Cosmetics - Microbiology - Detection of specified and non-specified microorganisms (ISO 18415:2007)

This International Standard gives general guidelines for the detection and identification of specified microorganisms in cosmetic products as well as for the detection and identification of other kinds of aerobic mesophilic non-specified microorganisms in cosmetic products. Microorganisms considered as specified in this International Standard might differ from country to country according to national practices or regulations. Most of them considered as specified microorganisms include one or more of the following species: Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Candida albicans.
In order to ensure product quality and safety for consumers, it is advisable to perform an appropriate microbiological risk analysis in order to determine the types of cosmetic product to which this International Standard is applicable. Products considered to present a low microbiological 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 microbial growth in a nonselective liquid medium (enrichment broth) suitable to detect microbial contamination, followed by isolation of microorganisms on non-selective agar media. Other methods can be appropriate depending on the level of detection required.
In this International Standard specific indications are given for identification of Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Candida albicans. Other microorganisms that grow under the conditions described in this International Standard, may be identified by using suitable tests according to a general scheme (see Annex A). Other standards (e.g., ISO 18416, ISO 21150, ISO 22717, ISO 22718) may be appropriate.
Because of the large variety of cosmetic products within this field of application, this method might not be suited in every detail, to some products (e.g. certain water-immiscible products). Other methods (e.g. automate

Kosmetik - Mikrobiologie - Nachweis von spezifizierten und nichtspezifizierten Mikroorganismen (ISO 18415:2007)

Diese Internationale Norm gibt allgemeine Anleitungen sowohl für den Nachweis und die Identifizierung von spezifizierten Mikroorganismen in kosmetischen Mitteln als auch für den Nachweis und die Identifizierung von anderen Arten nichtspezifizierter aerober mesophiler Mikroorganismen in kosmetischen Mitteln.
Die in dieser Internationalen Norm als spezifiziert angesehenen Mikroorganismen können in Übereinstimmung mit nationalen Praktiken oder Regelungen von Land zu Land unterschiedlich sein. Die meisten der als spezifiziert angesehenen Mikroorganismen schließen die folgenden Arten ein: Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus und Candida albicans.
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, auf die diese Internationale Norm anwendbar ist. Produkte, die ein geringes mikrobiologisches Risiko darstellen, umfassen Produkte mit geringer Wasseraktivität, alkoholisch-wässrige Produkte, Produkte mit extremen pH Werten usw.
Das in dieser Internationalen Norm beschriebene Verfahren basiert auf dem Nachweis von mikrobiellem Wachstum in einem nicht selektiven Flüssigmedium (Anreicherungsbouillon), geeignet für den Nachweis einer mikrobiellen Kontamination, gefolgt von einer Isolierung der Mikroorganismen auf einem nicht selektiven Agarmedium. In Abhängigkeit von der erforderlichen Nachweisgrenze können andere Verfahren geeignet sein.
In der vorliegenden Internationalen Norm sind spezielle Hinweise zur Identifizierung von Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus und Candida albicans angeführt. Andere Mikro-organismen, die unter den in dieser Internationalen Norm beschriebenen Bedingungen wachsen, können unter Anwendung geeigneter Untersuchungen nach einem allgemeinen Schema identifiziert werden (siehe Anhang A). Weitere Normen (z. B. ISO 18416, ISO 21150, ISO 22717, ISO 22718) können angebracht sein.
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). Ersatzweise können für die hier aufgeführten Untersuchungen andere Verfahren (z. B. automatisierte) zur Anwendung kommen, vorausgesetzt, dass deren Gleichwertigkeit nachgewiesen oder das Verfahren anderweitig validiert wurde.

Cosmétiques - Microbiologie - Détection des micro-organismes spécifiés et non spécifiés (ISO 18415:2007)

L'ISO 18415:2007 donne des lignes directrices générales pour la détection et l'identification de microorganismes spécifiés dans les produits cosmétiques, et aussi pour la détection et l'identification d'autres sortes de microorganismes mésophiles aérobies non spécifiés, 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 réglementations nationales. La plupart des microorganismes considérés comme spécifiés comprennent une ou plusieurs espèces, parmi les suivantes: Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus et Candida albicans.
La méthode décrite dans l'ISO 18415:2007 est fondée sur la détection d'une croissance microbienne dans un milieu liquide non sélectif (milieu liquide d'enrichissement) qui permet de détecter une contamination microbienne, suivie d'un isolement des microorganismes sur un milieu gélosé non sélectif. D'autres méthodes peuvent être appropriées, en fonction du niveau de détection requis.
Dans l'ISO 18415:2007, des indications spécifiques sont données pour l'identification de Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus et Candida albicans. D'autres microorganismes qui se développent dans les conditions décrites dans la présente Norme internationale, peuvent être identifiés en mettant en oeuvre des essais appropriés conformes à un schéma général.

