Cosmetics - Microbiology - Detection of Escherichia coli (ISO 21150:2015)

ISO 21150:2015 gives general guidelines for the detection and identification of the specified microorganism Escherichia coli in cosmetic products. Microorganisms considered as specified in this International Standard might differ from country to country according to national practices or regulations.
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 International Standard is applicable. Products considered to present a low microbiological (see ISO 29621) risk include those with low water activity, hydro-alcoholic products, extreme pH values, etc.
The method described in this International Standard is based on the detection of Escherichia coli in a non-selective liquid medium (enrichment broth), followed by isolation on a selective agar medium. Other methods may be appropriate, depending on the level of detection required.
NOTE          For the detection of Escherichia coli, subcultures can be performed on non-selective culture media followed by suitable identification steps (e.g. using identification kits).
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 International Standards (ISO 18415) may be appropriate. Other methods (e.g. automated) can be substituted for the test presented here provided that their equivalence has been demonstrated or the method has been otherwise shown to be suitable.

Kosmetische Mittel - Mikrobiologie - Nachweis von Escherichia coli (ISO 21150:2015)

Diese Internationale Norm gibt allgemeine Anleitungen für den Nachweis und die Identifizierung des festgelegten Mikroorganismus Escherichia coli in kosmetischen Mitteln. Die in dieser Internationalen Norm festgelegten Mikroorganismen können in Übereinstimmung mit nationalen Praktiken oder Regelungen von Land zu Land unterschiedlich sein.
Um die Qualität des Produkts und die Sicherheit für Verbraucher sicherzustellen, ist es ratsam, eine geeignete mikrobiologische Risikoanalyse zur Bestimmung der Arten von kosmetischen Mitteln durchzuführen, für die diese Internationale Norm anwendbar ist. Produkte, die ein geringes mikrobiologisches Risiko (siehe ISO 29621) darstellen, umfassen jene mit geringer Wasseraktivität, alkoholisch wässrige Produkte, Produkte mit extremen pH Werten usw.
Das in dieser Internationalen Norm beschriebene Verfahren basiert auf dem Nachweis von Escherichia coli in einem nicht selektiven Flüssigmedium (Anreicherungsbouillon), gefolgt von einer Isolation auf einem selektiven Agarmedium. In Abhängigkeit von der erforderlichen Nachweisgrenze können andere Verfahren angemessen sein.
ANMERKUNG   Für den Nachweis von Escherichia coli kann eine Subkultivierung auf nicht selektiven Nährmedien erfolgen, gefolgt von geeigneten Schritten zur Identifizierung (z. B. unter Anwendung von Identifizierungskits).
Wegen der großen Vielfalt an kosmetischen Mitteln innerhalb dieses Anwendungsbereichs, ist dieses Verfahren möglicherweise für einige Produkte nicht in allen Einzelheiten geeignet (z. B. bestimmte mit Wasser nicht mischbare Produkte). Andere Internationale Normen (ISO 18415) können geeignet sein. Ersatzweise können für die hier aufgeführten Untersuchungen andere Verfahren (z. B. automatisierte) zur Anwendung kommen, voraus¬gesetzt, dass deren Gleichwertigkeit nach¬gewiesen oder sich das Verfahren anderweitig als geeignet erwiesen hat.

Cosmétiques - Microbiologie - Détection d'Escherichia coli (ISO 21150:2015)

ISO 21150:2015 donne des lignes directrices générales pour la détection et l'identification du microorganisme spécifié Escherichia coli dans les produits cosmétiques. Les microorganismes considérés comme spécifiés dans la présente Norme internationale peuvent différer d'un pays à l'autre suivant les pratiques ou les réglementations nationales.
Pour garantir la qualité du produit et la sécurité des consommateurs, il est conseillé d'effectuer une analyse appropriée du risque microbiologique afin de déterminer les types de produits cosmétiques qui relèvent de la présente Norme internationale. Les produits considérés comme présentant un faible risque microbiologique (voir ISO 29621) comprennent ceux ayant une faible activité de l'eau, les produits hydroalcooliques, ceux ayant des valeurs de pH extrêmes, etc.
La méthode décrite dans la présente Norme internationale est basée sur la détection d'Escherichia coli dans un milieu liquide non sélectif (bouillon d'enrichissement), suivie d'un isolement sur un milieu gélosé sélectif. D'autres méthodes peuvent être appropriées en fonction du niveau de détection exigé.
NOTE          Pour la détection d'Escherichia coli, il est possible de réaliser des subcultures sur des milieux de culture non sélectifs, puis de procéder aux étapes appropriées d'identification (par exemple en utilisant des kits d'identification).
En raison de la grande variété de produits cosmétiques relevant de ce domaine d'application, il se peut que cette méthode ne soit pas adaptée en tous points à certains produits (par exemple aux produits non miscibles à l'eau). D'autres Normes internationales (ISO 18415) peuvent être appropriées. Il est possible de remplacer l'essai présenté 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.

