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
Principle
A sample of the product, either as delivered or diluted with hard water, is combined with a test suspension of fungi, such as yeast cells or mould spores, in the presence of an interfering substance This mixture is kept at a temperature of (20 ± 1) °C for a duration of 15 minutes ± 10 seconds, which are the required test conditions After this contact period, an aliquot is taken, and the fungicidal and/or fungistatic activity in this sample is promptly neutralized or suppressed using a validated method, with dilution-neutralization being the preferred approach.
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
The test evaluates fungicidal activity using vegetative cells of Candida albicans and spores of Aspergillus niger, while yeasticidal activity is assessed solely with vegetative cells of Candida albicans, adhering to obligatory test conditions.
5.1.3 Additional and optional contact times and temperatures are specified Additional test organisms can be used.
Materials and reagents
The fungicidal activity shall be evaluated using the following strains as test organisms: 1)
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:
The ATCC numbers represent the collection identifiers for strains provided by the American Type Culture Collection (ATCC) This information is intended to assist users of this standard and does not imply any endorsement of the named product by CEN.
Saccharomyces cerevisiae var diastaticus DSM 70487
When utilizing additional test organisms, it is essential to incubate them under optimal growth conditions, including temperature, time, atmosphere, and media, as detailed in the test report The suitability of these organisms must be verified if they do not match the specified strains Furthermore, if the additional test organisms are not classified at a reference center, their identification characteristics should be documented These organisms must also be maintained by the testing laboratory or national culture collection under a reference for a period of five years.
This standard specifies that all weights of chemical substances pertain to anhydrous salts While hydrated forms can be utilized as alternatives, it is essential to adjust the weights accordingly to account for the differences in molecular weight.
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 enhance reproducibility in culture media preparation, it is advisable to utilize commercially available dehydrated materials, ensuring strict adherence to the manufacturer's instructions for optimal results.
NOTE 2 For each culture medium and reagent, a limitation for use should be fixed
The water shall be freshly glass-distilled water and not demineralized water
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
!Malt extract agar, consisting of:
For optimal results, use food-grade malt extract, such as Cristomalt powder from Difal, or a similar product that is not overly purified and not solely composed of maltose, like malt extract from OXOID If there are issues achieving at least 75% spiny spores, refer to section 5.4.1.4.2 for guidance.
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 may be necessary to add neutralizer to the MEA Annex B gives guidance on the neutralizers that may be used."
Tryptone sodium chloride solution, consisting of:
Tryptone, pancreatic digest of casein 1,0 g
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
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
This European Standard provides information for user convenience and does not endorse the specified product Users may opt for equivalent products if they can demonstrate comparable results.
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:
To prepare solution A, dissolve 19.84 g of magnesium chloride (MgCl₂) and 46.24 g of calcium chloride (CaCl₂) in water and dilute to a final volume of 1,000 ml Sterilize the solution using membrane filtration or autoclaving; however, be aware that autoclaving may result in liquid loss, necessitating the addition of water to reach 1,000 ml under aseptic conditions Store the prepared solution in the refrigerator for a maximum of one month.
prepare solution B: dissolve 35,02 g sodium bicarbonate (NaHCO3) in water (5.2.2.2) and dilute to
1 000 ml Sterilize by membrane filtration (5.3.2.7) Store the solution in the refrigerator (5.3.2.8) for no longer than one week;
To prepare the solution, begin by adding 600 ml to 700 ml of water into a 1,000 ml volumetric flask, followed by the addition of 6.0 ml of solution A and 8.0 ml of solution B After mixing, dilute the mixture to a final volume of 1,000 ml with water Ensure that the pH of the hard water is maintained at 7.0 ± 0.2 when measured at (20 ± 1)°C If adjustments are needed, use a sodium hydroxide (NaOH) solution at approximately 40 g/l (about 1 mol/l) or a hydrochloric acid (HCl) solution at approximately 36.5 g/l (about 1 mol/l) to achieve the desired pH.
The hard water shall be freshly prepared under aseptic conditions and used within 12 h
When preparing product test solutions, the addition of the product to hard water results in varying final water hardness levels across test tubes However, the final hardness remains below 300 mg/l of calcium carbonate (CaCO3) in each test tube.
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
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 milk-powder in 1 000 ml water (5.2.2.2) Heat for 30 min at (105 ± 3) °C [or
The final concentration of reconstituted milk in the test procedure (5.5) is 10,0 g/l of reconstituted milk
Dehydrated yeast extract for bacteriology, shall be prepared as follows :
prepare a 100 g/l solution in water (5.2.2.2), adjust to pH 7,0 ± 0,2 with sodium hydroxide (NaOH);
The final concentration of yeast extract in the test procedure (5.5) is 10,0 g/l
5.2.2.8.6 Sucrose (beverage, soft drink industries)
Prepare a 100 g/l solution of sucrose in water (5.2.2.2), sterilize by membrane filtration (5.3.2.7)
The final concentration of sucrose in the test procedure (5.5) is 10,0 g/l
5.2.2.8.7 pH 5,0 and pH 9,0 buffer solutions (cleaning in place )
The test report will detail the buffer solution used and document the pH values It is essential to maintain the final pH in the test tubes, which includes the test organisms and product, within the range of 5.0 ± 0.2 or 9.0 ± 0.2.
5.2.2.8.8 Sodium dodecyl sulphate (cosmetic area )
Prepare a 50 g/l solution of sodium dodecyl sulphate (C12H25NaO4S) in water (5.2.2.2) Sterilize in the autoclave [5.3.2.1a)]
The final concentration of sodium dodecyl sulphate in the test procedure (5.5) is 5,0 g/l.
