EN 1657:2005

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 chemischer Desinfektionsmittel und Antiseptika für den Veterinärbereich - 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 vétérinaire
- Méthode d'essai et prescriptions (phase 2, étape 1)Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of fungicidal or yeasticidal activity of chemical disinfectants and antiseptics used in the veterinary area - Test method and requirements (phase 2, step 1)11.220VeterinarstvoVeterinary medicine11.080.20Dezinfektanti in antiseptikiDisinfectants and antisepticsICS:Ta slovenski standard je istoveten z:EN 1657:2005SIST EN 1657:2006en01-junij-2006SIST EN 1657:2006SLOVENSKI
STANDARDSIST EN 1657:20011DGRPHãþD

EUROPEAN STANDARDNORME EUROPÉENNEEUROPÄISCHE NORMEN 1657December 2005ICS 11.080.20; 71.100.35Supersedes EN 1657:2000
English VersionChemical disinfectants and antiseptics - Quantitative suspensiontest for the evaluation of fungicidal or yeasticidal activity ofchemical disinfectants and antiseptics used in the veterinaryarea - Test method and requirements (phase 2, step 1)Antiseptiques et désinfectants chimiques
- Essai quantitatifde suspension pour l'évaluation de l'activité fongicide oulevuricide des antiseptiques et des désinfectants chimiquesutilisés dans le domaine vétérinaire
- Méthode d'essai etprescriptions (phase 2, étape 1)Chemische Desinfektionsmittel und Antiseptika -Quantitativer Suspensionsversuch zur Bestimmung derfungiziden oder levuroziden Wirkung chemischerDesinfektionsmittel und Antiseptika für denVeterinärbereich - Prüfverfahren und Anforderungen(Phase 2, Stufe 1)This European Standard was approved by CEN on 28 July 2005.CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such nationalstandards may be obtained on application to the Central Secretariat or to any CEN member.This European Standard exists in three official versions (English, French, German). A version in any other language made by translationunder the responsibility of a CEN member into its own language and notified to the Central Secretariat has the same status as the officialversions.CEN members are the national standards bodies of Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France,Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia,Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMITÉ EUROPÉEN DE NORMALISATIONEUROPÄISCHES KOMITEE FÜR NORMUNGManagement Centre: rue de Stassart, 36
B-1050 Brussels© 2005 CENAll rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 1657:2005: ESIST EN 1657:2006

and, in particular, the following:.10 5.4 Preparation of test organism suspensions and product test solutions.11 5.4.1 Test organism suspensions (test and validation suspension).11 5.4.2 Product test solutions.14 5.5 Procedure for assessing the fungicidal or yeasticidal activity of the product.14 5.5.1 General.14 5.5.2 Dilution-neutralization method.16 5.5.3 Membrane filtration method.18 5.6 Experimental data and calculation.20 5.6.1 Explanation of terms and abbreviations.20 5.6.2 Calculation.20 5.7 Verification of methodology.23 5.7.1 General.23 5.7.2 Control of weighted mean counts.23 5.7.3 Basic limits.24 5.8 Expression of results and precision.24 5.8.1 Reduction.24 5.8.2 Control of active and non-active product test solution (5.4.2).24 5.8.3 Limiting test organism and fungicidal/yeasticidal concentration.24 5.8.4 Precision, replicates.25 5.9 Interpretation of results – conclusion.25 5.9.1 General.25 5.9.2 Fungicidal activity for general purposes.25 5.9.3 Fungicidal activity for specific purposes.25 5.9.4 Yeasticidal activity for general purposes.25 5.9.5 Yeasticidal activity for specific purposes.25 5.10 Test report.26 Annex A (informative)
Referenced strains in national collections.28 Annex B (informative)
Suitable neutralizers and rinsing liquids.29 Annex C (informative)
Graphical representation of test procedures.31 Validation.34 Annex D (informative)
Example of a typical test report.35 Annex E (informative)
Precision of the test result.39 Annex F (informative)
Information on the application and interpretation of European Standards on chemical disinfectants and antiseptics.41 Bibliography.43 SIST EN 1657:2006

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 European Standard and does not constitute an endorsement by CEN of the product named. SIST EN 1657:2006

b) for dry heat sterilization, a hot air oven capable of being maintained at (50180+) °C for a minimum holding time of 30 min, at (50170+) °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 (10 ± 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 ( ± 1) °C.
NOTE 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® mixer3) b)
Mechanical shaker 5.3.2.7 Membrane filtration apparatus, constructed of a material compatible with the substances to be filtered. The apparatus shall have a filter holder of at least 50 ml volume. It shall be suitable for use with filters of diameter 47 mm to 50 mm and 0,45 µm pore size for sterilization of hard water (5.2.2.7) and bovine albumin (5.2.2.8), 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.
2) Disposable sterile equipment is an acceptable alternative to reusable glassware. 3) 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 1657:2006

