SIST EN ISO 16212:2017
(Main)Cosmetics - Microbiology - Enumeration of yeast and mould (ISO 16212:2017)
Cosmetics - Microbiology - Enumeration of yeast and mould (ISO 16212:2017)
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 to determine the types of cosmetic products to which this document is
applicable. Products considered to present a low microbiological risk (see ISO 29621) include those
with low water activity or extreme pH values, hydro-alcoholic products, 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 substituted for the tests presented here provided that their equivalence has been
demonstrated or the method has been otherwise shown to be suitable.
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.
Kosmetische Mittel - Mikrobiologie - Zählung von Hefen und Schimmelpilzen (ISO 16212:2017)
Cosmétiques - Microbiologie - Dénombrement des levures et des moisissures (ISO 16212:2017)
L'ISO 16212:2017 donne des lignes directrices générales pour le dénombrement des levures et des moisissures présentes dans les cosmétiques par dénombrement des colonies en milieu gélosé sélectif après une incubation aérobie.
Pour garantir la qualité du produit et la sécurité des consommateurs, il est conseillé d'effectuer une analyse de risque microbiologique appropriée, afin de déterminer les types de produits cosmétiques qui relèvent de l'ISO 16212:2017. Les produits considérés comme présentant un faible risque microbiologique (voir l'ISO 29621) comprennent ceux ayant une faible activité de l'eau ou des valeurs de pH extrêmes, les produits hydro-alcooliques, etc.
En raison de la grande variété de produits cosmétiques entrant dans ce domaine d'application, la présente méthode pourrait ne pas être applicable en tout point à certains produits (par exemple à certains produits non miscibles à l'eau). Il est possible de remplacer les essais présentés ici par d'autres méthodes (par exemple des méthodes automatisées) sous réserve que leur équivalence ait été démontrée ou que la méthode ait été par ailleurs indiquée comme adéquate.
Les levures dénombrées peuvent être identifiées à l'aide d'essais d'identification appropriés, par exemple ceux décrits dans les normes indiquées dans la Bibliographie. Les moisissures dénombrées peuvent être identifiées en utilisant d'autres méthodes appropriées, si nécessaire.
Kozmetika - Mikrobiologija - Ugotavljanje števila kvasovk in plesni (ISO 16212:2017)
V tem dokumentu so podane splošne smernice za ugotavljanje števila kvasovk in plesni v kozmetičnih izdelkih s štetjem kolonij na selektivnem agarskem gojišču po aerobni inkubaciji.
Za zagotovitev kakovosti in varnosti izdelkov za stranke je priporočljivo izvesti ustrezno mikrobiološko analizo tveganja, s katero se določijo vrste kozmetičnih izdelkov, za katere se uporablja ta dokument. Izdelki, ki po ocenah predstavljajo nizko mikrobiološko tveganje (glej ISO 29621), vključujejo izdelke z nizko aktivnostjo vode ali s skrajnimi vrednostmi pH, hidro-alkoholne izdelke itd.
Zaradi velike raznolikosti kozmetičnih izdelkov na tem področju uporabe ta metoda morda ni primerna za nekatere izdelke (npr. tiste, ki se ne mešajo z vodo). Druge metode (npr. avtomatizirane) lahko nadomestijo v tem dokumentu opisani preskusi, če je bila dokazana njihova enakovrednost ali je bila metoda kako drugače opredeljena kot primerna.
Naštete kvasovke je mogoče identificirati z ustreznimi identifikacijskimi preskusi, na primer s preskusi, opisanimi v standardih, navedenih v bibliografiji. Naštete plesni je mogoče identificirati z drugimi ustreznimi metodami, če je to potrebno.