Apparatus and glassware
Ensure all glassware and apparatus components that will contact culture media, reagents, or samples are sterilized, except for those provided sterile This can be achieved through moist heat in an autoclave or dry heat in a hot air oven.
5.3.2 Usual microbiological laboratory equipment 2) and in particular, the following:
For effective sterilization, specific apparatus is required: a) for moist heat sterilization, an autoclave must be maintained at a temperature of 121 ± 3 °C for at least 15 minutes; b) for dry heat sterilization, a hot air oven should be set at 180 ± 5 °C for a minimum of 30 minutes, at 170 ± 5 °C for at least 1 hour, or at 160 ± 5 °C for a minimum of 2 hours.
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)
5.3.2.6 Shaker a) Electromechanical agitator, e.g Vortex® mixer 3) b) Mechanical shaker
5.3.2.7 Membrane filtration apparatus constructed of a material compatible with the substances to be filtered
The apparatus must include a filter holder with a minimum volume of 50 ml, compatible with filters ranging from 47 mm to 50 mm in diameter and featuring a pore size of 0.45 μm This setup is essential for the sterilization of hard water, bovine albumin, and sucrose, particularly when employing the membrane filtration method.
The vacuum source must ensure a consistent filtration flow rate to achieve an even distribution of micro-organisms on the membrane To avoid prolonged filtration, the device should be calibrated to filter 100 ml of rinsing liquid within 20 to 40 seconds.
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
2) Disposable sterile equipment is an acceptable alternative to reusable glassware
Vortex® is a commercially available product that serves as a suitable example This information is provided for the convenience of users of this standard and does not imply any endorsement by CEN.
5.3.2.11 Glass beads, 3 mm to 4 mm in diameter
5.3.2.13 Fritted filter, with porosity of 40 μm to 100 μm according to ISO 4793
Preparation of test organism suspensions and product test solutions
5.4.1 Test organism suspensions (test and validation suspension)
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
To establish the working culture of Candida albicans, initiate a subculture from the stock culture by streaking onto MEA slopes or plates and incubate for 42 to 48 hours Subsequently, prepare a second subculture from the first, following the same method and incubation period If desired, a third subculture can be generated from the second The second and any subsequent subcultures serve as the working cultures.
If preparing the second subculture on a specific day is not feasible, a 72-hour subculture can be utilized for further subculturing, as long as it has been maintained in the incubator throughout the 72-hour duration.
Never produce and use a fourth subculture
5.4.1.3.2 Aspergillus brasiliensis (previously A niger) (mould)
For the cultivation of Aspergillus brasiliensis (formerly A niger), utilize only the initial subculture grown on MEA in Petri dishes or flasks with ventilated caps, incubating for a duration of 7 to 9 days It is important to avoid further subculturing and to refrain from stacking the Petri dishes during incubation to ensure optimal temperature homogenization.
At the conclusion of the incubation period, all cultures should exhibit a dark brown or black surface Cultures displaying occasional small white or grey areas may still be retained for further observation (refer to Figure 1).
Figure 1 — Photos N°1: A brasiliensis ATCC 16404 after 7 d of incubation at 30 °C (right cultures)
Figure 2 — Photo N°2: A brasiliensis ATCC 16404 after 7 d of incubation at 30 °C (wrong culture)"
5.4.1.3.3 Other test organisms (yeasts or moulds)
Any deviations from the standard method of culturing yeast or mold, as well as the preparation of suspensions, must be documented in the test report, along with the justifications for these changes.
To prepare the Candida albicans test suspension, begin by adding 10 ml of diluent to a 100 ml flask containing 5 g of glass beads Transfer loopfuls of cells from the working culture into the diluent, ensuring the cells are dislodged by rubbing the loop against the flask's wet wall Shake the flask for 3 minutes using a mechanical shaker, then aspirate the suspension and transfer it to another tube Adjust the cell concentration to between 1.5 x 10^7 cfu/ml and 5.0 x 10^7 cfu/ml using the diluent, estimating the cfu as needed Finally, maintain the test suspension in a water bath at the specified test temperature and use it within 2 hours.
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
Colony-forming units per millilitre (cfu/ml) are determined by measuring the optical density of test organisms, which typically ranges from 0.200 to 0.350 A colorimeter can be used as an effective alternative for this measurement For accurate counting, prepare dilutions of the test suspension at 10^-5 and 10^-6 using the specified diluent, and ensure thorough mixing as outlined in the procedure.
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
When employing the spread plate technique, it is essential to distribute each 1.0 ml sample, divided into roughly equal portions, onto a minimum of two surface-dried plates that contain MEA (5.2.2.3).
For incubation and counting see 5.4.1.6
5.4.1.4.2 Aspergillus brasiliensis (previously A niger) (mould)
The procedure for preparing the Aspergillus brasiliensis 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
To prepare a conidiospore suspension, detach the spores from the culture surface using a glass rod or spatula, then transfer the suspension into a flask Gently shake the flask by hand for one minute while adding 5 g of glass beads Next, filter the suspension through a fritted filter Finally, conduct a microscopic examination at 400x magnification immediately after preparation to observe the results.
1) the presence of a high concentration (at least 75 % of spiny spores) of characteristic mature spores i.e spiny spores (versus smooth spores) [see Figure 3 and Figure 4];
2) the absence of spore germination (check at least ten fields of view);
3) if germinated spores are present, discard the suspension;
4) the absence of mycelia fragments (check at least ten fields of view)
If mycelia are present, proceed to a 2 nd fritted filtration
If mycelia are still present, discard the suspension
If the production of spiny spores falls below 75%, it may be attributed to the Aspergillus brasiliensis culture or the media utilized In such cases, it is essential to source the culture from an alternative collection and/or select a MEA from a different supplier.