Kozmetika - Mikrobiologija - Ugotavljanje prisotnosti bakterije Escherichia coli (ISO 21150:2015)

Ta mednarodni standard podaja splošne smernice za ugotavljanje prisotnosti in prepoznavanje
mikroorganizma Escherichia coli v kozmetičnih izdelkih. Mikroorganizmi, ki so upoštevani, kot so določeni v tem mednarodnem standardu, se lahko v različnih državah zaradi državnih praks ali predpisov razlikujejo. Za namen zagotovitve 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 mednarodni standard. Izdelki, za katere se meni, da predstavljajo nizko mikrobiološko (glejte ISO 29621) tveganje, vključujejo tiste z nizko aktivnostjo vode, hidro-alkoholne izdelke, skrajne vrednosti pH itd. Metoda, opisana v tem mednarodnem standardu, temelji na ugotavljanju prisotnosti Escherichia coli v neselektivnem tekočem gojišču (obogatitven bujon), ki mu sledi izolacija na selektivnem agarskem gojišču. Ustrezne so lahko tudi druge metode, odvisno od zahtevane ravni ugotavljanja prisotnosti. OPOMBA: Za namen ugotavljanja prisotnosti mikroorganizma Escherichia coli je mogoče precepljene kulture vzgojiti v neselektivnem gojišču kultur, čemur sledijo ustrezni koraki prepoznavanja (npr. uporaba kompletov za prepoznavanje).
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). Uporabljajo se lahko drugi mednarodni standardi (ISO 18415). Druge metode (npr. avtomatske) je mogoče zamenjati za tukaj predstavljene preskuse, če je bila dokazana njihova enakovrednost ali je bila metoda drugače dokazana za primerno.

General Information

Status
Published
Publication Date
15-Dec-2015
Withdrawal Date
29-Jun-2016
Technical Committee
Drafting Committee
Current Stage
6060 - Definitive text made available (DAV) - Publishing
Start Date
16-Dec-2015
Completion Date
16-Dec-2015

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Standards Content (Sample)

SLOVENSKI STANDARD
SIST EN ISO 21150:2016
01-marec-2016
1DGRPHãþD
SIST EN ISO 21150:2009
Kozmetika - Mikrobiologija - Ugotavljanje prisotnosti bakterije Escherichia coli
(ISO 21150:2015)
Cosmetics - Microbiology - Detection of Escherichia coli (ISO 21150:2015)
Kosmetische Mittel - Mikrobiologie - Nachweis von Escherichia coli (ISO 21150:2015)
Cosmétiques - Microbiologie - Détection d'Escherichia coli (ISO 21150:2015)
Ta slovenski standard je istoveten z: EN ISO 21150:2015
ICS:
07.100.40 Kozmetika - mikrobiologija Cosmetics microbiology
SIST EN ISO 21150:2016 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 21150:2016

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SIST EN ISO 21150:2016


EN ISO 21150
EUROPEAN STANDARD

NORME EUROPÉENNE

December 2015
EUROPÄISCHE NORM
ICS 07.100.99; 71.100.70 Supersedes EN ISO 21150:2009
English Version

Cosmetics - Microbiology - Detection of Escherichia coli
(ISO 21150:2015)
Cosmétiques - Microbiologie - Détection d'Escherichia Kosmetische Mittel - Mikrobiologie - Nachweis von
coli (ISO 21150:2015) Escherichia coli (ISO 21150:2015)
This European Standard was approved by CEN on 26 September 2015.

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, 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
© 2015 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 21150:2015 E
worldwide for CEN national Members.

