SIST EN ISO 16212:2017

SLOVENSKI STANDARD
oSIST prEN ISO 16212:2017
01-februar-2017
Kozmetika - Mikrobiologija - Ugotavljanje števila kvasovk in plesni (ISO/FDIS
16212:2017)
Cosmetics - Microbiology - Enumeration of yeast and mould (ISO/FDIS 16212:2017)
Kosmetische Mittel - Mikrobiologie - Zählung von Hefen und Schimmelpilzen (ISO/FDIS
16212:2017)
Cosmétiques - Microbiologie - Dénombrement des levures et des moisissures (ISO/FDIS
16212:2017)
Ta slovenski standard je istoveten z: prEN ISO 16212
ICS:
07.100.40 Kozmetika - mikrobiologija Cosmetics microbiology
oSIST prEN ISO 16212:2017 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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oSIST prEN ISO 16212:2017

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oSIST prEN ISO 16212:2017
FINAL
INTERNATIONAL ISO/FDIS
DRAFT
STANDARD 16212
ISO/TC 217
Cosmetics — Microbiology —
Secretariat: ISIRI
Enumeration of yeast and mould
Voting begins on:
2017­01­02
Cosmétiques — Microbiologie — Dénombrement des levures et des
moisissures
Voting terminates on:
2017­03­27
ISO/CEN PARALLEL PROCESSING
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 SUPPOR TING
DOCUMENTATION.
IN ADDITION TO THEIR EVALUATION AS
Reference number
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO­
ISO/FDIS 16212:2017(E)
LOGICAL, COMMERCIAL AND USER PURPOSES,
DRAFT INTERNATIONAL STANDARDS MAY ON
OCCASION HAVE TO BE CONSIDERED IN THE
LIGHT OF THEIR POTENTIAL TO BECOME STAN­
DARDS TO WHICH REFERENCE MAY BE MADE IN
©
NATIONAL REGULATIONS. ISO 2017

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oSIST prEN ISO 16212:2017
ISO/FDIS 16212:2017(E)

COPYRIGHT PROTECTED DOCUMENT
© ISO 2017, Published in Switzerland
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form
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copyright@iso.org
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oSIST prEN ISO 16212:2017
ISO/FDIS 16212:2017(E)

Contents Page
Foreword .iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principles . 2
4.1 General . 2
4.2 Plate count . 2
4.3 Membrane filtration . 2
5 Diluents, neutralizers and culture media . 3
5.1 General . 3
5.2 Neutralizing diluents and diluents . 3
5.3 Diluent for yeast suspension (tryptone sodium chloride solution) . 4
5.4 Culture media . 4
6 Apparatus and glassware . 5
7 Strain of microorganisms . 5
8 Handling of cosmetic products and laboratory samples . 6
9 Procedure. 6
9.1 General recommendation . 6
9.2 Preparation of the initial suspension . 6
9.2.1 General. 6
9.2.2 Water­miscible products. 6
9.2.3 Water­immiscible products . 6
9.3 Counting methods . 6
9.3.1 Dilutions for counting methods . 6
9.3.2 Plate­count methods . 7
10 Counting of colonies (plate counts and membrane filtration methods) .7
11 Expression of results . 8
11.1 Method of calculation for plate count . 8
11.2 Interpretation . 9
12 Neutralization of the antifungicidal properties of the product .10
12.1 General .10
12.2 Preparation of inoculum .11
12.3 Suitability of counting methods .11
12.3.1 Principle .11
12.3.2 Suitability test of the pour-plate method .11
12.3.3 Suitability of the surface spread method .11
12.3.4 Suitability of the membrane filtration method .11
13 Test report .12
Annex A (informative) Other neutralizing diluents .13
Annex B (informative) Other diluents .15
Annex C (informative) Other culture media .16
Annex D (informative) Neutralizers of antifungicidal activity of preservatives and
rinsing liquids .18
Bibliography .19
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Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non­governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation on the meaning of ISO specific terms and expressions related to conformity assessment,
as well as information about ISO’s adherence to the World Trade Organization (WTO) principles in the
Technical Barriers to Trade (TBT) see the following URL: www.iso.org/iso/foreword.html.
The committee responsible for this document is ISO/TC 217, Cosmetics.
This second edition cancels and replaces the first edition (ISO 16212:2008), of which it constitutes a
minor revision with the following changes:
— in 4.1, “validated” has been changed to “satisfactory”;
— in 9.3.2.1, “validated” has been changed to “demonstrated to be suitable”;
— in 9.3.2.3, “prepared as validated” has been changed to “demonstrated to be suitable”;
— in 11.2.1, “validated” has been changed to “demonstrated to be suitable”;
— in 12.3, “validation” has been changed to “suitability”;
— in 12.3.2, instances of “validation” have been changed to “suitability test”;
— in 12.3.3, the first instance of “validation” has been changed to “suitability” and the second instance
has been changed to “suitability test”;
— in 12.3.4, “validation” has been changed to “suitability”;
— in 12.3.4, “validated” has been changed to “satisfactory”;
— in 12.3.4, “validation” has been changed to “suitability test”;
— in Clause 13 f), “validation” has been changed to “suitability”.