Figure 3 — Photo N°3 Observation of conidiospores under light microscope: presence of smooth (a) and spiny (b) spores (wrong suspension)
Observation of conidiospores under a light microscope reveals a high concentration of characteristic mature spores with a spiny appearance Adjust the spore concentration in the suspension to between 1.5 x 10^7 cfu/ml and 5.0 x 10^7 cfu/ml using the specified diluent, estimating the number of cfu by appropriate methods It is essential to use the suspension within 4 hours, maintaining it in a water bath at a controlled temperature of 20 °C ± 1 °C Additionally, ensure that the temperature is adjusted according to the specified guidelines immediately before commencing the test.
The use of a cell counting device for adjusting the number of cells is highly recommended When using a suitable counting chamber, it is essential to follow the instructions explicitly
Each laboratory must generate calibration data to correlate the counts from the counting device with those obtained through the pour plate or spread plate technique Experienced laboratories have observed that a spore suspension count in the device should be 10% to 50% higher than the target count for optimal results For accurate counting, prepare 10^-5 and 10^-6 dilutions of the test suspension using the specified diluent and mix thoroughly.
Take a sample of 1,0 ml of each dilution in duplicate and inoculate using the pour plate or thespread plate technique
When employing the pour plate technique, transfer approximately 0.5 ml of each sample into separate Petri dishes, resulting in duplicates for a total of four plates Then, add 15 to 20 ml of melted MEA, which has been cooled to the appropriate temperature.
When employing the spread plate technique, distribute approximately one quarter of each 1.0 ml sample onto a minimum of four surface-dried plates containing MEA, ensuring that at least eight plates are used in total for duplication.
For incubation and counting see 5.4.1.6."
To prepare the validation suspension (Nv), dilute the test suspension as outlined in sections 5.4.1.4.1 and 5.4.1.4.2 with the specified diluent from section 5.2.2.4 This process aims to achieve a fungal count ranging from 3.0 x 10² cfu/ml to 1.6 x 10³ cfu/ml, which is approximately one-fourth of the total dilution.
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,
5.4.1.4.1c); with Aspergillus niger, 5.4.1.4.2d)] c) For incubation and counting, see 5.4.1.6
5.4.1.6 Incubation and counting of the test and the validation suspensions
Procedure for assessing the fungicidal or yeasticidal activity of the product
5.5.1.1 Experimental conditions (obligatory and additional)
In addition to the required temperature, contact time, interfering substances, and test organisms, further experimental conditions may be chosen based on the intended practical application of the product Specifically, the temperature (θ) should be measured in degrees Celsius (°C).
the obligatory temperature to be tested is θ= 20 °C;
additional temperatures may be chosen from 4 °C, 10 °C or 40 °C;
the allowed deviation for each chosen temperature is ± 1 °C; b) contact time t (in min):
the obligatory contact time to be tested is t = 15 min;
additional contact times may be chosen from 1 min, 5 min, 30 min or 60 min;
the allowed deviation for each chosen contact time is ± 10 s (except for 1 min: ± 5 s); c) interfering substance:
the obligatory interfering substance to be tested is 0,3 g/l bovine albumin (5.2.2.8.2) for clean conditions or 3 g/l bovine albumin (5.2.2.8.3) for dirty conditions according to practical applications;
additional interfering substances as given in 5.2.2.8 shall be chosen according to the field of application specified for the product; d) test organisms (5.2.1):
the obligatory test organisms for testing fungicidal activity are Candida albicans and Aspergillus niger;
the obligatory test organism for testing yeasticidal activity is Candida albicans
Additional test organisms may be tested
5.5.1.2 Choice of test method (dilution-neutralization or membrane filtration)
The preferred technique for this process is the dilution-neutralization method (5.5.2) To identify an appropriate neutralizer, it is essential to validate the dilution-neutralization method (5.5.2.3, 5.5.2.4, and 5.5.2.5 in conjunction with 5.5.2.6) using a neutralizer selected based on laboratory experience and existing literature.
If this neutralizer is not valid, repeat the validation test using an alternative neutralizer taking into account the information given in Annex B
If both neutralizers are found to be invalid, the membrane filtration method (5.5.3) may be used
NOTE 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 removal of fungicidal or fungistatic activity must be controlled and validated for the highest product test concentration across all test organisms and experimental conditions, including interfering substances, temperature, and contact time These procedures, which encompass experimental condition control, neutralizer or filtration control, and method validation, should be conducted simultaneously with the test using the same neutralizer or rinsing liquid employed in the test.
In the case of ready-to-use-products use water (5.2.2.2) instead of hard water
In cases where a neutralizer is added to MEA due to neutralization issues, it is essential that the MEA used for validation and control procedures contains the same quantity of the neutralizer for consistency in testing.
Before testing, ensure that all reagents, including product test solutions, test suspension, validation suspension, diluent, hard water, and interfering substances, are equilibrated to the test temperature θ using a water bath Verify that the temperature of the reagents is stabilized at θ.
