
(for hygienic
handrubs)
Dirty conditions
3,0 g/l Bovine Albumin
(for hygienic
handwashes)
≥ 4 lg for handrubs
Lg reduction
(decimal lg)
≥2 lg for handwashes

Where indicated, additional specific fungicidal or yeasticidal activity shall be determined applying other

interfering substances and test organisms (in accordance with 5.2.1, 5.2.2.8 and 5.5.1.1) in order to take

into account intended specific use conditions.
5 Test method
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 fungi (yeast cells or mould spores) in a solution of an

interfering substance. The mixture is maintained at the chosen test temperature for the adopted contact

time. At the end of this contact time, an aliquot is taken, and the fungicidal and/or the fungistatic

activity in this portion is immediately neutralized or suppressed by a validated method. The method of

choice is dilution-neutralization. If a suitable neutralizer cannot be found, membrane filtration is used.

The numbers of surviving fungi in each sample are determined and the reduction is calculated.

5.1.2 The test is performed using the vegetative cells of Candida albicans and the spores of Aspergillus

brasiliensis (fungicidal activity) or only the vegetative cells of Candida albicans (yeasticidal activity) as

test organisms.
5.1.3 Additional test organisms can be used.
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5.2 Materials and reagents
5.2.1 Test organisms

The fungicidal activity shall be evaluated using the following strains as test organisms:

— Candida albicans ATCC 10231
— Aspergillus brasiliensis ATCC 16404
The yeasticidal activity shall be evaluated using only Candida albicans.
NOTE See Annex A for strain references in some other culture collections.

The required incubation temperature for these test organisms is (30 ± 1) °C (5.3.2.3).

If required for specific applications, additional strains may be chosen from, e.g. for breweries:

— Saccharomyces cerevisiae ATCC 9763
— Saccharomyces cerevisiae var. diastaticus DSM 70487

If additional test organisms are used, they shall be incubated under optimum growth conditions

(temperature, time, atmosphere, media) noted in the test report. If the additional test organisms

selected do not correspond to the specified strains, their suitability for supplying the required inocula

shall be verified. 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 and reagents
5.2.2.1 General

All weights of chemical substances given in this 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 dehydrated material is used

for the preparation of culture media. The manufacturer's instructions relating to the preparation of

these products are to be rigorously followed.

NOTE For each culture medium and reagent, it is advised to fix a limitation for use.

5.2.2.2 Water
The water shall be freshly glass-distilled water and not demineralized water.
Sterilize in the autoclave [5.3.2.1 a)].

NOTE 1 Sterilization is not necessary if the water is used e.g. for preparation of culture media and subsequently

sterilized.

If distilled water of adequate quality is not available, water for injections can be used.

NOTE 2 See 5.2.2.7 for the procedure to prepare hard water.
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5.2.2.3 Malt extract agar (MEA)
Malt extract agar, consisting of:
Malt extract 30,0 g
Agar 15,0 g
Water (5.2.2.2) to 1 000,0 ml

The malt extract should be food grade (e.g. Cristomalt powder from Difal) or equivalent that is not

highly purified and not only based on maltose (e.g. Malt extract from OXOID) . However if there are

problems producing at least 75 % spiny spores see 5.4.1.4.2.

Sterilize in the autoclave [5.3.2.1 a)]. After sterilization, the pH of the medium shall be equivalent to

5,6 ± 0,2 when measured at (20 ± 1) °C.

NOTE In case of encountering problems with neutralization (5.5.1.2 and 5.5.1.3), it can be necessary to add

neutralizer to the MEA. Annex B gives guidance on the neutralizers that can be used.

5.2.2.4 Diluent
Tryptone sodium chloride solution, consisting of:
Tryptone, pancreatic digest of casein 1,0 g
Sodium chloride (NaCl) 8,5 g
Water (5.2.2.2) to 1 000,0 ml

Sterilize in the autoclave [5.3.2.1a)]. After sterilization, the pH of the diluent shall be equivalent to

7,0 ± 0,2 when measured at (20 ± 1) °C.
5.2.2.5 Neutralizer

The neutralizer shall be validated for the product being tested in accordance with 5.5.1.2, 5.5.1.3 and

5.5.2. It shall be sterile.

NOTE Information on neutralizers that have been found to be suitable for some categories of products is

given in Annex B.
5.2.2.6 Rinsing liquid (for membrane filtration)

The rinsing liquid shall be validated for the product being tested in accordance with 5.5.1.2, 5.5.1.3 and

5.5.3. It shall be sterile, compatible with the filter membrane and capable of filtration through the filter

membrane under the test conditions described in 5.5.3.

