SIST EN 13624:2013
(Main)Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of fungicidal or yeasticidal activity in the medical area - Test method and requirements (phase 2, step 1)
Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of fungicidal or yeasticidal activity in the medical area - Test method and requirements (phase 2, step 1)
This European Standard specifies a test method and the minimum requirements for 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 (97 % with a modified method for special cases) as some dilution is always produced by adding the test organisms and interfering substance. This European Standard applies to products that are used in the medical area in the fields of hygienic handrub, hygienic handwash, surgical handrub, surgical handwash, instrument disinfection by immersion, and surface disinfection by wiping, spraying, flooding or other means. This European Standard applies to areas and situations where disinfection or antisepsis is medically indicated. Such indications occur in patient care, for example: - in hospitals, in community medical facilities and in dental institutions; - in clinics of schools, of kindergartens and of nursing homes; and may occur in the workplace and in the home. It may also include services such as laundries and kitchens supplying products directly for the patients.
Chemische Desinfektionsmittel und Antiseptika - Quantitativer Suspensionsversuch zur Bestimmung der fungiziden oder levuroziden Wirkung im humanmedizinischen Bereich - Prüfverfahren und Anforderungen (Phase 2, Stufe 1)
Diese Europäische Norm legt ein Prüfverfahren und Mindestanforderungen für die fungizide oder levurozide (Hefen abtötende) Wirkung von chemischen Desinfektionsmitteln und Antiseptika fest, die bei Verdünnung mit Wasser standardisierter Härte bzw. – im Fall von gebrauchsfertigen Produkten – mit Wasser als homogenes physikalisch stabiles Präparat vorliegen. Die Produkte können nur in einer Konzentration bis zu 80 % (97 % bei einem modifizierten Verfahren in Sonderfällen) geprüft werden, da durch Zu¬gabe der Prüforganismen und der Belastungssubstanz stets eine gewisse Verdünnung bewirkt wird.
Diese Europäische Norm gilt für Produkte, die im humanmedizinischen Bereich für hygienische Hände-desinfektion und Händewaschung, chirurgische Händedesinfektion und Händewaschung, für die Instrumentendesinfektion durch Eintauchen sowie für die Oberflächendesinfektion mittels Wischen, Sprühen, Spülen o. ä. verwendet werden.
Diese Europäische Norm gilt für Bereiche und Bedingungen, wo eine Desinfektion oder Antisepsis aus medizinischen Gründen angezeigt ist. Derartige Gründe treten bei der Patientenbetreuung z. B. in
Krankenhäusern, kommunalen medizinischen Einrichtungen und im Dentalbereich;
medizinischen Einrichtungen in Schulen, Kindergärten und Heimen;
auf und können auch am Arbeitsplatz oder im privaten Bereich gegeben sein. Eingeschlossen sein können auch Einrichtungen wie Wäschereien und Küchen, die der direkten Versorgung der Patienten dienen.
ANMERKUNG 1 Das beschriebene Verfahren dient zur Bestimmung der Wirksamkeit kommerziell erhältlicher Zubereitungen oder Wirkstoffe unter den jeweiligen Anwendungsbedingungen.
ANMERKUNG 2 Dieses Verfahren entspricht einer Prüfung der Phase 2, Stufe 1.
EN 14885 legt detailliert die Beziehungen der unterschiedlichen Prüfungen untereinander und zu „Anwen¬dungs-empfehlungen“ fest.
Désinfectants chimiques et antiseptiques - Essai quantitatif de suspension pour l'évaluation de l'activité fongicide ou levuricide en médecine - Méthode d'essai et prescriptions (phase 2, étape 1)
La présente Norme européenne spécifie une méthode d’essai et les exigences minimales relatives à l’activité fongicide et levuricide des désinfectants chimiques et des produits antiseptiques 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 de l’eau. Les produits ne peuvent être soumis à l’essai qu’à une concentration inférieure ou égale à 80 % (97 % avec une méthode modifiée dans certains cas particuliers), car l’ajout des souches d’essai et de la substance interférente entraîne toujours une dilution.
La présente Norme européenne s’applique aux produits employés en médecine pour la friction et le lavage hygiéniques des mains, pour la friction et le lavage chirurgicaux des mains, pour la désinfection des instruments par immersion et pour la désinfection de surface par essuyage, pulvérisation, rinçage ou autre.
Le présent document s’applique dans les zones et situations où la désinfection ou l’antisepsie est médicalement préconisée. De telles indications se rencontrent dans le cadre des soins apportés aux patients, par exemple :
dans des hôpitaux, centres de soins médicaux et cabinets dentaires ;
dans des infirmeries d’écoles, de jardins d'enfants et de maisons de retraite ;
et peuvent aussi se rencontrer sur les lieux de travail ou à domicile. Elles peuvent également concerner des services tels que des blanchisseries et cuisines qui fournissent des produits directement aux patients.
NOTE 1 La méthode décrite est destinée à déterminer l’activité des formulations commerciales ou des substances actives dans leurs conditions d’utilisation.
NOTE 2 La présente méthode correspond à un essai de type phase 2, étape 1.