5.3.2.10 Petri dishes (plates), of size 90 mm to 100 mm. 5.3.2.11 Glass beads, 3 mm to 4 mm in diameter. 5.3.2.12 Volumetric flasks. 5.3.2.13 Fritted filter, with porosity of 40 µm to 100 µm according to ISO 4793. 5.3.2.14 Centrifuge (2 000 gN). 5.3.2.15 Roux bottles or similar flasks. 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 perform the controls and method validation. 5.4.1.2 Preservation and stock cultures of test organisms The test organisms and their stock cultures shall be prepared and kept in accordance with EN 12353. 5.4.1.3 Working culture of test organisms 5.4.1.3.1 Candida albicans (yeast) In order to prepare the working culture of Candida albicans (5.2.1), prepare a subculture from the stock culture (5.4.1.2) by streaking onto MEA (5.2.2.3) slopes or plates (5.3.2.10) and incubate (5.3.2.3). After 42 h to 48 h, prepare a second subculture from the first subculture in the same way and incubate for 42 h to 48 h. From this second subculture, a third subculture may be produced in the same way. The second and (if produced) third subcultures are the working cultures. If it is not possible to prepare the second subculture on a particular day, a 72 h subculture may be used for subsequent subculturing, provided that the subculture has been kept in the incubator (5.3.2.3) during the 72 h period. Never produce and use a fourth subculture. 5.4.1.3.2 Aspergillus niger (mould) For Aspergillus niger (5.2.1), use only the first subculture grown on MEA (5.2.2.3) in Roux bottles (5.3.2.15) and incubate for 9 d to 11 d. No further subculturing is needed. 5.4.1.3.3 Other test organisms (yeasts or moulds) For additional test organisms, any departure from this method of culturing the yeast or the mould or of preparing the suspensions shall be noted, giving the reasons in the test report. SIST EN 1657:2006

[5.5.1.1 a)] and use within 2 h. NOTE The use of a spectrophotometer for adjusting the number of cells is highly recommended (approximately 620 nm wavelength – cuvette 10 mm path length). Each laboratory should therefore produce calibration data for each test organism knowing that suitable values of optical density are generally found between 0,200 and 0,350. A colorimeter is a suitable alternative. c) For counting, prepare 10-5 and 10-6 dilutions of the test suspension using diluent (5.2.2.4). Mix [5.3.2.6a)]. Take a sample of 1,0 ml of each dilution in duplicate and inoculate using the pour plate or the spread plate technique. 1) When using the pour plate technique, transfer each 1,0 ml sample into separate Petri dishes and add 15 ml to 20 ml melted MEA (5.2.2.3), cooled to (45 ± 1) °C. 2) When using the spread plate technique, spread each 1,0 ml sample – divided into portions of approximately equal size – on an appropriate number (at least two) of surface dried plates containing MEA (5.2.2.3). For incubation and counting, see 5.4.1.6. 5.4.1.4.2 Aspergillus niger The procedure for preparing the Aspergillus niger test suspension is as follows: a) take the working culture (5.4.1.3.2) and suspend the spores in 10 ml of sterile 0,05 % (w/v) polysorbate 80 solution in water (5.2.2.2). Using a glass rod or spatula, detach the conidiospores from the culture surface. Transfer the suspension into a flask and gently shake by hand for one minute together with 5 g of glass beads (5.3.2.11). Filter the suspension through a fritted filter (5.3.2.13); b) carry out a microscopic examination under x 400 magnification immediately after the preparation and just before the test, to show the absence of mycelia fragments and spore germination (check at least ten fields of view for absence of both). If germinated spores are present, discard the suspension. If mycelia are present, set up a washing process (centrifugation) as follows. Transfer the filtered suspension to centrifuge tubes. The filtered suspension is centrifuged (5.3.2.14) at 2 000 gN for 20 min. The conidiospores are washed at least twice by resuspension in diluent (5.2.2.4) and subsequent centrifugation. If mycelia are still present, repeat the washing process;
4) cfu/ml = colony-forming unit(s) per millilitre. SIST EN 1657:2006

(in °C):  the obligatory temperature to be tested is θ = 10 °C;  additional temperature may be chosen from 4 °C, 20 °C or 40 °C;  the allowed deviation for each chosen temperature is ± 1 °C; SIST EN 1657:2006

5) For a graphical representation of this method, see Annex C. SIST EN 1657:2006
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