The neutralizer (5.2.2.5) or the rinsing liquid (5.2.2.6) and water (5.2.2.2) shall be equilibrated at a temperature of (20 ± 1) °C
In the case of ready-to-use-products, water (5.2.2.2) shall be additionally equilibrated to θ
5.5.1.5 Precautions for manipulation of test organisms
Do not touch the upper part of the test tube sides when adding the test or the validation suspensions (5.4.1)
5) For a graphical representation of this method see C.1
The test and the control and validation procedures (5.5.2.2 through 5.5.2.5) shall be carried out in parallel and separately for each experimental condition (5.5.1.1)
5.5.2.2 Test "Na" – determination of fungicidal or yeasticidal concentrations
To determine fungicidal or yeasticidal concentrations, begin by pipetting 1.0 ml of the interfering substance into a tube Next, add 1.0 ml of the test suspension and immediately start the stopwatch Mix the contents and place the tube in a water bath set to the selected test temperature for 2 minutes, with a tolerance of ± 10 seconds.
At the conclusion of the designated time, add 8.0 ml of one of the product test solutions and restart the stopwatch Mix the solution thoroughly and place the tube in a water bath maintained at a specific temperature for the selected contact time Just before the contact time ends, mix the solution again After the contact time, take a 1.0 ml sample of the test mixture and transfer it into a tube containing 8.0 ml of neutralizer and 1.0 ml of water Mix the contents and place the tube in a controlled water bath.
At a temperature of (20 ± 1)°C, allow a neutralization period of 5 minutes ± 10 seconds After this time, mix the solution thoroughly and promptly take a 1.0 ml sample of the neutralized test mixture "Na," which includes the neutralizer, product test solution, interfering substance, and test suspension This sample should be inoculated in duplicate using either the pour plate or spread plate technique.
1) When using the pour plate technique, pipette each 1,0 ml sample into separate Petri dishes and add
15 ml to 20 ml of melted MEA (5.2.2.3), cooled to (45 ± 1) °C
When employing the spread plate technique, it is essential to distribute each 1.0 ml sample into approximately equal portions across at least two surface-dried plates that contain MEA (5.2.2.3).
For incubation and counting, refer to section 5.5.2.6 Simultaneously conduct procedures a) and b) with the alternative product test solutions Additionally, implement procedures a) to c) while adhering to other mandatory and, if applicable, supplementary experimental conditions outlined in section 5.5.1.1.
5.5.2.3 Experimental conditions control "A" – validation of the selected experimental conditions and/or verification of the absence of any lethal effect in the test conditions
To validate the experimental conditions and ensure there are no lethal effects, follow this procedure: Pipette 1.0 ml of the interfering substance into a tube, then add 1.0 ml of the validation suspension Start the stopwatch immediately, mix thoroughly, and place the tube in a water bath controlled at θ for 2 minutes ± 10 seconds.
At the conclusion of the designated time, add 8.0 ml of hard water, or water for ready-to-use products, and restart the stopwatch Mix the solution and place the tube in a water bath maintained at a specific temperature for a set duration Just before the time is up, mix the solution again After the time has elapsed, take a duplicate sample of 1.0 ml from this mixture, referred to as "A," and inoculate it using either the pour plate or spread plate technique.
For incubation and counting see 5.5.2.6
5.5.2.4 Neutralizer control "B" – verification of the absence of toxicity of the neutralizer
To verify the absence of toxicity in the neutralizer, pipette 8.0 ml of the neutralizer and 1.0 ml of water into a tube, then add 1.0 ml of the validation suspension Start a stopwatch upon addition, mix the contents, and incubate the tube in a water bath at (20 ± 1)°C for 5 minutes ± 10 seconds, mixing again just before the time is up Finally, take a 1.0 ml sample of the mixture in duplicate and inoculate using either the pour plate or spread plate technique.
For incubation and counting see 5.5.2.6
5.5.2.5 Method validation "C" – dilution-neutralization validation
To validate the dilution neutralization method, begin by pipetting 1.0 ml of the interfering substance into a tube, followed by 1.0 ml of the diluent Start a stopwatch and add 8.0 ml of the product test solution at the highest concentration Mix thoroughly and place the tube in a water bath at a controlled temperature for a specified time Just before the time elapses, mix again After the time is complete, transfer 1.0 ml of the mixture into a tube containing 8.0 ml of neutralizer, restarting the stopwatch upon addition Mix and incubate in a water bath at (20 ± 1)°C for 5 minutes Then, add 1.0 ml of the validation suspension, starting the stopwatch again, and mix Incubate for 30 minutes at the same temperature, mixing just before the end Finally, take a 1.0 ml sample of the mixture in duplicate and inoculate using either the pour plate or spread plate technique.
For incubation and counting see 5.5.2.6
5.5.2.6 Incubation and counting of the test mixture and the control and validation mixtures
To incubate and count the test mixture along with the control and validation mixtures, follow these steps: Incubate the plates for 42 to 48 hours, and discard any plates that are uncountable for any reason Finally, count the colony-forming units (cfu) on the plates to determine the total cfu.
Experimental data and calculation
5.6.1 Explanation of terms and abbreviations
5.6.1.1 Overview of the different suspensions and test mixtures
N and Nv represent the fungal suspensions, Na represents the fungicidal test mixture, A (experimental conditions control), B (neutralizer or filtration control), C (method validation) represent the different control test mixtures
N , Nv , N 0 , Nv 0 , Na and A , B and C represent the number of cells counted per ml in the different test mixtures in accordance with Table 1
Table 1 — Number of cells counted per ml in the different test mixtures
Number of cells per ml in the fungal suspensions
Number of cells per ml in the test mixtures at the beginning of the contact time (time = 0 )
Number of survivors per ml in the test mixtures at the end of the contact time t (A) or 5 min (B) or 30 min (C)
N 0 (= N/10) Na (before neutralization or filtration)
All experimental data are reported as Vc values:
in the dilution-neutralization method (test and controls), a Vc value is the number of colony-forming units counted per 1,0 ml sample
in the membrane filtration method, a Vc value is the number of colony-forming units counted per 0,1 ml sample of test mixture “Na” and per 1,0 ml sample in the controls
The first step in the calculation is the determination of the Vc values, the second the calculation of N, N 0, Na,
Nv, Nv 0, A, B and C The third step is the calculation of the reduction R (5.8.1)
The Vc values are established based on specific colony count limits for fungi on agar plates, which typically range from 15 to 150 colonies for moulds and 15 to 300 colonies for yeasts According to European Standards, a 10% deviation is permissible, adjusting the limits to 14-165 for moulds and 14-330 for yeasts For membrane counts, the upper limits differ, set at 50 for moulds and 150 for yeasts, with the 10% deviation resulting in adjusted limits of 55 for moulds and 165 for yeasts.