L’EN 14885 précise de manière 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 na kvasovke v medicini - Preskusna metoda in zahteve (faza 2, stopnja 1)
Ta evropski standard določa preskusno metodo in minimalne zahteve za fungicidno delovanje ali delovanje na kvasovke kemičnih razkužil in antiseptikov, ki tvorijo homogen, fizikalno stabilen pripravek, če so razredčeni s trdo vodo ali, pri proizvodih, ki so pripravljeni za uporabo, z vodo. Proizvode je mogoče preskušati samo pri 80-odstotni ali nižji koncentraciji (s prilagojeno metodo v posebnih primerih 97-odstotni), ker dodajanje preskusnih organizmov in moteče snovi vedno povzroči razredčenje. Ta evropski standard velja za proizvode, ki se uporabljajo na področju zdravstva pri higienskem drgnjenju rok, higienskem umivanju rok, kirurškem drgnjenju rok, kirurškem umivanju rok, dezinfekciji instrumentov s potapljanjem in površinski dezinfekciji z brisanjem, pršenjem, zalivanjem ali na druge načine. Ta evropski standard se uporablja za področja in primere, ko obstajajo zdravniške indikacije za dezinfekcijo ali antisepso. Te indikacije se pojavljajo pri negi bolnikov, na primer: – v bolnišnicah, skupnih zdravstvenih in zobozdravstvenih ustanovah; – v klinikah šol, vrtcev in domov za starejše; in lahko nastanejo na delovnem mestu ali doma. Vključujejo lahko tudi storitve, kot so pralnice in kuhinje, ki proizvode neposredno dostavljajo bolnikom.
General Information
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Standards Content (Sample)
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Chemische Desinfektionsmittel und Antiseptika - Quantitativer Suspensionsversuch zur Bestimmung der fungiziden oder levuroziden Wirkung im humanmedizinischen Bereich - Prüfverfahren und Anforderungen (Phase 2, Stufe 1)Désinfectants chimiques et antiseptiques - Essai quantitatif de suspension pour l'évaluation de l'activité fongicide ou levuricide en médecine - Méthode d'essai et prescriptions (phase 2, étape 1)Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of fungicidal or yeasticidal activity in the medical area - Test method and requirements (phase 2, step 1)11.080.20Dezinfektanti in antiseptikiDisinfectants and antisepticsICS:Ta slovenski standard je istoveten z:EN 13624:2013SIST EN 13624:2013en,fr,de01-november-2013SIST EN 13624:2013SLOVENSKI
STANDARDSIST EN 13624:20041DGRPHãþD
SIST EN 13624:2013
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 13624
September 2013 ICS 11.080.20 Supersedes EN 13624:2003English Version
Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of fungicidal or yeasticidal activity in the medical area - Test method and requirements (phase 2, step 1) Désinfectants chimiques et antiseptiques - Essai quantitatif de suspension pour l'évaluation de l'activité fongicide ou levuricide en médecine - Méthode d'essai et prescriptions (phase 2, étape 1)
Chemische Desinfektionsmittel und Antiseptika - Quantitativer Suspensionsversuch zur Bestimmung der fungiziden oder levuroziden Wirkung im humanmedizinischen Bereich - Prüfverfahren und Anforderungen (Phase 2, Stufe 1) This European Standard was approved by CEN on 3 August 2013.
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, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre:
Avenue Marnix 17,
B-1000 Brussels © 2013 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 13624:2013: ESIST EN 13624:2013
EN 13624:2013 (E) 2 Contents Page Foreword .3 Introduction .4 1 Scope .5 2 Normative references .5 3 Terms and definitions .5 4 Requirements .5 5 Test method .7 5.1 Principle .7 5.2 Materials and reagents .7 5.3 Apparatus and glassware . 10 5.4 Preparation of test organism suspensions and product test solutions . 12 5.5 Procedure for assessing the fungicidal and yeasticidal activity of the product . 17 5.6 Experimental data and calculation. 24 5.7 Verification of methodology . 30 5.8 Expression of results and precision . 30 5.9 Interpretation of results – conclusion . 31 5.10 Test report . 32 Annex A (informative)
Referenced strains in national collections . 35 Annex B (informative)
Neutralizers and rinsing liquids . 36 Annex C (informative)
Graphical representation of test procedures . 38 C.1 Dilution-neutralization method . 38 C.2 Membrane filtration method . 40 C.3 Dilution-neutralization method (modified method for ready-to-use products) . 42 C.4 Membrane filtration method (modified method for ready-to-use products) . 44 Annex D (informative)
Example of a typical test report . 46 Annex E (informative)
Precision of the test result . 50 Annex ZA (informative)
Relationship between this European Standard and the Essential Requirements of EU Directive 93/42/EEC . 53 Bibliography . 54
SIST EN 13624:2013
EN 13624:2013 (E) 3 Foreword This document (EN 13624:2013) 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 March 2014, and conflicting national standards shall be withdrawn at the latest by March 2014. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights. This document supersedes EN 13624:2003. The document was revised to adapt it to the latest state of science, to correct errors and ambiguities, to harmonise the structure and wording with other tests of CEN/TC 216 existing or in preparation and to improve the readability of the standard and thereby make it more understandable. The following is a list of significant technical changes since the last edition: The Scope was expanded for the following fields of application within the medical area, i.e. products for surgical and/or hygienic handrub and/or handwash and disinfectants for other surfaces than instrument surfaces. “Obligatory test conditions” were replaced by “minimum test conditions” (test temperatures and contact times can be chosen within limits) that have to be performed to pass the test. An additional modified method is described to test ready-to-use products in a higher concentration than 80 %, i.e. 97 %. The quality of the cultured conidiospores of Aspergillus brasiliensis is described in greater detail (media, limits and the control methods) resulting from work done in WG 3 of CEN/TC 216. The neutralization time was shortened to 10 s for products with contact times of 10 min or less. The Annex ZA was reformulated to more accurately describe the relationship with the Medical Device Directive. Data obtained using the former version of EN 13624 may still be used, if the quality of the conidiospores of Aspergillus brasiliensis had been controlled and had met the requirements in this standard (5.4.1.4.2). This document has been prepared under a mandate given to CEN by the European Commission and the European Free Trade Association, and supports essential requirements of EU Directive(s). For relationship with EU Directive(s), see informative Annex ZA, which is an integral part of this document. 