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SIST EN ISO 21150:2016
EN ISO 21150:2015 (E)
Contents Page
European foreword . 3

2

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SIST EN ISO 21150:2016
EN ISO 21150:2015 (E)
European foreword
This document (EN ISO 21150:2015) 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 June 2016, and conflicting national standards shall be
withdrawn at the latest by June 2016.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent
rights.
This document supersedes EN ISO 21150:2009.
This document has been prepared under a mandate given to CEN by the European Commission and the
European Free Trade Association.
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, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and the United Kingdom.
Endorsement notice
The text of ISO 21150:2015 has been approved by CEN as EN ISO 21150:2015 without any modification.
3

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SIST EN ISO 21150:2016
INTERNATIONAL ISO
STANDARD 21150
Second edition
2015-12-01
Cosmetics — Microbiology —
Detection of Escherichia coli
Cosmétiques — Microbiologie — Détection d’Escherichia coli
Reference number
ISO 21150:2015(E)
©
ISO 2015

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SIST EN ISO 21150:2016
ISO 21150:2015(E)

COPYRIGHT PROTECTED DOCUMENT
© ISO 2015, 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
Ch. de Blandonnet 8 • CP 401
CH-1214 Vernier, Geneva, Switzerland
Tel. +41 22 749 01 11
Fax +41 22 749 09 47
copyright@iso.org
www.iso.org
ii © ISO 2015 – All rights reserved

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SIST EN ISO 21150:2016
ISO 21150:2015(E)

Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Diluents and culture media . 2
5.1 General . 2
5.2 Diluent for the bacterial suspension (tryptone sodium chloride solution) . 3
5.2.1 General. 3
5.2.2 Composition . 3
5.2.3 Preparation . 3
5.3 Culture media . 3
5.3.1 General. 3
5.3.2 Agar medium for the suitability test (see Clause 11) [soybean-casein
digest agar medium (SCDA) or tryptic soy agar (TSA)] . 3
5.3.3 Enrichment broth . 4
5.3.4 Selective agar medium for isolation of Escherichia coli . 4
5.3.5 Selective agar medium for confirmation of Escherichia coli . 5
6 Apparatus and glassware . 6
7 Strains of microorganisms . 6
8 Handling of cosmetic products and laboratory samples . 6
9 Procedure. 6
9.1 General recommendation . 6
9.2 Preparation of the initial suspension in the enrichment broth . 6
9.2.1 General. 6
9.2.2 Water-miscible products. 7
9.2.3 Water-immiscible products . 7
9.2.4 Filterable products . 7
9.3 Incubation of the inoculated enrichment broth . 7
9.4 Detection and identification of Escherichia coli . 7
9.4.1 Isolation . 7
9.4.2 Identification of Escherichia coli . 7
10 Expression of the results (detection of Escherichia coli) . 8
11 Neutralization of the antimicrobial properties of the product . 8
11.1 General . 8
11.2 Preparation of inoculum . 8
11.3 Suitability of the detection method . 8
11.3.1 Procedure . 8
11.3.2 Interpretation of suitability test results . 9
12 Test report . 9
Annex A (informative) Other enrichment broths .10
Annex B (informative) Neutralizers of antimicrobial activity of preservatives and
rinsing liquids .13
Bibliography .14
© ISO 2015 – All rights reserved iii

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SIST EN ISO 21150:2016
ISO 21150:2015(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 meaning of ISO specific terms and expressions related to conformity
assessment, as well as information about ISO’s adherence to the WTO principles in the Technical
Barriers to Trade (TBT) see the following URL: Foreword - Supplementary information
The committee responsible for this document is ISO/TC 217, Cosmetics.
This second edition cancels and replaces the first edition (ISO 21150:2006), which has been
technically revised.
iv © ISO 2015 – All rights reserved

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ISO 21150:2015(E)

Introduction
Microbiological examinations of cosmetic products are carried out according to an appropriate
microbiological risk analysis in order to ensure their quality and safety for consumers.
Microbiological risk analysis depends on several parameters such as the following:
— potential alteration of cosmetic products;
— pathogenicity of microorganisms;
— site of application of the cosmetic product (hair, skin, eyes, mucous membranes);
— type of users (adults, children under 3 years).
For cosmetics and other topical products, the detection of skin pathogens such as Staphylococcus
aureus, Pseudomonas aeruginosa and Candida albicans may be relevant because they can cause skin
or eye infections. The detection of other kinds of microorganism might be of interest since these
microorganisms (including indicators of faecal contamination e.g. Escherichia coli) suggest hygienic
failure during the manufacturing process.
© ISO 2015 – All rights reserved v