The lower limit of 14 is established due to increased variability in smaller sample volumes (1 ml or 0.1 ml), which can lead to inaccurate results This limit pertains specifically to the sample rather than the counting on a single plate; for instance, a 1 ml sample with counts of 3 cfu, 8 cfu, and 5 cfu across three plates results in a Vc value of 16 Conversely, the upper limits of 55, 165, and 330 indicate the imprecision in counting confluent colonies and the effects of nutrient depletion on growth, and these limits apply only to the counting on individual plates For the counting of the test suspension "N," the validation suspension "Nv," and all counts using the dilution-neutralization method, it is essential to determine and document the Vc values based on the number of plates utilized per 1 ml sample.
NOTE If more than one plate per 1 ml sample has been used to determine the Vc value, the countings per plate should be noted
If the count on a single plate exceeds 165 (or 330), it should be reported as “>165” (or “>330”) In cases where multiple plates are used for a 1 ml sample and at least one plate shows a count greater than 165 (or 330), the Vc value should be reported as “> sum of the counts” (for example, if the counts are “> 165, 132, 144”, report it as “> 441”).
If a Vc value is lower than 14, report the number but substitute by “165, >165
Na duplicate Vc values (membrane-filtration, Aspergillus niger): 40, >55
Na duplicate Vc values (two spread plates per 1,0 ml sample, Candida albicans): > 660, 600
5.6.2.5 Calculation of Nv and Nv 0
The number of cells per milliliter in the validation suspension (Nv) is ten times greater than the counts represented by the Vc values, attributed to a dilution factor of 10^-1.
At the start of the contact time (time 0), Nv 0 represents the number of cells per ml in mixtures "A," "B," and "C." This value is calculated as one-tenth of the average Vc values of Nv, as referenced in section 5.4.1.6c).
Calculate Nv and Nv 0 using the following Equations:
Nv 0= c/n (4) where c is the sum of Vc values taken into account; n is the number of Vc values taken into account
The numbers of survivors in the experimental conditions are represented by A, B, and C, corresponding to the control "A" (5.5.2.3 or 5.5.3.3), neutralizer control "B" (5.5.2.4), and filtration control "B" (5.5.3.4), as well as method validation "C" (5.5.2.5 or 5.5.3.5) These values are measured at the end of the contact time \( t \) for A, and at defined intervals of 5 minutes for B and 30 minutes for C The results reflect the mean of the Vc values for the mixtures A, B, and C.
Calculate A , B and C using the following Equation (5):
A, B, C = c/n (5) where c is the sum of Vc values taken into account; n is the number of Vc values taken into account.
Verification of methodology
all results meet the criteria of 5.7.3; and
the requirements of 5.8.2 are fulfilled
5.7.2 Control of weighted mean counts
For results derived from the weighted mean of two consecutive dilutions, the ratio of the mean values must fall between 5 and 15 Any results that are below this lower limit are recorded as the lower limit value of 14, while results exceeding the upper limit are noted as the upper limit value.
EXAMPLE For N 10 -5 dilution: > 165 + 150 cfu; 10 -6 dilution: 20 + 25 cfu; (165 + 150) / (20 + 25) = 315/45 = 7,0 therefore between 5 and 15
When the plate counts exceed the established limits for determining V c values, it is essential to calculate the weighted mean using only the V c values that fall within the counting limits for the N calculation.
For each test organism check that: a) N is between 1,5 × 10 7 and 5,0 × 10 7 (7,17 ≤ lg N ≤ 7,70)
N 0 is between 1,5 × 10 6 and 5,0 × 10 6 (6,17 ≤ lg N 0 ≤ 6,70) b) Nv0 is between 30 and 160 (3,0 × 10 1 and 1,6 × 10 2 )
(Nv is between 3,0 × 10 2 and 1,6 × 10 3 ) c) A,B,C are equal to or greater than 0,5 × Nv 0. d) control of weighted mean counts (5.7.2): quotient is not lower than 5 and not higher than 15
For A brasiliensis (previously A niger) spore suspension check the presence of a high concentration of spiny spores (at least 75 %)."
Expression of results and precision
The reduction ( R = N 0 /Na ) is expressed in logarithm
For each test organism record the number of cfu/ml in the test suspension N (5.6.2.3) and in the test Na (5.6.2.4) Calculate N 0 (5.6.2.3)
For each product concentration and each experimental condition, calculate and record the decimal log reduction (lg) separately using the Equation (6):
For the controls and validation of the dilution-neutralization method or membrane filtration method, record Nv 0
(5.6.2.5), the results of A, B and C (5.6.2.6) and their comparison with Nv 0[5.7.3c)]
5.8.2 Control of active and non-active product test solution (5.4.2)
At least one concentration per test [5.5.2.2a) - c) or 5.5.3.2a) - c)] shall demonstrate a reduction of 4 lg or more and at least one concentration shall demonstrate a lg reduction of less than 4
5.8.3 Limiting test organism and fungicidal/yeasticidal concentration
For each test organism, identify the minimum concentration of the product that meets the criteria (lg R ≥ 4) The limiting test organism is defined as the one that requires the highest concentration, indicating it is the least susceptible to the product under the specified experimental conditions.