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, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. SIST EN 13624:2013
EN 13624:2013 (E) 4
Introduction This European Standard specifies a suspension test for establishing whether a chemical disinfectant or an antiseptic has a fungicidal or yeasticidal activity in the area and fields described in the scope. This laboratory test takes into account practical conditions of application of the product including contact time, temperature, test organisms and interfering substances, i.e. conditions which may influence its action in practical situations. Each utilisation concentration of the chemical disinfectant or antiseptic found by this test corresponds to the chosen experimental conditions. SIST EN 13624:2013
EN 13624:2013 (E) 5 1 Scope This European Standard 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 (97 % with a modified method for special cases) as some dilution is always produced by adding the test organisms and interfering substance.
This European Standard applies to products that are used in the medical area in the fields of hygienic handrub, hygienic handwash, surgical handrub, surgical handwash, instrument disinfection by immersion, and surface disinfection by wiping, spraying, flooding or other means. This European Standard applies to areas and situations where disinfection or antisepsis is medically indicated. Such indications occur in patient care, for example: in hospitals, in community medical facilities and in dental institutions; in clinics of schools, of kindergartens and of nursing homes; and may occur in the workplace and in the home. It may also include services such as laundries and kitchens supplying products directly for the patients. NOTE 1 The method described is intended to determine the activity of commercial formulations or active substances under the conditions in which they are used. NOTE 2 This method corresponds to a phase 2 step 1 test. EN 14885 specifies in detail the relationship of the various tests to one another and to “use recommendations”. 2 Normative references The following documents, in whole or in part, are normatively referenced in this document and are indispensable for its application. 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 4793:1980, 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. 4 Requirements The product shall demonstrate at least a 4 decimal log (lg) reduction (for hygienic handwash at least a 2 lg reduction), when tested in accordance with Table 1 and Clause 5. SIST EN 13624:2013
EN 13624:2013 (E) 6 Table 1 — Minimum and additional test conditions Test conditions Hygienic handrub and handwash Surgical handrub and handwash Instrument disinfection Surface disinfection Minimum spectrum of test organisms
Candida albicans (vegetative cells) Candida albicans (vegetative cells) a) fungicidal activity: Aspergillus brasiliensis (conidiospores) Candida albicans (veg. cells) b) yeasticidal activity: Candida albicans (veg. cells) a) fungicidal activity: Aspergillus brasiliensis (conidiospores) Candida albicans (veg. cells) b) yeasticidal activity: Candida albicans (veg. cells)
additional Any relevant test organism Test temperature according to the manufacturer's recommendation, but at/ between 20 °C 20 °C 20 °C and 70 °C 4 °C and 30 °C Contact time according to the manufacturer's recommendation,
but between
but no longer than 30 s and 60 s 1 min and 5 min 60 min 5 min or 60 mina Interfering substance clean conditions 0,3 g/l bovine albumin solution (hygienic handrub)b 0,3 g/l bovine albumin solution (surgical handrub)b 0,3 g/l bovine albumin solution 0,3 g/l bovine albumin solution dirty conditions 3,0 g/l bovine albumin solution plus 3,0 ml/l erythrocytes (hygienic handwash)c 3,0 g/l bovine albumin solution plus 3,0 ml/l erythrocytes (surgical and handwash)c and/or 3,0 g/l bovine albumin solution plus 3,0 ml/l erythrocytes and/or 3,0 g/l bovine albumin solution plus 3,0 ml/l erythrocytes b) additional — — any relevant substance any relevant substance NOTE For the additional conditions, the concentration defined as a result can be lower than the one obtained under the minimum test conditions. a The contact times for surface disinfectants stated in this table are chosen on the basis of the practical conditions of the product. The recommended contact time for the use of the product is within the responsibility of the manufacturer. Products intended to disinfect surfaces that are likely to come into contact with the patient and/or the medical staff and surfaces, which are frequently touched by different people, leading to the transmission of microorganisms to the patient, shall be tested with a contact time of maximum 5 min. The same applies where the contact time of the product shall be limited for practical reasons. Products for other surfaces than stated above may be tested with a contact time of maximum 60 min.
b Hygienic and surgical handrub shall be tested as a minimum under clean conditions.
c Hygienic and surgical handwash shall be tested as a minimum under dirty conditions.