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SIST EN ISO 21150:2016

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SIST EN ISO 21150:2016
INTERNATIONAL STANDARD ISO 21150:2015(E)
Cosmetics — Microbiology — Detection of Escherichia coli
1 Scope
This International Standard gives general guidelines for the detection and identification of the specified
microorganism Escherichia coli in cosmetic products. Microorganisms considered as specified in this
International Standard might differ from country to country according to national practices or regulations.
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
International Standard is applicable. Products considered to present a low microbiological (see
ISO 29621) risk include those with low water activity, hydro-alcoholic products, extreme pH values, etc.
The method described in this International Standard is based on the detection of Escherichia coli in
a non-selective liquid medium (enrichment broth), followed by isolation on a selective agar medium.
Other methods may be appropriate, depending on the level of detection required.
NOTE For the detection of Escherichia coli, subcultures can be performed on non-selective culture media
followed by suitable identification steps (e.g. using identification kits).
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 International
Standards (ISO 18415) may be appropriate. Other methods (e.g. automated) can be substituted for the
test presented here provided that their equivalence has been demonstrated or the method has been
otherwise shown to be suitable.
2 Normative references
The following documents, in whole or in part, are normatively referenced in this document and are
indispensable for its application. For dated references, only the edition cited applies. For undated
references, the latest edition of the referenced document (including any amendments) applies.
ISO 21148:2005, 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.
3.1
product
portion of an identified cosmetic product received in the laboratory for testing
3.2
sample
portion of the product (at least 1 g or 1 ml) that is used in the test to prepare the initial suspension
3.3
initial suspension
suspension (or solution) of the sample in a defined volume of an appropriate enrichment broth
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SIST EN ISO 21150:2016
ISO 21150:2015(E)

3.4
sample dilution
dilution of the initial suspension
3.5
specified microorganism
aerobic mesophilic bacterium or yeast that is undesirable in a cosmetic product and is recognized as a
skin pathogen species that may be harmful for human health or as an indication of hygienic failure in
the manufacturing process
3.6
Escherichia coli
gram-negative rod, motile, smooth colonies
Note 1 to entry: The main characteristics for identification are catalase positive, oxidase negative, fermentation
of lactose, production of indole, growth on selective medium containing bile salts with characteristic colonies.
Note 2 to entry: Escherichia coli can be isolated from the moist environmental sources (air, water, soil) and is a
faecal contamination indicator.
3.7
enrichment broth
non-selective liquid medium containing suitable neutralizers and/or dispersing agents and
demonstrated to be suitable for the product under test
4 Principle
The first step of the procedure is to perform an enrichment by using a non-selective broth medium to
increase the number of microorganisms without the risk of inhibition by the selective ingredients that
are present in selective/differential growth media.
The second step of the test (isolation) of the test is performed on a selective medium followed by
identification tests.
The possible inhibition of microbial growth by the sample shall be neutralized to allow the detection
[1]
of viable microorganisms. In all cases and whatever the methodology, the neutralization of the
antimicrobial properties of the product shall be checked and demonstrated (see Clause 11).
5 Diluents and culture media
5.1 General
General instructions are given in ISO 21148. When water is mentioned in this International Standard,
use distilled water or purified water as specified in ISO 21148.
The enrichment broth is used to disperse the sample and to increase the initial microbial population. It
may contain neutralizers if the specimen to be tested has antimicrobial properties. The efficacy of the
neutralization shall be demonstrated (see Clause 11). Information relative to suitable neutralizers is
given in Annex B.
The enrichment broth (5.3.3.1) or any of the ones listed in Annex A is suitable for checking the presence
of Escherichia coli in accordance with this International Standard provided that they have been
demonstrated to be suitable in accordance with Clause 11.
Other diluents and culture media may be used if it has been demonstrated that they are suitable for use.
2 © ISO 2015 – All rights reserved

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ISO 21150:2015(E)