NOTE Information on rinsing liquids that have been found to be suitable for some categories of products is

given in Annex B.
5.2.2.7 Hard water for dilution of products
For the preparation of 1 000 ml of hard water, the procedure is as follows:

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. Equivalent products may be used if they can be shown to lead to the

same results.
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— prepare solution A: dissolve 19,84 g magnesium chloride (MgCl ) and 46,24 g calcium chloride

(CaCl ) in water (5.2.2.2) and dilute to 1 000 ml. Sterilize by membrane filtration (5.3.2.7) or in the

autoclave [5.3.2.1a)]. Autoclaving - if used - may cause a loss of liquid. In this case make up to

1 000 ml with water (5.2.2.2) under aseptic conditions. Store the solution in the refrigerator

(5.3.2.8) for no longer than one month;

— prepare solution B: dissolve 35,02 g sodium bicarbonate (NaHCO ) in water (5.2.2.2) and dilute to

1 000 ml. Sterilize by membrane filtration (5.3.2.7). Store the solution in the refrigerator (5.3.2.8)

for no longer than one week;

— place 600 ml to 700 ml of water (5.2.2.2) in a 1 000 ml volumetric flask (5.3.2.12) and add 6,0 ml

(5.3.2.9) of solution A, then 8,0 ml of solution B. Mix and dilute to 1 000 ml with water (5.2.2.2). The

pH of the hard water shall be 7,0 ± 0,2, when measured at (20 ± 1) °C (5.3.2.4). If necessary, adjust

the pH by using a solution of approximately 40 g/l (about 1 mol/l) of sodium hydroxide (NaOH) or

approximately 36,5 g/l (about 1 mol/l) of hydrochloric acid (HCl).

The hard water shall be freshly prepared under aseptic conditions and used within 12 h.

NOTE When preparing the product test solutions (5.4.2), the addition of the product to the hard water

produces a different final water hardness in each test tube. In any case, the final hardness is lower than 375 mg/l

of calcium carbonate (CaCO ) in the test tube.
5.2.2.8 Interfering substance
5.2.2.8.1 General

The interfering substance shall be chosen according to the conditions of use laid down for the product.

The interfering substance shall be sterile and prepared at 10 times its final concentration in the test.

The ionic composition (e.g. pH, calcium and/or magnesium hardness) and chemical composition (e.g.

mineral substances, protein, carbohydrates, lipids and detergents) shall be defined.

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 0,3 g of bovine albumin fraction V (suitable for microbiological purposes) in 100 ml of water

(5.2.2.2).

Sterilize by membrane filtration (5.3.2.7), keep in the refrigerator (5.3.2.8) and use within one month.

The final concentration of bovine albumin in the test procedure (5.5) is 0,3 g/l.

5.2.2.8.3 Dirty conditions (bovine albumin solution – high concentration)

Dissolve 3,0 g of bovine albumin fraction V (suitable for microbiological purposes) in 100 ml of water

(5.2.2.2).

Sterilize by membrane filtration (5.3.2.7), keep in the refrigerator (5.3.2.8) and use within one month.

The final concentration of bovine albumin in the test procedure (5.5) is 3,0 g/l.

5.2.2.8.4 Milk (dairies ...)

Skimmed milk, guaranteed free of antibiotics and additives and reconstituted at a rate of 100 g powder

per litre of water (5.2.2.2), shall be prepared as follows:

— prepare a solution of 10,0 % (v/v) in water (5.2.2.2) by adding 10 parts of reconstituted milk to 90

parts of water. Heat for 30 min at (105 ± 3) °C [or 5 min at (121 ± 3 °C)].
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The final concentration of reconstituted milk in the test procedure (5.5) is 1,0 % (v/v)of reconstituted

milk.
5.3 Apparatus and glassware
5.3.1 General

Sterilize all glassware and parts of the apparatus that will come into contact with the culture media and

reagents or the sample, except those which are supplied sterile, by one of the following methods:

a) by moist heat, in the autoclave [5.3.2.1 a)]
b) by dry heat, in the hot air oven [5.3.2.1 b)]

5.3.2 Usual microbiological laboratory equipment and in particular, the following:

5.3.2.1 Apparatus for steriliz
...

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