SIST EN 13624:2013
EN 13624:2013 (E) 7
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 temperature and the contact time specified in Clause 4 and 5.5.1.1. At the end of this contact time, an aliquot is taken; the fungicidal and/or the fungistatic action 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. NOTE Handwash products are always prediluted with hard water (5.2.2.7). The resulting solution is regarded as a ready-to-use product (5.4.2). 5.1.2 The test is performed using the vegetative cells of Candida albicans and the conidiospores of Aspergillus brasiliensis (fungicidal activity) or only the vegetative cells of Candida albicans (yeasticidal activity) as test-organisms (Clause 4, Table 1). 5.1.3 Additional contact times and temperatures are specified (Clause 4, Table 1). Additional interfering substances and test organisms may be used. 5.2 Materials and reagents 5.2.1 Test organisms The fungicidal activity shall be evaluated using the following strains as test organisms selected according to Clause 4 (Table 1)1): Candida albicans ATCC 10231; Aspergillus brasiliensis (former “A.niger”) ATCC 16404. The yeasticidal activity shall be evaluated using only Candida albicans.
NOTE See Annex A for strain reference in some other culture collections. The required incubation temperature for these test organisms is (30 ± 1) °C (5.3.2.3). 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.
1) The ATCC numbers are the collection numbers of strains supplied by these culture collections. This information is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of the product named. SIST EN 13624:2013
EN 13624:2013 (E) 8 5.2.2 Culture media and reagents 5.2.2.1 General All weights of chemical substances given in this European Standard refer to the anhydrous salts. Hydrated forms may be used as an alternative, but the weights required shall be adjusted to allow for consequent molecular weight differences. The reagents shall be of analytical grade and/or appropriate for microbiological purposes. They shall be free from substances that are toxic or inhibitory to the test organisms. To improve reproducibility, it is recommended that commercially available dehydrated material is used for the preparation of culture media. The manufacturer's instructions relating to the preparation of these products should be rigorously followed.
For each culture medium and reagent, a time limitation for use should be fixed.
All specified pH values are measured at (20 ± 1) °C. 5.2.2.2 Water The water shall be freshly glass-distilled water and not demineralised water. If distilled water of adequate quality is not available, water for injections (see bibliographic reference [1]) can be used. Sterilise in the autoclave [5.3.2.1 a)]. Sterilisation is not necessary if the water is used, e.g. for preparation of culture media and subsequently sterilised. See 5.2.2.7 for the procedure to prepare hard water. 5.2.2.3 Malt extract agar (MEA) Malt extract agar, consisting of:
Malt extract (food grade (e.g. Christomalt powder from Difal) or an equivalent extract that is not highly purified and not only based on maltose (e.g. Malt extract from OXOID))2) 30,0 g
Agar 15,0 g
Water (5.2.2.2) to 1 000,0 ml
Sterilise in the autoclave (5.3.1). After sterilisation, the pH (5.3.2.4) of the medium shall be equivalent to 5,6 ± 0,2. In case of an encountering (problems with neutralization (5.5.1.2 and 5.5.1.3), it may be necessary to add neutralizer to MEA. Annex B gives guidance on the neutralizers that may be used. It is recommended not to use neutralizer that causes opalescence in the agar. If there are problems with producing at least 75 % spiny conidiospores, see 5.4.1.4.2. 5.2.2.4 Diluent Tryptone sodium chloride solution, consisting of:
2) This Malt extract from OXOID is an example of a suitable product available commercially. This information is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of this product. SIST EN 13624:2013
EN 13624:2013 (E) 9
Tryptone, pancreatic digest of casein 1,0 g
Sodium chloride (NaCl) 8,5 g
Water (5.2.2.2) to 1 000,0 ml Sterilise in the autoclave [5.3.2.1 a)]. After sterilisation, the pH (5.3.2.4) of the diluent shall be equivalent to 7,0 ± 0,2. 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 l of hard water, the procedure is as follows: prepare solution A: dissolve 19,84 g magnesium chloride (MgCl2) and 46,24 g calcium chloride (CaCl2) in water (5.2.2.2) and dilute to 1 000 ml. Sterilise by membrane filtration (5.3.2.7) or in the autoclave [5.3.2.1 a)]. Autoclaving – if used – may cause a loss of liquid. In this case make up to 1 000 ml with water (5.2.2.2) under aseptic conditions. Store the solution in the refrigerator (5.3.2.8) for no longer than one month; prepare solution B: dissolve 35,02 g sodium bicarbonate (NaHCO3) in water (5.2.2.2) and dilute to 1 000 ml. Sterilise 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 (5.3.2.4) of the hard water shall be 7,0 ± 0,2. If necessary, adjust the pH by using a solution of approximately 40 g/l (about 1 mol/l) of sodium hydroxide (NaOH) or approximately 36,5 g/l (about 1 mol/l) of hydrochloric acid (HCl).