5.2 Diluent for the bacterial suspension (tryptone sodium chloride solution)
5.2.1 General
The diluent is used for the preparation of bacterial suspension used for the suitability test procedure
(see Clause 11).
5.2.2 Composition
— Tryptone, pancreatic digest of casein 1,0 g
— Sodium chloride 8,5 g
— Water 1 000 ml
5.2.3 Preparation
Dissolve the components in water by mixing while heating. Dispense into suitable containers. Sterilize
in the autoclave at 121 °C for 15 min.
After sterilization and cooling down, the pH shall be equivalent to 7,0 ± 0,2 when measured at room
temperature.
5.3 Culture media
5.3.1 General
Culture media may be prepared using the descriptions provided below or from dehydrated culture
media, according to the instructions from the manufacturer. The instructions provided by the supplier
of the media should be followed.
NOTE Ready-to-use media can be used when their composition and/or growth yields are comparable to
those of the formulae given herein.
5.3.2 Agar medium for the suitability test (see Clause 11) [soybean-casein digest agar medium
(SCDA) or tryptic soy agar (TSA)]
5.3.2.1 Composition
— pancreatic digest of casein 15,0 g
— papaic digest of soybean meal 5,0 g
— sodium chloride 5,0 g
— agar 15,0 g
— water 1 000 ml
5.3.2.2 Preparation
Dissolve the components or the dehydrated complete medium in the water by mixing while heating.
Dispense the medium into suitable containers. Sterilize in the autoclave at 121 °C for 15 min.
After sterilization and cooling down, the pH shall be equivalent to 7,3 ± 0,2 when measured at room
temperature.
© ISO 2015 – All rights reserved 3

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ISO 21150:2015(E)

5.3.3 Enrichment broth
5.3.3.1 Eugon LT 100 broth
5.3.3.1.1 General
This medium contains ingredients which neutralize inhibitory substances present in the sample:
lecithin and polysorbate 80 and dispersing agent: octoxynol 9.
5.3.3.1.2 Composition
— pancreatic digest of casein 15,0 g
— papaic digest of soybean meal 5,0 g
— L-cystine 0,7 g
— sodium chloride 4,0 g
— sodium sulfite 0,2 g
— glucose 5,5 g
— egg lecithin 1,0 g
— polysorbate 80 5,0 g
— octoxynol 9 1,0 g
— water 1 000 ml
5.3.3.1.3 Preparation
Dissolve the components, polysorbate 80, octoxynol 9 and egg lecithin, successively into boiling water
until their complete dissolution. Dissolve the other components by mixing while heating.
Dispense the medium into suitable containers. Sterilize in the autoclave at 121 °C for 15 min. After
sterilization and cooling down, the pH shall be equivalent to 7,0 ± 0,2 when measured at room temperature.
5.3.3.2 Other enrichment broths
Other enrichment broths may be used as appropriate (see Annex A).
5.3.4 Selective agar medium for isolation of Escherichia coli
5.3.4.1 MacConkey agar medium
5.3.4.1.1 Composition
— pancreatic digest of gelatin 17,0 g
— pancreatic digest of casein 1,5 g
— peptic digest of animal tissue 1,5 g
— lactose 10,0 g
— bile salts mixture 1,5 g
4 © ISO 2015 – All rights reserved

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ISO 21150:2015(E)

— sodium chloride 5,0 g
— agar 13,5 g
— neutral red 30,0 mg
— crystal violet 1,0 mg
— water 1 000 ml
5.3.4.1.2 Preparation
Dissolve all solid components in the water and boil for 1 min to effect solution.
Dispense in suitable containers and sterilize at 121 °C for 15 min.
The pH, after sterilization and cooling down, shall be equivalent to 7,1 ± 0,2 when measured at
room temperature.
5.3.5 Selective agar medium for confirmation of Escherichia coli
5.3.5.1 Levine eosin-methylene blue agar medium
5.3.5.1.1 Composition
— pancreatic digest of gelatin 10,0 g
— potassium hydrogen phosphate (K HPO ) 2,0 g
2 4
— agar 15,0 g
— lactose 10,0 g
— eosin Y 400 mg
— methylene blue 65 mg
— water 1 000 ml
5.3.5.1.2 Preparation
Dissolve the pancreatic digest of gelatin, the potassium hydrogen phosphate, and the agar in the water,
with warming, and allow to cool. Just prior to use, liquefy the gelled agar solution, add the remaining
ingredients, as solutions in the following amounts, and mix; for each 100 ml of the liquefied agar solution
— 5 ml of 20 % lactose solution,
— 2 ml of 2 % eosin Y solution, and
— 2 ml of 0,033 % methylene blue solution.
The finished medium may not be clear.
Dispense in suitable containers and sterilize at 121 °C for 15 min.
The pH, after sterilization and cooling down, shall be equivalent to 7,1 ± 0,2 when measured at
room temperature.
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ISO 21150:2015(E)