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 16212:2017
01-november-2017
1DGRPHãþD
SIST EN ISO 16212:2011
Kozmetika - Mikrobiologija - Ugotavljanje števila kvasovk in plesni (ISO
16212:2017)
Cosmetics - Microbiology - Enumeration of yeast and mould (ISO 16212:2017)
Kosmetische Mittel - Mikrobiologie - Zählung von Hefen und Schimmelpilzen (ISO
16212:2017)
Cosmétiques - Microbiologie - Dénombrement des levures et des moisissures (ISO
16212:2017)
Ta slovenski standard je istoveten z: EN ISO 16212:2017
ICS:
07.100.40 Kozmetika - mikrobiologija Cosmetics microbiology
SIST EN ISO 16212:2017 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN ISO 16212:2017
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SIST EN ISO 16212:2017
EN ISO 16212
EUROPEAN STANDARD
NORME EUROPÉENNE
July 2017
EUROPÄISCHE NORM
ICS 07.100.40 Supersedes EN ISO 16212:2011
English Version
Cosmetics - Microbiology - Enumeration of yeast and
mould (ISO 16212:2017)
Cosmétiques - Microbiologie - Dénombrement des Kosmetische Mittel - Mikrobiologie - Zählung von
levures et des moisissures (ISO 16212:2017) Hefen und Schimmelpilzen (ISO 16212:2017)
This European Standard was approved by CEN on 26 April 2017.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this
European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references
concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN
member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by
translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management
Centre has the same status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2017 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 16212:2017 E
worldwide for CEN national Members.
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SIST EN ISO 16212:2017
EN ISO 16212:2017 (E)
Contents Page
European foreword . 3
2
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SIST EN ISO 16212:2017
EN ISO 16212:2017 (E)
European foreword
This document (EN ISO 16212:2017) has been prepared by Technical Committee ISO/TC 217
"Cosmetics" in collaboration with Technical Committee CEN/TC 392 “Cosmetics” the secretariat of
which is held by AFNOR.
This European Standard shall be given the status of a national standard, either by publication of an
identical text or by endorsement, at the latest by January 2018 and conflicting national standards shall
be withdrawn at the latest by January 2018.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
This document supersedes EN ISO 16212:2011.
According to the CEN-CENELEC Internal Regulations, the national standards organizations of the
following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,
Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia,
France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta,
Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and the United Kingdom.
Endorsement notice
The text of ISO 16212:2017 has been approved by CEN as EN ISO 16212:2017 without any modification.
3
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SIST EN ISO 16212:2017
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SIST EN ISO 16212:2017
INTERNATIONAL ISO
STANDARD 16212
Second edition
2017-06
Cosmetics — Microbiology —
Enumeration of yeast and mould
Cosmétiques — Microbiologie — Dénombrement des levures et des
moisissures
Reference number
ISO 16212:2017(E)
©
ISO 2017
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SIST EN ISO 16212:2017
ISO 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
or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior
written permission. Permission can be requested from either ISO at the address below or ISO’s member body in the country of
the requester.
ISO copyright office
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Tel. +41 22 749 01 11
Fax +41 22 749 09 47
copyright@iso.org
www.iso.org
ii © ISO 2017 – All rights reserved
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SIST EN ISO 16212:2017
ISO 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 Water-miscible products. 6
9.2.3 Water-immiscible products . 6
9.3 Counting methods . 6
9.3.1 Dilutions for counting methods . 6
9.3.2 Plate-count 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
© ISO 2017 – All rights reserved iii
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SIST EN ISO 16212:2017
ISO 16212:2017(E)
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 non-governmental, 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 voluntary nature of standards, 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: w w w . i s o .org/ iso/ foreword .html.
This document was prepared by Technical Committee ISO/TC 217, Cosmetics.