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oSIST prEN ISO 16212:2017
FINAL DRAFT INTERNATIONAL STANDARD ISO/FDIS 16212:2017(E)
Cosmetics — Microbiology — Enumeration of yeast and
mould
1 Scope
This document gives general guidelines for enumeration of yeast and mould present in cosmetics by
counting the colonies on selective agar medium after aerobic incubation.
In order to ensure product quality and safety for consumers, it is advisable to perform an appropriate
microbiological risk analysis so as to determine the types of cosmetic products to which this document
is applicable. Products considered to present a low microbiological risk include those with low water
activity, hydro-alcoholic products, products with extreme pH values, etc.
Because of the large variety of cosmetic products within this field of application, this method might
not be suited to some products in every detail (e.g. certain water-immiscible products). Other methods
(e.g. automated) can be used for the test presented here provided that their equivalence has been
demonstrated or the method has been otherwise validated.
Yeast enumerated can be identified using suitable identification tests, for example, tests described
in the standards listed in the Bibliography. Mould enumerated can be identified by other appropriate
methods, if necessary.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
ISO 21148, Cosmetics — Microbiology — General instructions for microbiological examination
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
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— IEC Electropedia: available at http://www.electropedia.org/
— ISO Online browsing platform: available at http://www.iso.org/obp
3.1
yeast
single-cell fungus, which multiplies mainly vegetatively by budding, able to grow under the test
conditions specified in this document
3.2
mould
mycelium forming microfungus, including spores and conidia, able to grow under the test conditions
specified in this document
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3.3
product
portion of an identified cosmetic product received in the laboratory for testing
3.4
sample
portion of the product (at least 1 g or 1 ml) which is used in the test to prepare the initial suspension
3.5
initial suspension
suspension (or solution) of a sample in a defined volume of an appropriate liquid (diluent, neutralizer,
broth or combination thereof)
3.6
sample dilution(s)
dilution(s) of the initial suspension
4 Principles
4.1 General
This method involves enumeration of colonies on a selective agar medium. The possible inhibition of
fungal growth by the sample shall be neutralized to allow the detection of viable microorganism (see
Reference [5]). In all cases and whatever the methodology, the neutralization of the antifungicidal
properties of the product shall be checked and satisfactory (see References [6], [8] and [9]).
4.2 Plate count
Plate count consists of the following steps.
a) Preparation of poured plates or spread plates, using a specified culture medium, and inoculation of
the plates using a defined quantity of the initial suspension or dilution of the product.
b) Aerobic incubation of the plates at 25 °C ± 2,5 °C for 3 d to 5 d.
c) Counting of the number of colony-forming units (CFU) and calculation of the amount of yeast and
mould per millilitre or per gram of product.
NOTE An alternative condition for incubation is 22,5 °C ± 2,5 °C for 5 d to 7 d using the culture medium
without antibiotic.
4.3 Membrane filtration
Membrane filtration consists of the following steps.
a) Transfer of a suitable amount of the sample, prepared by a valid method, in the filtration apparatus,
wetted with a small volume of an appropriate sterile diluent. Immediate filtration and washing
according to the validated procedure. Transfer of the membrane filter onto the surface of the
specified agar medium as specified in ISO 21148.
b) Aerobic incubation of the membranes at 25 °C ± 2,5 °C for 3 d to 5 d.
c) Counting of the number of colony-forming units (CFU) and calculation of the amount of yeast and
mould per millilitre or per gram of product.
NOTE An alternative condition for incubation is 22,5 °C ± 2,5 °C for 5 d to 7 d using the culture medium
without antibiotic.
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5 Diluents, neutralizers and culture media
5.1 General
General specifications are given in ISO 21148. When water is mentioned in a formula, use distilled
water or purified water as specified in ISO 21148.
The following diluents, neutralizers and culture media are suitable for enumeration of yeasts and
moulds. Other diluents, neutralizers and culture media may be used if they have been demonstrated to
be suitable for use.
5.2 Neutralizing diluents and diluents
5.2.1 General
The diluent is used to disperse the sample. It may contain neutralizers if the sample to be tested
has antifungicidal properties. The efficacy of the neutralization shall be demonstrated before the
determination of the count (see Clause 12). Information relative to suitable neutralizers is given in
Annex D.