6 Apparatus and glassware
Use the laboratory equipment, apparatus and glassware described in ISO 21148.
7 Strains of microorganisms
For the verification of the test conditions suitability, the following representative strain is used:
1) 2) 3) 4) 5)
Escherichia coli ATCC 8739 [equivalent strain: CIP 53.126 or NCIMB 8545 or NBRC 3972 or KCTC
2571 or other equivalent national collection strain].
The culture should be reconstituted according to the procedures provided by the supplier of the
reference strain.
[12]
The strain can be kept in the laboratory according to 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 and samples 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 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 in an appropriate solubilizing agent, the
time which elapses between the end of preparation and the moment the inoculum comes into contact
with the enrichment broth shall not exceed 45 min, unless specifically mentioned in the established
protocols or documents.
9.2 Preparation of the initial suspension in the enrichment broth
9.2.1 General
The enrichment is prepared from a sample (3.2) of at least 1 g or 1 ml of the well-mixed product under
test, which is dispersed in at least 9 ml of enrichment broth.
Note S, the exact weight or volume of the sample.
The method shall be checked to ensure that the composition (neutralizer eventually added) and the
volume of the broth perform satisfactorily (see 11.3).
NOTE In some cases, and when possible, filtration of the cosmetic product through a membrane that is
afterwards immersed in the enrichment broth, facilitates the neutralization of the antimicrobial properties of
the product (see 11.3.)
1
...

SLOVENSKI STANDARD
kSIST FprEN ISO 21150:2015
01-september-2015
Kozmetika - Mikrobiologija - Ugotavljanje prisotnosti bakterije Escherichia coli
(ISO/FDIS 21150:2015)
Cosmetics - Microbiology - Detection of Escherichia coli (ISO/FDIS 21150:2015)
Kosmetische Mittel - Mikrobiologie - Nachweis von Escherichia coli (ISO/FDIS
21150:2015)
Cosmétiques - Microbiologie - Détection d'Escherichia coli (ISO/FDIS 21150:2015)
Ta slovenski standard je istoveten z: FprEN ISO 21150
ICS:
07.100.99 Drugi standardi v zvezi z Other standards related to
mikrobiologijo microbiology
71.100.70 .R]PHWLND7RDOHWQL Cosmetics. Toiletries
SULSRPRþNL
kSIST FprEN ISO 21150:2015 en,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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kSIST FprEN ISO 21150:2015

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kSIST FprEN ISO 21150:2015
FINAL
INTERNATIONAL ISO/FDIS
DRAFT
STANDARD 21150
ISO/TC 217
Cosmetics — Microbiology —
Secretariat: ISIRI
Detection of Escherichia coli
Voting begins on:
2015­06­25
Cosmétiques — Microbiologie — Détection d’Escherichia coli
Voting terminates on:
2015­08­25
Please see the administrative notes on page iii
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 21150:2015(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 2015

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kSIST FprEN ISO 21150:2015
ISO/FDIS 21150:2015(E)

COPYRIGHT PROTECTED DOCUMENT
© ISO 2015, 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
Ch. de Blandonnet 8 • CP 401
CH­1214 Vernier, Geneva, Switzerland
Tel. +41 22 749 01 11
Fax +41 22 749 09 47
copyright@iso.org
www.iso.org
ii © ISO 2015 – All rights reserved

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kSIST FprEN ISO 21150:2015
ISO/FDIS 21150:2015(E)

ISO/CEN PARALLEL PROCESSING
This final draft has been developed within the International Organization for Standardization (ISO), and pro­
cessed under the ISO-lead mode of collaboration as defined in the Vienna Agreement. The final draft was
established on the basis of comments received during a parallel enquiry on the draft.
This final draft is hereby submitted to the ISO member bodies and to the CEN member bodies for a parallel
two­month approval vote in ISO and formal vote in CEN.
Positive votes shall not be accompanied by comments.
Negative votes shall be accompanied by the relevant technical reasons.
© ISO 2015 – All rights reserved iii