This second edition cancels and replaces the first edition (ISO 16212:2008), of which it constitutes a
minor revision. The changes compared to the previous edition are as follows:
— in the Scope, “see ISO 29621” has been added and the reference has been added to the Bibliography;
— in the Scope, “used” has been changed to “substituted” and “validated” has been changed to “shown
to be suitable”;
— in 4.1, “validated” has been changed to “demonstrated”;
— in 4.3, “by a valid method” has been changed to “as described in Clause 12” and “validated procedure”
has been replaced by “described procedure”;
— in 5.1, “specifications” has been changed to “instructions”;
— in 5.2.3.1.2, “peptone” has been changed to “peptic digest of animal tissue”;
— in Clause 7, “validation” has been changed to “suitability”;
— 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 according to” has been changed to “demonstrated to be suitable for”;
— in 12.3, “validation” has been changed to “suitability”;
— in 12.3.2, instances of “validation” have been changed to “suitability test” and “validated” has been
changed to “satisfactory”;
— in 12.3.3, the first instance of “validation” has been changed to “suitability” and the second instance
has been changed to “suitability test”; “validated” has been changed to “satisfactory”;
iv © ISO 2017 – All rights reserved
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SIST EN ISO 16212:2017
ISO 16212:2017(E)
— in 12.3.4, the first instance of “validation” has been changed to “suitability” and the second instance
has been changed to “suitability test”; “validated” has been changed to “satisfactory”;
— in Clause 13 f), “validation” has been changed to “suitability”;
— in A.1, B.1 and C.1, “validated” has been changed to “demonstrated to be suitable”.
© ISO 2017 – All rights reserved v
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SIST EN ISO 16212:2017
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SIST EN ISO 16212:2017
INTERNATIONAL STANDARD ISO 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 to determine the types of cosmetic products to which this document is
applicable. Products considered to present a low microbiological risk (see ISO 29621) include those
with low water activity or extreme pH values, hydro-alcoholic products, 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 substituted for the tests presented here provided that their equivalence has been
demonstrated or the method has been otherwise shown to be suitable.
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:
— ISO Online browsing platform: available at http:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
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
© ISO 2017 – All rights reserved 1
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SIST EN ISO 16212:2017
ISO 16212:2017(E)
3.3
product
portion of an identified cosmetic product received in the laboratory for testing
3.4
sample
portion of the product (3.3) (at least 1 g or 1 ml) that is used in the test to prepare the initial suspension
3.5
initial suspension
suspension (or solution) of the sample (3.4) in a defined volume of an appropriate enrichment broth
3.6
sample dilution
dilution of the initial suspension (3.5)
4 Principles
4.1 General
This method involves enumeration of colonies on a selective agar medium. The possible inhibition of
[5]
fungal growth by the sample shall be neutralized to allow the detection of viable microorganisms .
In all cases and whatever the methodology, the neutralization of the antifungicidal properties of the
[6][8][9]
product shall be checked and demonstrated .
4.2 Plate count
Plate count consists of the following steps.
— 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.
— Aerobic incubation of the plates at 25 °C ± 2,5 °C for 3 d to 5 d.
— 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.
— Transfer a suitable amount of the sample, prepared as described in Clause 12, in the filtration
apparatus, wetted with a small volume of an appropriate sterile diluent. Filter immediately and
wash according to the described procedure (see 12.3.4). Transfer the membrane filter onto the
surface of the specified agar medium as specified in ISO 21148.
— Aerobic incubation of the membranes at 25 °C ± 2,5 °C for 3 d to 5 d.
— 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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SIST EN ISO 16212:2017
ISO 16212:2017(E)
5 Diluents, neutralizers and culture media
5.1 General
General instructions are given in ISO 21148. When water is mentioned in this document, 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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SIST EN ISO 16212:2017
ISO 16212:2017(E)
5.2.3.1.2 Preparation
Dissolve 1 g of peptic digest of animal tissue 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
4 © ISO 2017 – All rights reserved
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SIST EN ISO 16212:2017
ISO 16212:2017(E)
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 non-contaminated 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 suitability 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 13624:2013, 5.4.1.4 ).
The culture should be reconstituted according to the procedures provided by the supplier of the
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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SIST EN ISO 16212:2017
ISO 16212:2017(E)
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
...
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)
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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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oSIST prEN ISO 16212:2017
ISO/FDIS 16212:2017(E)
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
...
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