5.2.2 Neutralizing diluent
5.2.2.1 Fluid casein digest–soy lecithin–polysorbate 20 medium (SCDLP 20 broth)
5.2.2.1.1 Composition
Pancreatic digest of casein 20,0 g
Soy lecithin 5,0 g
Polysorbate 20 40 ml
Water 960 ml
5.2.2.1.2 Preparation
Dissolve the polysorbate 20 in 960 ml of water by mixing while heating in a water bath at 49 °C ± 2 °C.
Add pancreatic digest of casein and soy lecithin. Heat for about 30 min to effect solution. Mix and
dispense the medium into suitable containers. Sterilize in the autoclave at 121 °C for 15 min. After
sterilization, the pH shall be equivalent to 7,3 ± 0,2 when measured at room temperature.
5.2.2.2 Other neutralizing diluents
Other neutralizing diluents may be used as appropriate (see Annex A and Annex D).
5.2.3 Diluent
5.2.3.1 Fluid A
5.2.3.1.1 Composition
Peptic digest of animal tissue 1,0 g
Water 1 000 ml
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5.2.3.1.2 Preparation
Dissolve 1 g of peptone in water to make 1 l. Heat with frequent agitation. Dispense into suitable
containers. Sterilize in the autoclave at 121 °C for 15 min. After sterilization, the pH shall be equivalent
to 7,1 ± 0,2 when measured at room temperature.
5.2.3.2 Other diluents
Other diluents may be used as appropriate (see Annex B).
5.3 Diluent for yeast suspension (tryptone sodium chloride solution)
5.3.1 Composition
Tryptone, pancreatic digest of casein 1,00 g
Sodium chloride 8,50 g
Water 1 000 ml
5.3.2 Preparation
Dissolve the components in the water by mixing while heating. Dispense into suitable containers.
Sterilize in the autoclave at 121 °C for 15 min. After sterilization, the pH shall be equivalent to 7,0 ± 0,2
when measured at room temperature.
5.4 Culture media
5.4.1 General
Culture media may be prepared as follows or from dehydrated culture media according to the
manufacturer’s instructions. Ready-to-use media may be used when their composition and/or growth
yields are comparable to those of the formulae given herein.
5.4.2 Sabouraud dextrose chloramphenicol agar medium (SDCA)
5.4.2.1 Composition
Dextrose 40,0 g
Peptic digest of animal tissue 5,0 g
Pancreatic digest of casein 5,0 g
Chloramphenicol 0,050 g
Agar 15,0 g
Water 1 000 ml
5.4.2.2 Preparation
Dissolve the components (including the chloramphenicol) or the dehydrated complete medium in the
water by mixing while heating. Dispense the medium into suitable containers. Sterilize in an autoclave
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at 121 °C for 15 min. After sterilization, the pH shall be equivalent to 5,6 ± 0,2 when measured at room
temperature.
NOTE For known and non­contaminated products (with bacteria), the media are used without
chloramphenicol.
5.4.3 Other media
Other media may be used as appropriate (see Annex C).
5.4.4 Agar medium for cultivation of reference strain: Sabouraud dextrose agar medium (SDA)
5.4.4.1 Composition
Dextrose 40,0 g
Peptic digest of animal tissue 5,0 g
Pancreatic digest of casein 5,0 g
Agar 15,0 g
Water 1 000 ml
5.4.4.2 Preparation
Dissolve the components or the dehydrated complete medium in the water by mixing while heating.
Dispense the medium into suitable containers. Sterilize in an autoclave at 121 °C for 15 min. After
sterilization, the pH shall be equivalent to 5,6 ± 0,2 when measured at room temperature.
6 Apparatus and glassware
The laboratory equipment, apparatus and glassware are described in ISO 21148.
7 Strain of microorganisms
For testing the efficacy of neutralizers, one yeast reference strain is used:
1) 2) 3) 4)
— Candida albicans ATCC 10231 or equivalent strain (IP 48.72 or NCPF 3179 or NBRC 1594 or
5) 6)
KCTC 17205 or TISTR 5779) or other equivalent national collection strain.
The selected yeast strain being considered more susceptible to antifungicidal activity than moulds
may be accepted as representative of fungi (yeast and mould) for the validation of the methodology.
However, in case of specific needs, the test for the efficacy of neutralizers may be performed with an
additional mould reference strain, using a suitable protocol for the preparation of a calibrated inoculum
[3]
(e.g. see EN 13624:2003, 5.4.1.4 ).
The culture should be reconstituted according to the procedures provided by the supplier of
reference strain.
1) ATCC = American Type Culture Collection.
2) IP = Institut Pasteur.
3) NCPF = National Collection of Pathogenic Fungi.
4) NBRC = Biological Resource Center, NITE.
5) KCTC = Korean Collection for Type Cultures.
6) TISTR = Thailand Institute of Scientific and Technological Research.