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kSIST FprEN ISO 21150:2015
ISO/FDIS 21150:2015(E)

Contents Page
Foreword .v
Introduction .vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Diluents and culture media . 2
5.1 General . 2
5.2 Diluent for the bacterial suspension (tryptone sodium chloride solution) . 2
5.2.1 Composition . 3
5.2.2 Preparation . 3
5.3 Culture media . 3
5.3.1 General. 3
5.3.2 Agar medium for the suitability test (see Clause 11) [soybean-casein
digest agar medium (SCDA) or tryptic soy agar (TSA)] . 3
5.3.3 Enrichment broth . 3
5.3.4 Selective agar medium for isolation of Escherichia coli . 4
5.3.5 Selective agar medium for confirmation of Escherichia coli . 5
6 Apparatus and glassware . 5
7 Strains of microorganisms . 5
8 Handling of cosmetic products and laboratory samples . 6
9 Procedure. 6
9.1 General recommendations. 6
9.2 Preparation of the initial suspension in the enrichment broth . 6
9.2.1 General. 6
9.2.2 Water­miscible products. 6
9.2.3 Water­immiscible products . 7
9.2.4 Filterable products . 7
9.3 Incubation of the inoculated enrichment broth . 7
9.4 Detection and identification of Escherichia coli . 7
9.4.1 Isolation . 7
9.4.2 Identification of Escherichia coli . 7
10 Expression of the results (detection of Escherichia coli) . 8
11 Neutralization of the antimicrobial properties of the product . 8
11.1 General . 8
11.2 Preparation of inoculum . 8
11.3 Suitability of the detection method . 8
11.3.1 Procedure . 8
11.3.2 Interpretation of suitability test results . 9
12 Test report . 9
Annex A (informative) Other enrichment broths .10
Annex B (informative) Neutralizers of antimicrobial activity of preservatives and
rinsing liquids .13
Bibliography .14
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kSIST FprEN ISO 21150:2015
ISO/FDIS 21150:2015(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 WTO principles in the Technical Barriers
to Trade (TBT) see the following URL: Foreword - Supplementary information
The committee responsible for this document is ISO/TC 217, Cosmetics.
This second editions cancels and replaces the first edition (ISO 21150:2006), which has been
technically revised.
© ISO 2015 – All rights reserved v

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kSIST FprEN ISO 21150:2015
ISO/FDIS 21150:2015(E)

Introduction
Microbiological examinations of cosmetic products are to be carried out according to an appropriate
microbiological risk analysis in order to ensure their quality and safety for consumers.
Microbiological risk analysis depends on several parameters such as the following:
— potential alteration of cosmetic products;
— pathogenicity of microorganisms;
— site of application of the cosmetic product (hair, skin, eyes, mucous membranes, etc.);
— type of users (adults, children under 3 years, etc.).
For cosmetics and other topical products, the detection of skin pathogens such as Staphylococcus
aureus, Pseudomonas aeruginosa and Candida albicans may be relevant. The detection of other kinds
of microorganisms might be of interest since these microorganisms (including indicators of faecal
contamination, e.g. Escherichia coli) suggest hygienic failure during manufacturing process.
vi © ISO 2015 – All rights reserved

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kSIST FprEN ISO 21150:2015
FINAL DRAFT INTERNATIONAL STANDARD ISO/FDIS 21150:2015(E)
Cosmetics — Microbiology — Detection of Escherichia coli
1 Scope
This International Standard gives general guidelines for the detection and identification of the specified
microorganism Escherichia coli in cosmetic products. Microorganisms considered as specified in this
International Standard might differ from country to country according to national practices or regulations.
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 International
Standard is applicable. Products considered to present a low microbiological (see ISO 29621) risk include
those with low water activity, hydro-alcoholic products, extreme pH values, etc.
This International Standard specifies a method that is based on the detection of Escherichia coli in a non­
selective liquid medium (enrichment broth), followed by isolation on a selective agar medium. Other
methods may be appropriate depending on the level of detection required.
NOTE For the detection of Escherichia coli, subcultures can be performed on non­selective culture media
followed by suitable identification steps (e.g. using identification kits).
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 International
Standards may be appropriate. Other methods (e.g. automated) can be substituted for the test presented
here provided that their equivalence has been demonstrated or the method has been otherwise validated.
2 Normative references
The following documents, in whole or in part, are normatively referenced in this document and are
indispensable for its application. 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
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
product
portion of an identified cosmetic product received in the laboratory for testing
3.2
sample
portion of the product (at least 1 g or 1 ml) which is used in the test to prepare the initial suspension
3.3
initial suspension
suspension (or solution) of the sample in a defined volume of an appropriate enrichment broth
3.4
sample dilution
dilution of the initial suspension
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kSIST FprEN ISO 21150:2015
ISO/FDIS 21150:2015(E)