The lowest concentration of the product active on the limiting test organism is the fungicidal concentration determined according to this European Standard
The minimum concentration of the product that effectively inhibits Candida albicans, defined as the yeasticidal concentration, is established according to European Standard guidelines, with a requirement of lg R ≥ 4.
The methodology's precision, as determined by a statistical analysis from a collaborative study for EN 1657, recommends conducting seven replicates for a precision of ± 1 lg in reduction (refer to Annex E) Replication involves performing the complete test procedure with separately prepared test and validation suspensions, which may be limited to the test organism The mean of the replicate results must show a reduction of at least 4 lg and should be calculated and recorded accordingly.
Interpretation of results - conclusion
According to the chosen experimental conditions (obligatory or obligatory and additional) the fungicidal concentrations determined according to this European Standard may differ (Clause 4)
5.9.2 Fungicidal activity for general purposes
The product meets the EN 1650 standard if it achieves a reduction of at least 4 log within 15 minutes at 20 °C, using a selected interfering substance that simulates clean or dirty conditions, with test organisms being Candida albicans and Aspergillus niger.
The fungicidal concentration for general purposes is the concentration active on the limiting strain
5.9.3 Fungicidal activity for specific purposes
The fungicidal concentration for a specific purpose is defined as the concentration of a tested product that achieves a reduction of at least 4 log in a valid test under selected conditions To qualify, the product must meet the EN 1650 standard for fungicidal activity under mandatory testing conditions It is important to note that the fungicidal concentration for specific purposes may be lower than that established for general purposes.
5.9.4 Yeasticidal activity for general purposes
The product meets the EN 1650 standard for yeasticidal activity if it achieves a minimum reduction of 4 log within 15 minutes at 20 °C, using an appropriate interfering substance that simulates either clean or dirty conditions, with Candida albicans as the test organism.
The yeasticidal concentration for general purposes is the concentration active on Candida albicans
5.9.5 Yeasticidal activity for specific purposes
The yeasticidal concentration for a specific purpose is defined as the concentration of a tested product that achieves a minimum reduction of 4 log in a valid test under selected conditions To meet this criterion, the product must comply with the EN 1650 standard for yeasticidal activity under mandatory testing conditions It is important to note that the yeasticidal concentration for specific purposes may be lower than that established for general purposes.
Test report
The test report shall refer to this document (EN 1650) mentioning if fungicidal activity or only yeasticidal activity has been tested
The test report shall state, at least, the following information: a) identification of the testing laboratory; b) identification of the client; c) identification of the sample:
2) batch number and — if available — expiry date;
3) manufacturer – if not known: supplier;
6) product diluent recommended by the manufacturer for use;
7) active substance(s) and their concentration(s) (optional);
8) appearance of the product; d) test method and its validation:
1) If the dilution-neutralization method is used, full details of the test for validation of the neutralizer shall be given;
When employing the membrane filtration method, it is essential to provide comprehensive details of the procedure undertaken to validate its use Additionally, the experimental conditions must be clearly outlined.
1) date(s) of test (period of analysis);
2) diluent used for product test solution (hard water or distilled water);
3) product test concentrations (= desired test concentrations according to 5.4.2);
8) stability and appearance of the mixture during the procedure (note the formation of any precipitate or flocculant);
11) identification of the fungal strains used; f) test results:
2) evaluation of fungicidal or yeasticidal activity;
3) number of replicates per test organism; g) special remarks; h) conclusion; i) locality, date and identified signature
NOTE An example of a typical test report is given in Annex D
Referenced strains in national collections
Examples of neutralizers of the residual antimicrobial activity of chemical disinfectants and antiseptics and rinsing liquids
Important Neutralizers of the residual antimicrobial activity of chemical disinfectants and antiseptics and rinsing liquids shall be validated according to the prescriptions of the standard
Chemical compounds able to neutralize residual antimicrobial activity
Examples of suitable neutralizers and of rinsing liquids (for membrane filtration methods) a)
Quaternary ammonium compounds and fatty amines
Lecithin, Saponin, Polysorbate 80, Sodium dodecyl sulphate, Ethylene oxide condensate of fatty alcohol (non- ionic surfactants) b)
- Polysorbate 80, 30 g/l + sodium dodecyl sulphate, 4 g/l + lecithin, 3 g/l
- Ethylene oxide condensate of fatty alcohol, 3 g/l + lecithin, 20 g/l + polysorbate 80, 5 g/l
Rinsing liquid : tryptone, 1 g/l + NaCl, 9 g/l; polysorbate 80, 5 g/l
Lecithin c) , Saponin, Polysorbate 80 - Polysorbate 80, 30 g/l + saponin, 30 g/l + lecithin, 3 g/l