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The strain may be kept in the laboratory in accordance with EN 12353.
8 Handling of cosmetic products and laboratory samples
If necessary, store products to be tested at room temperature.
Do not incubate, refrigerate or freeze products (3.3) and samples (3.4) before or after analysis.
Sampling of cosmetic products to be analysed should be carried out as described in ISO 21148. Analyse
samples as described in ISO 21148 and according to the procedure described in Clause 9.
9 Procedure
9.1 General recommendation
Use sterile material, equipment and aseptic techniques to prepare the sample, initial suspension and
dilutions. In the case of the preparation of the initial suspension, the time that elapses between the end
of the preparation and the moment the inoculum comes into contact with the culture medium shall not
exceed 45 min, unless specifically mentioned in the established protocols or documents.
9.2 Preparation of the initial suspension
9.2.1 General
The initial suspension is prepared from a sample of at least 1 g or 1 ml of the well­mixed product
under test.
Note S, the exact mass or volume of the sample.
The initial suspension is usually a 1:10 dilution. Larger volumes of diluent may be required if high levels
of contamination are expected and/or if antifungicidal properties are still present in the 1:10 dilution.
9.2.2 Water-miscible products
Transfer the sample, S, of product to an appropriate volume (e.g. 9 ml) of neutralizing diluent (5.2.2) or
diluent (5.2.3).
Note d, the dilution factor.
9.2.3 Water-immiscible products
Transfer the sample, S, of product to a suitable container containing a suitable quantity of solubilizing
agent (e.g. polysorbate 80 solution). Disperse the sample within the solubilizing agent and add an
appropriate volume (e.g. 9 ml) of neutralizing diluent (5.2.2) or diluent (5.2.3).
Note d, the dilution factor.
9.3 Counting methods
9.3.1 Dilutions for counting methods
Usually, the initial suspension is the first counted dilution. If needed, additional serial dilutions (e.g.
1:10 dilution) may be performed from the initial suspension using the same diluent (according to the
expected level of contamination of the product).
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Generally, counting is performed using at least two Petri dishes, but it is possible to use only one Petri
dish in case of routine testing, or if counts are performed on successive dilutions of the same sample or
according to previous results.
9.3.2 Plate-count methods
9.3.2.1 Pour-plate method
In Petri dishes 85 mm to 100 mm in diameter, add 1 ml of the initial suspension and/or sample dilution
prepared as demonstrated to be suitable (see Clause 12) and pour 15 ml to 20 ml of the melted agar
medium (5.4.2) kept in a water bath at no more than 48 °C. If larger Petri dishes are used, the amount of
agar medium is increased accordingly.
Mix the initial suspension and/or sample dilution with the medium, carefully rotating or tilting the
plates sufficiently to disperse them. Allow the mixture in the Petri dishes to solidify on a horizontal
surface at room temperature.
9.3.2.2 Surface spread method
In Petri dishes 85 mm to 100 mm in diameter, put 15 ml to 20 ml of the melted agar medium (5.4.2) kept
in a water bath at no more than 48 °C. If larger Petri dishes are used, the volume of the agar is increased
accordingly.
Allow plates to cool and solidify, for example, in a microbiological cabinet or in an incubator. Spread
over the surface of the medium a measured volume of not less than 0,1 ml of the initial suspension
and/or sample dilution prepared as described in Clause 12.
9.3.2.3 Membrane filtration method
Use a membrane having a nominal pore size ≤0,45 µm.
Transfer a suitable amount of the initial suspension or of the sample dilution demonstrated to be
suitable (preferably representing at least 1 g or 1 ml of the product) onto the membrane.
Filter immediately and wash the membrane (follow the suitability test procedure; see Clause 12).
Transfer the membrane onto the surface of the agar medium (5.4.2).
9.3.2.4 Incubation
Unless otherwise stated, invert the inoculated dishes and place them in the incubator set at 25 °C ± 2,5 °C
for 3 d to 5 d or use the alternative condition (see Notes in 4.2 and 4.3). After incubation, the dishes shall,
if possible, be examined immediately. Alternatively, they may be stored, unless otherwise specified, for
up to a maximum of 24 h in the refrigerator at 5 °C ± 3 °C.
NOTE 1 In certain cases, where there is a potential for confusing particles from the product with counted
colonies, it can be useful to prepare duplicate dishes containing the same sample dilutions and agar medium
which are stored in the refrigerator for comparison with incubated dishes.
NOTE 2 An intermediate check can be performed where both yeast and mould are suspected.
10 Counting of colonies (plate counts and membrane filtration methods)
After incubation, count the colonies
— in Petri dishes containing 15 colonies to 150 colonies; if less than 15 colonies are counted, see 11.2.3;
— on membranes cont
...