EN 1657:2016

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 1657:2016SIST EN 1657:2016en,fr,de01-november-2016SIST EN 1657:2016SLOVENSKI
STANDARDSIST EN 1657:2006/AC:2007SIST EN 1657:20061DGRPHãþD

EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 1657
April
t r s x ICS
y sä s r rä u w Supersedes EN
s x w yã t r r wEnglish Version
Chemical disinfectants and antiseptics æ Quantitative suspension test for the evaluation of fungicidal or yeasticidal activity of chemical disinfectants and antiseptics used in the veterinary area æ Test method and Antiseptiques et désinfectants chimiques æ Essai quantitatif de suspension pour l 5évaluation de l 5activité fongicide ou levuricide des antiseptiques et des désinfectants chimiques utilisés dans le domaine
Chemische Desinfektionsmittel und Antiseptika æ Quantitativer Suspensionsversuch zur Bestimmung der fungiziden oder levuroziden Wirkung chemischer Desinfektionsmittel und Antiseptika für den Veterinärbereich æ Prüfverfahren und Anforderungen This European Standard was approved by CEN on
t u January
t r s xä
egulations which stipulate the conditions for giving this European Standard the status of a national standard without any alterationä Upætoædate lists and bibliographical references concerning such national standards may be obtained on application to the CENæCENELEC Management Centre or to any CEN memberä
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t r s x CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Membersä Refä Noä EN
s x w yã t r s x ESIST EN 1657:2016

low level soiling high level soiling 3,0 g/l bovine albumin 10 g/l yeast extract plus 10 g/l bovine albumin 3,0 g/l bovine albumin 10 g/l yeast extract plus 10 g/l bovine albumin 10,0 g/l of reconstituted skimmed milk additional any relevant substance any relevant substance any relevant substance The obligatory contact times for disinfectants stated in Table 1 were chosen to enable comparison of standard conditions. NOTE For the additional conditions, the concentration defined as a result can be lower than the one obtained under the obligatory test conditions. a The recommended contact time for the use of the product is within the responsibility of the manufacturer. Any additional specific fungicidal 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 10°C ± 1 °C for 30 min ± 10 s or 30°C ± 1 °C for 5 min ± 10 s or 30 s± 5 s for teat disinfectants (obligatory test conditions). At the end of this contact time, an aliquot is taken, and the fungicidal/yeasticidal and/or the fungistatic/yeastistatic 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. SIST EN 1657:2016

(formerly A.niger) 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 (see 5.3.2.3). The same temperature shall be used for all incubations performed during a test and its control and validation. 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 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 limitation for use should be fixed. 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)].
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 1657:2016

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. SIST EN 1657:2016

3) Disposable sterile equipment is an acceptable alternative to reusable glassware. SIST EN 1657:2016

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 1657:2016

Figure 1 — Photo No 1: A. brasiliensis ATCC 16404 after 7 d of incubation at 30°C SIST EN 1657:2016

Figure 2 — Photo No.2: Example of inappropriate (not usable) culture of A. brasiliensis ATCC 16404 after 7 d of incubation at 30°C 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. 5.4.1.4 Test suspension (“N”) 5.4.1.4.1 Candida albicans The procedure for preparing the Candida albicans test suspension is as follows: a) take 10 ml of diluent (5.2.2.4) and place in a 100 ml flask with 5 g of glass beads (5.3.2.11). Take the working culture (5.4.1.3.1) and transfer loopfuls of the cells into the diluent (5.2.2.4). The cells should be suspended in the diluent rubbing the loop against the wet wall of the flask to dislodge the cells before immersing in the diluent. Shake the flask for 3 min using a mechanical shaker [5.3.2.6b)]. Aspirate the suspension from the glass beads and transfer to a tube; b) adjust the number of cells in the suspension to 1,5 × 107 cfu/ml5) to 5,0 × 107 cfu/ml using diluent (5.2.2.4), estimating the number of cfu by any suitable means. Maintain this test suspension in the water bath at the test temperature 5.5.1.1 a)] and use within 2 h. 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 colourimeter 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.
5) cfu/ml = colony-forming unit(s) per millilitre SIST EN 1657:2016