3.5
specified microorganism
aerobic mesophilic bacteria or yeast which is undesirable in a cosmetic product because it can cause skin
or eye infection, or it can be recognized as an indicator of hygienic failure in the manufacturing process
3.6
Escherichia coli
Gram­negative rod, motile, smooth colonies
Note 1 to entry: The main characteristics for identification are catalase positive, oxidase negative, fermentation
of lactose, production of indole, growth on selective medium containing bile salts with characteristic colonies.
Note 2 to entry: Escherichia coli can be isolated from the moist environmental sources (air, water, soil) and is a
faecal contamination indicator.
3.7
enrichment broth
non­selective liquid medium containing suitable neutralizers and/or dispersing agents and demonstrated
to be suitable for the product under test
4 Principle
The first step of the procedure is to perform an enrichment by using a non-selective broth medium to
increase the number of microorganisms without the risk of inhibition by the selective ingredients that
are present in selective/differential growth media.
The second step (isolation) of the test is performed on a selective medium followed by identification tests.
The possible inhibition of microbial growth by the sample shall be neutralized to allow the detection
[5]
of viable microorganisms. In all cases and whatever the methodology, the neutralization of the
antimicrobial properties of the product shall be checked and demonstrated (see Clause 11).
5 Diluents and culture media
5.1 General
Use the general instructions given in ISO 21148. When water is mentioned in this International Standard,
use distilled water or purified water as specified in ISO 21148.
The enrichment broth is used to disperse the sample and to increase the initial microbial population. It
may contain neutralizers if the specimen to be tested has antimicrobial properties. The efficacy of the
neutralization shall be demonstrated (see Clause 11). Information relative to suitable neutralizers is
given in Annex B.
The enrichment broth (5.3.3.1) or any of the ones listed in Annex A is suitable for checking the presence
of Escherichia coli according to this International Standard provided that they have been demonstrated
to be suitable in accordance with Clause 11.
Other diluents and culture media may be used if they have been demonstrated to be suitable for use.
5.2 Diluent for the bacterial suspension (tryptone sodium chloride solution)
The diluent is used for the preparation of bacterial suspension used for the suitability test procedure
(see Clause 11).
2 © ISO 2015 – All rights reserved

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kSIST FprEN ISO 21150:2015
ISO/FDIS 21150:2015(E)

5.2.1 Composition
— Tryptone, pancreatic digest of casein 1,0 g
— Sodium chloride 8,5 g
— Water 1 000 ml
5.2.2 Preparation
Dissolve the components in water by mixing while heating. Dispense into suitable containers. Sterilize
in the autoclave at 121 °C for 15 min.
After sterilization and cooling down, the pH shall be equivalent to 7,0 ± 0,2 when measured at room
temperature.
5.3 Culture media
5.3.1 General
Culture media may be prepared using the descriptions provided below or from dehydrated culture
media, according to the instructions from the manufacturer. The instructions provided by the supplier
of the media should be followed.
NOTE Ready-to-use media can be used when their composition and/or growth yields are comparable to
those of the formulae given herein.
5.3.2 Agar medium for the suitability test (see Clause 11) [soybean-casein digest agar medium
(SCDA) or tryptic soy agar (TSA)]
5.3.2.1 Composition
— Pancreatic digest of casein 15,0 g
— Papaic digest of soybean meal 5,0 g
— Sodium chloride 5,0 g
— Agar 15,0 g
— Water 1 000 ml
5.3.2.2 Preparation
Dissolve the components or the dehydrated complete medium in the water by mixing while heating.
Dispense the medium into suitable containers. Sterilize in the a
...

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