The hard water shall be freshly prepared under aseptic conditions and used within 12 h. NOTE When preparing the product test solutions (5.4.2), the addition of the product to the hard water produces different final water hardness in each test tube. In any case the final hardness expressed as calcium carbonate (CaCO3) is in the test tube lower than 375 mg/l. 5.2.2.8 Interfering substance 5.2.2.8.1 General The interfering substance shall be chosen according to the conditions of use laid down for the product. The interfering substance shall be sterile and prepared at 10 times its final concentration in the test (50 times in the case of the modified method, see 5.2.2.8.4). SIST EN 13624:2013
EN 13624:2013 (E) 10 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 0,30 g of bovine albumin fraction V (suitable for microbiological purposes) in 100 ml of diluent (5.2.2.4). Sterilise by membrane filtration (5.3.2.7), keep in a refrigerator (5.3.2.8) and use within one month. The final concentration of the bovine albumin in the test procedure (5.5) shall be 0,3 g/l. 5.2.2.8.3 Dirty conditions (mixture of bovine albumin solutions – high concentration with sheep erythrocytes) Dissolve 3,00 g of bovine albumin fraction V (suitable for microbiological purposes) in 97 ml of diluent (5.2.2.4). Sterilise by membrane filtration (5.3.2.7). Prepare at least 8,0 ml fresh defibrinated sheep blood (5.2.2.9). Centrifuge the erythrocytes at 800 gN for 10 min (5.3.2.13). After discarding the supernatant, resuspend erythrocytes in diluent (5.2.2.4). Repeat this procedure at least 3 times, until the supernatant is colourless.
Resuspend 3 ml of the packed sheep erythrocytes in the 97 ml of sterilised bovine albumin solution (see above). To avoid later contamination this mixture should be split in portions probably needed per day and kept in separate containers for a maximum of 7 d in a refrigerator (5.3.2.8). The final concentration of bovine albumin and sheep erythrocytes in the test procedure (5.5) shall be 3 g/l and 3 ml/l respectively. 5.2.2.8.4 Clean and dirty conditions for the modified method for ready-to-use products (5.5.4) Follow the procedures for preparation according to 5.2.2.8.2 and 5.2.2.8.3, but prepare the interfering substance in fivefold higher concentrations. a) Clean conditions (5.2.2.8.2) – dissolve 1,50 g bovine albumin (instead of 0,3 g) in 100 ml of diluent; b) Dirty conditions (5.2.2.8.3) – dissolve 15,0 g bovine albumin (instead of 3,0 g) in 85 ml of diluent (instead of 97 ml). Prepare at least 40 ml (instead of 8,0 ml) sheep blood. Resuspend 15 ml (instead of 3,0 ml) of the packed sheep erythrocytes in 85 ml of sterilised bovine albumin solution (see above). 5.2.2.9 Defibrinated sheep blood The defibrinated sheep blood should be sterile (aseptic blood-letting and preparation), pooled from more than one sheep and can be acquired from a commercial supplier. 5.3 Apparatus and glassware 5.3.1 General Sterilise all glassware and parts of the apparatus that will come into contact with the culture media and reagents or the sample, except those which are supplied sterile, by one of the following methods: SIST EN 13624:2013
EN 13624:2013 (E) 11 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 equipment3) and, in particular, the following: 5.3.2.1 Apparatus for moist and dry heat sterilisation: a) for moist heat sterilisation, an autoclave capable of being maintained at (30121+) °C for a minimum holding time of 15 min;
b) for dry heat sterilisation, a hot air oven capable of being maintained at (50180+) °C for a minimum holding time of 30 min, at (170 50+) °C for a minimum holding time of 1 h or at (50160+) °C for a minimum holding time of 2 h. 5.3.2.2 Water baths, capable of being controlled at (20 ± 1) °C, at (45 ± 1) °C (to maintain melted MEA in case of pour plate technique) and at additional test temperatures ± 1 °C (5.5.1). 5.3.2.3 Incubator, capable of being controlled at (30 ± 1) °C. 5.3.2.4 pH-meter, having an inaccuracy of calibration of no more than ± 0,1 pH units at (20 ± 1) °C. A puncture electrode or a flat membrane electrode should be used for measuring the pH of the agar media (5.2.2.3). 5.3.2.5 Stopwatch. 5.3.2.6 Shakers. a) Electromechanical agitator, e.g. Vortex® mixer4); b) Mechanical shaker. 5.3.2.7 Membrane filtration apparatus, constructed of a material compatible with the substances to be filtered, with a filter holder of at least 50 ml volume, and suitable for use of filters of diameter 47 mm to 50 mm and 0,45 µm pore size for sterilisation of hard water (5.2.2.7), bovine albumin (5.2.2.8.2, 5.2.2.8.3 and 5.2.2.8.4), and if the membrane filtration method is used (5.5.3). The vacuum source used shall give an even filtration flow rate. In order to obtain a uniform distribution of the micro-organisms over the membrane and to prevent overlong filtration, the device shall be set so as to obtain the filtration of 100 ml of rinsing liquid in 20 s to 40 s. 5.3.2.8 Refrigerator, capable of being controlled at 2 °C to 8 °C. 5.3.2.9 Graduated pipettes, of nominal capacities 10 ml and 1 ml and 0,1 ml, or calibrated automatic pipettes. 5.3.2.10 Petri dishes, (plates) of size 90 mm to 100 mm.
3) Disposable sterile equipment is an acceptable alternative to reusable glassware. 4) Vortex® is an example of a suitable product available commercially. This information is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of this product. SIST EN 13624:2013
EN 13624:2013 (E) 12 5.3.2.11 Glass beads (diameter 3 mm to 4 mm). 5.3.2.12 Volumetric flasks. 5.3.2.13 Centrifuge (800 gN).