Rinsing liquid : tryptone, 1 g/l + NaCl, 9 g/l; polysorbate 80, 5 g/l
(Chlorine, iodine, hydrogen peroxide, peracetic acid, hypochlorites, etc…)
Catalase [for hydrogen peroxide or products releasing hydrogen peroxide]
- Sodium thiosulphate, 3 g/l to 20 g/l + polysorbate 80, 30 g/l + lecithin, 3 g/l
- Polysorbate 80, 30 g/l + lecithin, 3 g/l + L-histidine, 1 g/l (or + glycine, 1 g/l)
- Polysorbate 80, 30 g/l + saponin, 30 g/l + L-histidine, 1 g/l (or + glycine, 1 g/l)
Rinsing liquid : polysorbate 80, 5 g/l + L-histidine, 0,5 g/l (or + glycine, 1 g/l)
Phenolic and related compounds: orthophenylphenol, phenoxyethanol, triclosan, phenylethanol, etc…
Ethylene oxide condensate of fatty alcohol b)
- Ethylene oxide condensate of fatty alcohol, 7 g/l + lecithin, 20 g/l, + polysorbate 80, 4 g/l
Rinsing liquid : tryptone, 1 g/l + NaCl, 9 g/l; polysorbate 80, 5 g/l
Alcohols Lecithin, Saponin, Polysorbate 80 e) - Polysorbate 80, 30 g/l + saponin, 30 g/l + lecithin, 3 g/l
Rinsing liquid : tryptone, 1 g/l + NaCl, 9 g/l; polysorbate 80, 5 g/l
Analytical quality products, such as TWEEN 80, are commercially available and comply with the European Pharmacopoeia, although this does not imply endorsement by CEN The pH of the tested product may require adjustment using a neutralizer or rinsing liquid, which can be prepared with a phosphate buffer (e.g., 0.25 mol/l potassium dihydrogen phosphate adjusted to pH 7.2 ± 0.2 with sodium hydroxide) The carbon chain length of the products ranges from C 12 to C 18 carbon atoms, with egg-derived components being preferable over soya The toxic effects of sodium thiosulphate vary among different microorganisms For neutralizing short-chain alcohols (less than C 5), simple dilution may suffice, but caution is advised if the alcohol-based products contain additional antimicrobial agents.
NOTE 1 Other neutralizer mixtures may be required for products containing more than one antimicrobial agent
NOTE 2 The concentrations of the various neutralizing compounds or of the neutralizer as such may not be adequate to neutralize high concentrations of the products
Graphical representations of dilution-neutralization method and membrane filtration method
Example of a typical test report
NOTE 1 All names and significations in Annex D are imaginary apart from those used in this European Standard
NOTE 2 Test reports for yeasticidal activity should be entitled “EN 1650, YEASTICIDAL ACTIVITY”, and be presented in the same format
NOTE 3 Only the test results of one replicate for Aspergillus niger and for Candida albicans are given as an example -
Fax ++011-57 83 62-19 e-mail:h.h.Q.lab@net.com
EN 1650, FUNGICIDAL ACTIVITY (obligatory and additional conditions) Client: Centipede Formulations Inc., Markkleeberg / Euroland
Name of the product: Z Batch number: 91-71-51
Manufacturer or - if not known - supplier: Centipede Formulations Inc (manufacturer)
Storage conditions (temp and other): Room temperature, darkness
Appearance of the product: Liquid, clear, yellowish
Active substance(s) and their concentration(s): Not indicated
Product diluent recommended by the manufacturer for use: Potable water
Date of delivery of the product: 2006-03-16 Dates of tests: see “Test results” (attached)
Product diluent: hard water Concentrations of the product tested: see “Test results” (attached)
Obligatory conditions: test-organisms: Candida albicans ATCC 10231 and Aspergillus niger ATCC 16404; test temperature: 20 °C; contact time: 15 min; interfering substance: 3,0 g/l bovine albumin = dirty conditions;
Additional conditions: test organism: Saccharomyces yyy;
Test temperature: 10 °C; contact time: 60 min; interfering substance: 10,0 g/l reconstituted milk;
Special remarks regarding the results:
All controls and validation were within the basic limits
At least one concentration of the product demonstrated a lg reduction of less than 4 lg
No precipitate during the test procedure (test mixtures were homogeneous)
Test results: see attached sheets
For the product Z (batch 91-71-51), the fungicidal concentration for general purposes determined according to the
EN 1650 standard (obligatory conditions) under dirty conditions is:
In a study involving a 1% (v/v) concentration, the mean reduction observed across six replicates with the limiting test organism Aspergillus niger was 1.2 x 10^4 Additionally, Candida albicans was tested once and demonstrated a reduction of 4 log or more at a concentration lower than that required for Aspergillus niger.
For the product Z (batch 91-71-51), the yeasticidal concentration for specific purposes determined according to the
EN 1650 standard at 10°C, with 60 min contact time, with milk as interfering substance using Saccharomyces yyy as test organism is:
Alexandra May, MD, PhD, Scientific Director
! Test results (fungicidal suspension test)
EN 1650 (Phase 2, step 1) Product-name: Z Batch No.: 91-71-51.…….….………
Dilution neutralization method Pour plate Spread plate Number of plates / ml
Membrane filtration method Rinsing liquid: ………
The test was conducted at a temperature of 20 °C, using bovine albumin at a concentration of 3.0 g/l as an interfering substance The test organism employed was Aspergillus niger ATCC 16404, with an incubation temperature of 30 °C The internal laboratory number for this test is QS 58/00, dated August 5, 2006, and was conducted by the responsible person, Fang, who also provided their signature The diluent used for the product test solutions was hard water, and the appearance of the product test solutions was clear.
