SIST EN 1650:2019
(Main)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)
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.
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.
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
Relations
Standards Content (Sample)
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.
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SIST EN 1650:2019
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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.
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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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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.
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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.
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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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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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EN 1650:2019 (E)
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.
10
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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
1)
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:
1)
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.
11
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— prepare solution A: dissolve 19,84 g magnesium chloride (MgCl ) and 46,24 g calcium chloride
2
(CaCl ) in water (5.2.2.2) and dilute to 1 000 ml. Sterilize by membrane filtration (5.3.2.7) or in the
2
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
3
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.
3
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)].
12
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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)]
2)
5.3.2 Usual microbiological laboratory equipment and in particular, the following:
5.3.2.1 Apparatus for steriliz
...
SLOVENSKI STANDARD
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01-junij-2018
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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); Deutsche und
Englische Fassung prEN 1650:2016
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Ta slovenski standard je istoveten z: prEN 1650
ICS:
71.100.35 Kemikalije za dezinfekcijo v Chemicals for industrial and
industriji in doma domestic disinfection
purposes
oSIST prEN 1650:2018 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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oSIST prEN 1650:2018
DRAFT
EUROPEAN STANDARD
prEN 1650
NORME EUROPÉENNE
EUROPÄISCHE NORM
May 2018
ICS 71.100.35 Will supersede 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 Quantitativer Suspensionsversuch zur Bestimmung der
l¿activité fongicide ou levuricide des antiseptiques et fungiziden oder levuroziden Wirkung chemischer
des désinfectants chimiques utilisés dans le domaine Desinfektionsmittel und Antiseptika in den Bereichen
de l¿agro-alimentaire, dans l¿industrie, dans les Lebensmittel, Industrie, Haushalt und öffentliche
domaines domestiques et en collectivité - Méthode Einrichtungen - Prüfverfahren und Anforderungen
d¿essai et prescriptions (Phase 2, Stufe 1); Deutsche und Englische Fassung
prEN 1650:2016
This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee
CEN/TC 216.
If this draft becomes a European Standard, 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.
This draft European Standard was established by CEN 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, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and United Kingdom.
Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are
aware and to provide supporting documentation.
Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without
notice and shall not be referred to as a European Standard.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2018 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 1650:2018 E
worldwide for CEN national Members.
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prEN 1650:2018 (E)
Contents Page
European foreword . 4
Introduction . 5
1 Scope . 6
2 Normative references . 7
3 Terms and definitions . 7
4 Requirements . 8
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
5.3.1 General . 13
)
5.3.2 Usual microbiological laboratory equipment and in particular, the following: . 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 . 25
5.6.1 Explanation of terms and abbreviations . 25
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 . 29
5.7.4 Microscopic observation . 29
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 . 30
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 . 33
Annex B (informative) Examples of neutralizers of the residual antimicrobial activity of
chemical disinfectants and antiseptics and rinsing liquids . 34
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Annex C (informative) Graphical representations of dilution-neutralization method and
membrane filtration method . 36
C.1 Dilution – neutralization method . 36
C.2 Membrane filtration method . 38
Annex D (informative) Example of a typical test report . 40
Annex E (informative) Precision of the test result . 45
Bibliography . 48
3
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European foreword
This document (prEN 1650:2018) has been prepared by Technical Committee CEN/TC 216 “Chemical
disinfectants and antiseptics”, the secretariat of which is held by AFNOR.
This document is currently submitted to the CEN Enquiry.
This document will supersede EN 1650:2008+A1:2013.
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.
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Introduction
This European Standard specifies 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.
5
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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.
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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”.
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.
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 http://www.iso.org/obp
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4 Requirements
The product shall demonstrate a reduction of at least a 4 decimal log (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.
Table 1 — Experimental conditions for general purpose disinfection
Test Conditions Yeasticidal activity Fungicidal activity
Test organism Candida albicans
(see 5.2.1)
Candida albicans
Aspergillus brasiliensis
obligatory (ex A. niger)
Example of
Saccharomyces cerevisiae (for breweries)
any relevant test
additional test
organism
Saccharomyces cerevisiae var. diastaticus (for breweries)
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 and between 5 andto to 60 min
in a range from 1 min
Contact time
(from 1 min to 5 min at intervals of 1 min and from 5 min
to 60 min only with
to 60 min at intervals of 5 min) at only with 5 min intervals
5 min 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
Log reduction
from a water
≥ 4Log ≥ 4Log
control (decimal
log)
The recommended contact time for the use of the product is within the responsibility of the manufacturer.
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Table 2 — Experimental 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
(for hygienic
handrubs)
Dirty conditions
3,0 g/l Bovine Albumin
(for hygienic
handwashes)
Log reduction
≥ 4Lg for handrubs
from a water
control (decimal
2Lg for handwashes
log)
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
1)
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 DSM 1333
— 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, a limitation for use is to be fixed.
1)
The ATCC numbers are the collection numbers of strains supplied by the American Type Culture Collection
(ATCC). This information is given for the convenience of users of this standard and does not constitute an
endorsement by CEN of the product named.
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5.2.2.2 Water
The water shall be freshly glass-distilled water and not demineralized water.
Sterilize in the autoclave [5.3.2.1a)].
NOTE 1 Sterilization is not necessary if the water is used e.g. for preparation of culture media and subsequently
sterilized.
NOTE 2 If distilled water of adequate quality is not available, water for injections (see bibliographic reference
[1]
) can be used.
NOTE 3 See 5.2.2.7 for the procedure to prepare hard water.
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
2)
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 [5.3.2.1a)]. 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.
2)
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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oSIST prEN 1650:2018
prEN 1650:2018 (E)
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:
— prepare solution A: dissolve 19,84 g magnesium chloride (MgCl ) and 46,24 g calcium chloride
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(CaCl ) in water (5.2.2.2) and dilute to 1 000 ml. Sterilize by membrane filtration (5.3.2.7) or in
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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
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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.
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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.
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oSIST prEN 1650:2018
prEN 1650:2018 (E)
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 100 g m
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