5.3.2.14 Flasks with ventilated caps: Roux bottles or similar flasks. 5.3.2.15 Fritted filters: Porosity of 40 µm to 100 µm according to ISO 4793:1980, Clause 2. 5.4 Preparation of test organism suspensions and product test solutions 5.4.1 Test organism suspensions (test and validation suspension) 5.4.1.1 General For each test organism, two different suspensions have to be prepared: the “test suspension” to perform the test and the “validation suspension” to per
...
SLOVENSKI STANDARD
oSIST prEN 13624:2010
01-april-2010
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RFHQMHYDQMHIXQJLFLGQHJDGHORYDQMDDOLGHORYDQMDNHPLþQLKUD]NXåLOLQDQWLVHSWLNRY
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Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation
of fungicidal or yeasticidal activity in the medical area - Test method and requirements
(phase 2, step 1)
Chemische Desinfektionsmittel und Antiseptika - Quantitativer Suspensionsversuch zur
Bestimmung der fungiziden oder levuroziden Wirkung im humanmedizinischen Bereich -
Prüfverfahren und Anforderungen (Phase 2, Stufe 1)
Désinfectants chimiques et antiseptiques - Essai quantitatif de suspension pour
l'évaluation de l'activité fongicide ou levuricide en médecine - Méthode d'essai et
prescriptions (phase 2, étape 1)
Ta slovenski standard je istoveten z: prEN 13624
ICS:
11.080.20 Dezinfektanti in antiseptiki Disinfectants and antiseptics
oSIST prEN 13624:2010 en,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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oSIST prEN 13624:2010
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oSIST prEN 13624:2010
EUROPEAN STANDARD
DRAFT
prEN 13624
NORME EUROPÉENNE
EUROPÄISCHE NORM
January 2010
ICS 11.080.20 Will supersede EN 13624:2003
English Version
Chemical disinfectants and antiseptics - Quantitative suspension
test for the evaluation of fungicidal or yeasticidal activity in the
medical area - Test method and requirements (phase 2, step 1)
Désinfectants chimiques et antiseptiques - Essai quantitatif Chemische Desinfektionsmittel und Antiseptika -
de suspension pour l'évaluation de l'activité fongicide ou Quantitativer Suspensionsversuch zur Bestimmung der
levuricide en médecine - Méthode d'essai et prescriptions fungiziden oder levuroziden Wirkung im
(phase 2, étape 1) humanmedizinischen Bereich - Prüfverfahren und
Anforderungen (Phase 2, Stufe 1)
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 Management Centre has the
same status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland,
France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,
Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland 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
Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2010 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 13624:2010: E
worldwide for CEN national Members.
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prEN 13624:2010 (E)
Contents Page
Foreword .4
Introduction .4
1 Scope .4
2 Normative references .5
3 Terms and definitions .5
4 Requirements .5
5 Test method .7
5.1 Principle .7
5.2 Materials and reagents .7
5.2.1 Test organisms .7
5.2.2 Culture media and reagents .8
5.3 Apparatus and glassware . 10
5.3.1 General . 10
5.3.2 Usual microbiological laboratory equipment . 10
5.4 Preparation of test organism suspensions and product test solutions . 12
5.4.1 Test organism suspensions (test and validation suspension) . 12
5.4.2 Product test solutions . 14
5.5 Procedure for assessing the fungicidal activity of the product . 15
5.5.1 General . 15
5.5.2 Dilution-neutralization method . 16
5.5.3 Membrane filtration method . 19
5.5.4 Modified method for ready-to-use products . 21
5.6 Experimental data and calculation. 22
5.6.1 Explanation of terms and abbreviations . 22
5.6.2 Calculation . 23
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
5.8 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 and yeasticidal concentration . 29
5.8.4 Precision, repetitions . 29
5.9 Interpretation of results – conclusion . 30
5.9.1 General . 30
5.9.2 Fungicidal activity for instrument disinfection products . 30
5.9.3 Fungicidal activity for surface disinfection products . 30
5.9.4 Yeasticidal activity for handrub and handwash products . 30
5.9.5 Yeasticidal activity for instrument disinfection products . 30
5.9.6 Yeasticidal activity for surface disinfection products . 30
5.9.7 Qualification for certain fields of application . 30
5.10 Test report . 31
Annex A (informative) Referenced strains in national collections . 33
Annex B (informative) Neutralizers and Rinsing Liquids . 34
Annex C (informative) Graphical representation of test procedures . 36
C.1 Dilution-neutralization method . 36
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C.2 Membrane filtration method . 38
C.3 Dilution-neutralization method (modified method for ready-to-use products) . 40
C.4 Membrane filtration method (modified method for ready-to-use products) . 42
Annex D (informative) Example of a typical test report . 44
Annex E (informative) Precision of the test result . 50
Annex ZA (informative) Relationship between this European Standard and the Essential
Requirements of EU Directive 93/42/EEC . 53
Bibliography . 54
3
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Foreword
This document (prEN 13624:2010) 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 13624:2003.
This document has been prepared under a mandate given to CEN by the European Commission and the
European Free Trade Association, and supports essential requirements of EU Directive.