Presence of a high concentration of A brasiliensis spiny spores in the spore suspension yes no
Conditions control (A) Neutralizer or filtration control (B) Method validation (C) Product conc.: 10 ml/l
30 ≤ x of Nv0 ≤ 160 ? yes no x of A is ≥ 0,5x x of Nv0? yes no x of B is ≥ 0,5x x of Nv0? yes no x of C is ≥ 0,5x x of Nv0? yes no
Test suspension and Test Test-suspension
Conc of the product % Vc1 Vc2 Na = x x10 lg Na lg R Contact time (min)
Remarks: Counting per plate for (N): 10 -5 = 60 + 79, 72+ 82, 10 -6 = 4 + 10, 2 + 15
Vc = count per ml (one plate or more) x = average of Vc1and Vc2 (1 + 2 duplicate) x wm = weighted mean of x
R = reduction (lg R = lg N0 – lg Na)
Test results (fungicidal suspension test)
EN 1650 (Phase 2, step 1) Product-name: Z Batch No.: 91-71-51.…….……… Remarks:
Dilution neutralization method Pour plate Spread plate Number of plates 2 / ml Neutralizer: Lecithin 3,0 g/l in diluent ……… Membrane filtration method Rinsing liquid: ………
Test temperature: 10°C Interfering substances: bovine albumin 3,0 g/l ……… Test organism: Candida albicans ATCC 10231 ………… Incubation temperature:.30°C
Internal lab no: QS 58/08 Date of test:.2006-08-27 Responsible person: Fang Signature: Fang Diluent used for product test solutions: hard water Appearance of the product test solutions: clear………
Conditions control (A) Neutralizer or filtration control
30 ≤ x of Nv0 ≤ 160 ? yes no x of A is ≥ 0,5x x of Nv0? yes no x of B is ≥ 0,5x x of Nv0? yes no x of C is ≥ 0,5x x of Nv0? yes no
Test suspension and Test Test-suspension
(N and N 0 ): N Vc1 Vc2 x wm = 259,54 x 10 5 ; lg N = 7,41
Conc of the product % Vc1 Vc2 Na = x x10 lg Na lg R contact- time (min)
Remarks: Counts per spread plate (2 / ml): 1 Nv0 38+38, 34+38 2 A 60+22, 52+29 3 B 32+49, 32+40 4 C 34 +
Vc = count per ml (one plate or more) x = average of Vc1and Vc2 (1 + 2 duplicate) x wm = weighted mean of x
R = reduction (lg R = lg N0 – lg Na)
Precision of the test result
A collaborative study conducted from 1997 to 1999, known as ANDISTAND, aimed to assess the precision of a testing method across various laboratories This research included 12 laboratories from different European countries, with each lab performing three replicates of the test.
Although the interfering substance was different (EN 1657-high soiling) the results can be extrapolated to this European Standard (EN 1650)
The study utilized dilution neutralization and membrane filtration methods to test the efficacy of sodium dichlorisocyanurate and phenol against Aspergillus niger and Candida albicans Detailed results and statistical evaluations are documented in CEN/TC 216 HWG N 121 + N.
The agreement between laboratories, expressed in terms of fungicidal effect (reduction R = 4 lg), is very good at low and high concentrations, but less good at intermediate levels
The theoretical reduction factors were used to replace uncountable data, particularly when counts fell below the lower limit of 15 viable colonies These factors varied significantly, ranging from a maximum of 3.33 × 10^4, indicating countable data, to 5.00 × 10^8, representing no surviving colonies, which corresponded to log reductions of 4.52 and 6.70, respectively To account for variability, three theoretical reduction factors were selected for each replicate, specifically 4.5, 5.6, and 6.7 in logarithmic terms.
When colony counts exceeded the upper limit of 150 viable colonies, the reduction factor ranged from 1.00 × 10^0 (indicating no reduction) to 1.00 × 10^3 (the minimum countable value), resulting in log reduction values of 0.00 and 3.00, respectively The selected theoretical reduction factors for this analysis were 0.0, 1.5, and 3.0.
All these theoretical values were collected and are shown in Table E.1
Table E.1 — Theoretical reduction factors used for fungi (in logarithmic terms)
Below the lower limit (15) Above the upper limit (150)
Variance analyses highlighted significant results in reduction factors under various tested conditions, focusing on both "within" and "between" laboratory variability Notably, differences in reduction factors were primarily observed at medium and high dilutions of the tested products Some significant findings were influenced by outliers, with certain laboratories reporting markedly different reduction factors compared to their peers In other instances, identifying outliers proved challenging, yet significant differences stemmed from substantial "within lab" variability.
The estimated standard deviation for sodium dichlorisocyanurate ranged from 0.89 to 2.42, while for phenol, it varied from 0.47 to 1.89, indicating that the "between" variability was generally higher than the "within" variability, which ranged from 0.63 to 1.80 However, in certain instances, the "between" dispersion was lower than the "within" dispersion, likely due to the data replacement techniques employed, which may have overestimated the "within" variability and underestimated the "between" variation.
The estimation of precision for the reduction factors was based on "inside" variability, considering two hypotheses: a "worst" case with maximum variability (σ = 1.66) and an average dispersion (σ = 1.23) The precision also varied with sample size and confidence level, with a 90% probability used for calculations With three replicates, the "worst" case resulted in a precision of ± 2.80, while the "mean" case yielded ± 2.07 To achieve a precision target of ± 1 lg reduction, ten replicates were necessary for the "worst" case and seven for the "mean" case.
X Sample size (number of replicates)
Figure E.1— Precision of the reduction factor obtained with fungi (in logarithmic terms)
Table E.2 — Precision of the reduction factors obtained for fungi (in logarithmic terms)
The precision of the tests may vary when different test organisms, products, or interfering substances are evaluated Nonetheless, it is expected that the precision will generally remain within a similar range across these variations.
[1] European Pharmacopoeia (EP), Edition 1997 supplement 2000, Water for injections