Figure 3 — Photo No 3: Observation of conidiospores under light microscope: presence of smooth (a) and spiny (b) spores (insufficient spiny spores) SIST EN 1657:2016

Figure 4 — Photo No 4: Observation of conidiospores under light microscope: High concentration of characteristic mature spores with spiny aspect (sufficient spiny spores) c) Adjust the number of spores in the suspension to 1,5 × 107 cfu/ml to 5,0 × 107 cfu/ml using the diluent (5.2.2.4), estimating the number of cfu by any suitable means. Use the suspension within 4 h, maintain in a water bath controlled at 20°C ± 1°C (5.3.2.2). In any case, adjust the temperature according to 5.5.1.4 only immediately before the start of the test (5.5.2 or 5.5.3). The use of a cell counting device for adjusting the number of cells is highly recommended. When using a suitable counting chamber, follow the instructions explicitly. Each laboratory should therefore produce calibration data to establish the relationship between the counts obtained using the counting device and the counts (5.4.1.6) obtained by the pour plate or the spread plate technique. Experienced laboratories found a better fit to the required number of spores when the spore suspension count in the device was 10 % to 50 % higher than the number aimed at. d) 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 about half of each 1,0 ml sample into separate Petri dishes (i.e. in duplicate = four plates) and add 15 ml to 20 ml of melted MEA (5.2.2.3), cooled to (45 ± 1) °C. 2) When using the spread plate technique, spread about one quarter of each 1,0 ml sample on an appropriate number (at least four) of surface dried plates containing MEA (5.2.2.3) (i.e. in duplicate – at least eight plates). For incubation and counting, see 5.4.1.6. 5.4.1.5 Validation suspension (“Nv”) a) To prepare the validation suspension, dilute the test suspension (5.4.1.4.1 and 5.4.1.4.2) with the diluent (5.2.2.4) to obtain the fungal count of 3,0 × 102 cfu/ml to 1,6 × 103 cfu/ml [about one-fourth (1 + 3) of the 10 «4 dilution]. b) For counting, prepare a 10 «1 dilution with diluent (5.2.2.4). Mix [5.3.2.6a)]. Take a sample of 1,0 ml in duplicate and inoculate using the pour plate or the spread plate technique [with Candida albicans, 5.4.1.4.1 c); with Aspergillus brasiliensis, 5.4.1.4.2 d)].For incubation and counting, see 5.4.1.6. SIST EN 1657:2016

contact time t (in min): The obligatory and additional contact times to be tested are specified in Clause 4, Table 1. The allowed deviation for each chosen time is ± 10 s or ±5 s for contact times of 1 min or less. c)
interfering substance: The obligatory interfering substance to be tested is 3,0 g/l bovine albumin (5.2.2.8.2) for low level soiling or 10 g/l bovine albumin plus 10 g/l yeast extract (5.2.2.8.3) for high level soiling or skimmed milk 10 g/l for teat disinfectants (5.2.2.8.4) according to Clause 4, Table 1 and practical applications. Additional interfering substances may be tested according to specific fields of application. d) test organisms: Aspergillus brasiliensis and Candida albicans (Clause 4, Table 1 and 5.2.1). Additional test organisms may be tested. 5.5.1.2 Choice of test method (dilution-neutralization or membrane filtration) The method of choice is the dilution-neutralization method (5.5.2). To determine a suitable neutralizer, carry out the validation of the dilution neutralization method (5.5.2.3, 5.5.2.4 and 5.5.2.5 in connection with 5.5.2.6) using a neutralizer, chosen according to laboratory experience and/or published data. If this neutralizer is not valid, repeat the validation test using an alternative neutralizer taking into account the information given in Annex B. In special circumstances, it may be necessary to add neutralizer to MEA (5.2.2.3). 5.5.1.3 General instructions for validation and control procedures The neutralization and/or removal of the fungicidal and/or fungistatic activity of the product shall be controlled and validated – only for the highest product test concentration – for each of the used test organisms and for each experimental condition (interfering substance, temperature, contact time). These procedures (experimental condition control, neutralizer or filtration control and method validation) shall be performed at the same time with the test and with the same neutralizer – or rinsing liquid – used in the test. SIST EN 1657:2016
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