For relationship with EU Directive, see informative Annex ZA, which is an integral part of this document.
Introduction
This European Standard specifies a suspension test for establishing whether a chemical disinfectant or an
antiseptic has a fungicidal or yeasticidal activity in the area and fields described in the scope.
This laboratory test takes into account practical conditions of application of the product including contact time,
temperature, test organisms and interfering substances, i. e. conditions which may influence its action in
practical situations.
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 the chosen experimental conditions. However, for some applications the instructions of use of
a product may differ and therefore additional test conditions need to be used.
1 Scope
This European Standard 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 (97 % with a modified method for special cases) as some
dilution is always produced by adding the test organisms and interfering substance.
This European Standard applies to products that are used in the medical area in the fields of hygienic
handrub, hygienic handwash, surgical handrub, surgical handwash, instrument disinfection by immersion, and
surface disinfection by wiping, spraying, flooding or other means.
This European Standard applies to areas and situations where disinfection or antisepsis is medically
indicated. Such indications occur in patient care, for example:
in hospitals, in community medical facilities and in dental institutions;
in clinics of schools, of kindergartens and of nursing homes;
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and may occur in the workplace and in the home. It may also include services such as laundries and kitchens
supplying products directly for the patients.
NOTE 1 The method described is intended to determine the activity of commercial formulations or active substances
under the conditions in which they are used.
NOTE 2 This method corresponds to a phase 2 step 1 test.
EN 14885 specifies in detail the relationship of the various tests to one another and to “use
recommendations”.
2 Normative references
The following referenced documents are indispensable for the application 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, mycobactericidal, sporicidal and fungicidal 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.
4 Requirements
The product shall demonstrate at least a 4 decimal log (lg) reduction (for hygienic and surgical hand wash at
least a 2 lg reduction), when tested in accordance with table 1 and clause 5 under simulated clean conditions
(0,3 g/l bovine albumin solution) or dirty conditions (3 g/l bovine albumin solution, plus 3 ml/l washed sheep
erythrocytes). For instrument disinfectants recommended by the manufacturer to be used at higher
temperatures the product may demonstrate the 4 lg reduction only at the recommended temperature.
5
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Table 1 — Obligatory and additional test conditions
Test conditions Hygienic handrub Surgical handrub Instrument Surface
and handwash and handwash disinfection disinfection
Test organism Candida albicans Candida albicans a1) fungicidal a1) fungicidal
a) obligatory (vegetative cells) (vegetative cells) activity: Aspergillus activity: Aspergillus
niger (spores) niger (spores)
Candida albicans Candida albicans
(eg. cells) (eg. cells)
a2) yeasticidal a2) yeasticidal
activity: Candida activity: Candida
albicans (veg. cells) albicans (veg. cells)
b) additional Any relevant test organism
Test temperature 20 °C 20 °C At the lowest 20 °C
a) obligatory recommended
temperature, min.
20 °C, max. 60 °C
b) additional according to the manufacturer's recommendation, but not higher than
20 °C 20 °C 60 °C 30 °C
Contact time 60 s 5 min 60 min 5 min or 60 min*
a) obligatory
b) additional according to the manufacturer's recommendation, but no longer than
60 s ** 5 min 60 min 5 min or 60 min*
Interfering 0,3 g/l bovine 0,3 g/l bovine 0,3 g/l bovine 0,3 g/l bovine
substance albumin solution albumin solution albumin solution albumin solution
a) obligatory clean (hygienic handrub) (surgical handrub)
conditions
dirty conditions 3,0 g/l bovine 3,0 g/l bovine and/or and/or
albumin solution albumin solution 3,0 g/l bovine 3,0 g/l bovine
plus 3,0 ml plus 3,0 ml albumin solution albumin solution
eryhtrocytes erythrocytes plus 3,0 ml plus 3,0 ml
(hygienic (surgical and erythrocytes erythrocytes
handwash) handwash)
b) additional — — any relevant any relevant
substance substance
* The obligatory contact times for surface disinfectants stated in table 1 are chosen on the basis of exacted
practical application of the product. The referenced test conditions are by no means intended as
requirements for the use of a product, nor as requirements for the evaluation and acceptance of products by
regulatory authorities. The recommended contact time for the use of the product is within the responsibility of
the manufacturer. Products intended to disinfect surfaces that are likely to come into contact with the patient
and / or the medical staff, which could lead to the transmission of micro-organisms to the patient, and where
the contact time of the product shall be limited for practical reasons, shall be tested with an obligatory contact
time of 5 min. Products for other surfaces than stated above, could be tested with an obligatory contact time
of 60 min.
** For hygienic handwash 75 s.
NOTE For the additional conditions, the concentration defined as a result can be lower than the one
obtained under the obligatory test conditions.
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Table 2 — Obligatory and additional test conditions.
Any additional specific fungicidal or yeasticidal activity shall be determined in accordance with 5.2.1 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 temperature and the contact time specified in clause 4 and
5.5.1.1. At the end of this contact time, an aliquot is taken; the fungicidal and/or the fungistatic action 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.
NOTE 1 Handwash products are always prediluted with hard water (5.2.2.7). The resulting solution is
regarded as a ready-to-use product (5.4.2).
5.1.2 The test is performed using the vegetative cells of Candida albicans and the spores of Aspergillus
niger (fungicidal activity) or only the vegetative cells of Candida albicans (yeasticidal activity) as test-
organisms (clause 4, table 1).
5.1.3 Additional and optional contact times and temperatures are specified (clause 4, table 1). Additional
interfering substances and test organisms may be used.
5.2 Materials and reagents
5.2.1 Test organisms
The fungicidal activity shall be evaluated using the following strains as test organisms selected according to
1)
clause 4 (table 1) :
Candida albicans ATCC 10231;
Aspergillus niger ATCC 16404.
The yeasticidal activity shall be evaluated using only Candida albicans.
NOTE See annex A for strain reference in some other culture collections.
The required incubation temperature for these test organisms is (30 ± 1) °C.
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.
1) The ATCC numbers are the collection numbers of strains supplied by these culture collections. This information is
given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of the
product named.
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5.2.2 Culture media and reagents
5.2.2.1 General
All weights of chemical substances given in this European Standard refer to the anhydrous salts. Hydrated
forms may be used as an alternative, but the weights required shall be adjusted to allow for consequent
molecular weight differences.
The reagents shall be of analytical grade and/or appropriate for microbiological purposes. They shall be free
from substances that are toxic or inhibitory to the test organisms.
NOTE 1 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 should be
rigorously followed.
NOTE 2 For each culture medium and reagent, a time limitation for use should be fixed.
5.2.2.2 Water
The water shall be freshly glass-distilled water and not demineralized water. If distilled water of adequate
quality is not available, water for injections (see bibliographic reference [1]) can be used.
Sterilize in the autoclave [5.3.2.1 a)]. Sterilization is not necessary if the water is used e. g. for preparation of
culture media and subsequently sterilized.
NOTE 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
Soya peptone, papaic digest of Soybean Meal 3,0 g
Agar 15,0 g
Water (5.2.2.2) to 1000,0 ml
Sterilize in the autoclave (5.3.1). After sterilization, the pH of the medium shall be equivalent to 5,6 ± 0,2 when
measured at (20 ± 1) °C (5.3.2.4).
NOTE In case of an encountering (problems with neutralization (5.5.1.2 and 5.5.1.3) it may be necessary to add
neutralizer to MEA. Annex B gives guidance on the neutralizers that may be used. It is recommended not to use
neutralizer that causes opalescence in the agar.
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 1000,0 ml
Sterilize in the autoclave [5.3.2.1 a)]. 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.
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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 l of hard water, the procedure is as follows:
prepare solution A: dissolve 19,84 g magnesium chloride (MgCl ) and 46,24 g calcium chloride (CaCl ) in
2
2
water (5.2.2.2) and dilute to 1000 ml. Sterilize by membrane filtration (5.3.2.7) or in the autoclave
[5.3.2.1 a)]. Autoclaving – if used - may cause a loss of liquid. In this case make up to 1000 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
1000 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 1000 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 1000 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 hours.
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 300 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 0,30 g of bovine albumin fraction V (suitable for microbiological purposes) in 100 ml of diluent
(5.2.2.4).
Sterilize by membrane filtration (5.3.2.7), keep in a refrigerator (5.3.2.8) and use within one month.
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The final concentration of the bovine albumin in the test procedure (5.5) shall be 0,3 g/l.
5.2.2.8.3 Dirty conditions (Mixture of bovine albumin solutions – high concentration with sheep
erythrocytes)
Dissolve 3,00 g of bovine albumin fraction V (suitable for microbiological purposes) in 97 ml of diluent
(5.2.2.4) .
Sterilize by membrane filtration (5.3.2.7).
Prepare at least 8,0 ml fresh sterile defibrinated sheep blood (5.2.2.9). Centrifuge the erythrocytes at 800 g
N
for 10 min (5.3.2.13). After discarding the supernatant, resuspend erythrocytes in diluent (5.2.2.4). Repeat this
procedure at least 3 times, until the supernatant is colourless.
Resuspend 3 ml of the packed sheep erythrocytes in the 97 ml of sterilized bovine albumin solution (see
above). To avoid later contamination this mixture should be split in portions probably needed per day and kept
in separate containers for a maximum of 7 days in a refrigerator (5.3.2.8).
The final concentration of bovine albumin and sheep erythrocytes in the test procedure (5.5) shall be 3 g/l and
3 ml/l respectively.
5.2.2.8.4 Clean and dirty conditions for the modified method for ready-to-use products (5.5.4)
Follow in general the procedures for preparation according to 5.2.2.8.2 and 5.2.2.8.3, but prepare the
interfering substance in fivefold higher concentrations.
a) Clean conditions (5.2.2.8.2) – dissolve 1,50 g bovine albumin (instead of 0,3 g) in 100 ml of diluent;
b) Dirty conditions (5.2.2.8.3) – dissolve 15,0 g bovine albumin (instead of 3,0 g) in 85 ml of diluent (instead
of 97 ml).
Prepare at least 40 ml (instead of 8,0 ml) sheep blood. Resuspend 15 ml (instead of 3,0 ml) of the packed
sheep erythrocytes in 85 ml of sterilized bovine albumin solution (see above).
5.2.2.9 Sterile defibrinated sheep blood
The sterile defibrinated sheep blood can be acquired from a commercial supplier or p
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