Kozmetika - Mikrobiologija - Ugotavljanje prisotnosti specifičnih in nespecifičnih mikroorganizmov (ISO 18415:2007)

Ta mednarodni standard podaja splošne smernice za ugotavljanje prisotnosti in identifikacijo specifičnih mikroorganizmov v kozmetičnih izdelkih ter za ugotavljanje prisotnosti in identifikacijo drugih vrst mezofilnih nespecifičnih mikroorganizmov v kozmetičnih izdelkih. Mikroorganizmi, ki v tem mednarodnem standardu veljajo za specifične, se lahko razlikujejo od države do države v skladu z nacionalnimi praksami ali predpisi. Večina od mikroorganizmov, ki veljajo za specifične, vključujejo eno ali več naslednjih vrst: Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus in Candida albicans. Za zagotavljanje kakovosti izdelka in varnosti za potrošnike je priporočljivo opraviti ustrezno analizo mikrobiološkega tveganja, da se določijo vrste kozmetičnih izdelkov, za katere velja ta mednarodni standard. Izdelki, pri katerih je nizko mikrobiološko tveganje, so izdelki z nizko aktivnostjo vode, hidroalkoholni izdelki, izdelki z ekstremnimi vrednostmi pH itd. Metoda, opisana v tem mednarodnem standardu, temelji na ugotavljanju prisotnosti mikrobne rasti v neselektivnem tekočem gojišču (obogatitveno gojišče), primernem za ugotavljanje mikrobne kontaminacije, temu pa sledi izolacija mikroorganizmov na neselektivnem agarskem gojišču. Lahko so primerne tudi druge metode, odvisno od zahtevane ravni detekcije. V tem mednarodnem standardu so posebej navedena navodila za identifikacijo vrst Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus in Candida albicans. Drugi mikroorganizmi, ki rastejo v pogojih, opisanih v tem mednarodnem standardu, se lahko identificirajo z uporabo ustreznih preskusov v skladu s splošno shemo (glej dodatek A). Ustrezajo lahko drugi standardi (npr. ISO 18416, ISO 21150, ISO 22717, ISO 22718). Zaradi velike raznolikosti kozmetičnih izdelkov v tem okviru uporabe ta metoda za nekatere izdelke morda ni primerna v vsaki podrobnosti (npr. za nekatere izdelke, ki se ne mešajo z vodo). Preskus, ki je tu predstavljen, lahko nadomestijo druge metode (npr. avtomatske), če je bila dokazana njihova enakovrednost ali da je bila metoda kako drugače validirana.

General Information

Status
Withdrawn
Public Enquiry End Date
19-Feb-2011
Publication Date
15-Sep-2011
Withdrawal Date
18-Sep-2017
Current Stage
9900 - Withdrawal (Adopted Project)
Start Date
18-Sep-2017
Due Date
11-Oct-2017
Completion Date
19-Sep-2017

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SLOVENSKI STANDARD
SIST EN ISO 18415:2011
01-oktober-2011

.R]PHWLND0LNURELRORJLMD8JRWDYOMDQMHSULVRWQRVWLVSHFLILþQLKLQQHVSHFLILþQLK

PLNURRUJDQL]PRY ,62

Cosmetics - Microbiology - Detection of specified and non-specified microorganisms

(ISO 18415:2007)
Kosmetik - Mikrobiologie - Nachweis von spezifizierten und nichtspezifizierten
Mikroorganismen (ISO 18415:2007)

Cosmétiques - Microbiologie - Détection des micro-organismes spécifiés et non spécifiés

(ISO 18415:2007)
Ta slovenski standard je istoveten z: EN ISO 18415:2011
ICS:
07.100.40 Kozmetika - mikrobiologija Cosmetics microbiology
SIST EN ISO 18415:2011 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 18415:2011
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SIST EN ISO 18415:2011
EUROPEAN STANDARD
EN ISO 18415
NORME EUROPÉENNE
EUROPÄISCHE NORM
June 2011
ICS 07.100.99; 71.100.70
English Version
Cosmetics - Microbiology - Detection of specified and non-
specified microorganisms (ISO 18415:2007)

Cosmétiques - Microbiologie - Détection des micro- Kosmetik - Mikrobiologie - Nachweis von spezifizierten und

organismes spécifiés et non spécifiés (ISO 18415:2007) nichtspezifizierten Mikroorganismen (ISO 18415:2007)

This European Standard was approved by CEN on 12 May 2011.

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, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland,

Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.

EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: Avenue Marnix 17, B-1000 Brussels

© 2011 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 18415:2011: E

worldwide for CEN national Members.
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SIST EN ISO 18415:2011
EN ISO 18415:2011 (E)
Contents Page

Foreword ..............................................................................................................................................................3

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SIST EN ISO 18415:2011
EN ISO 18415:2011 (E)
Foreword

The text of ISO 18415:2007 has been prepared by Technical Committee ISO/TC 217 “Cosmetics” of the

International Organization for Standardization (ISO) and has been taken over as EN ISO 18415:2011 by

Technical Committee CEN/TC 392 “Cosmetics” the secretariat of which is held by NEN.

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 December 2011, and conflicting national standards shall be withdrawn

at the latest by December 2011.

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.

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, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia,

Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain,

Sweden, Switzerland and the United Kingdom.
Endorsement notice

The text of ISO 18415:2007 has been approved by CEN as a EN ISO 18415:2011 without any modification.

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SIST EN ISO 18415:2011
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SIST EN ISO 18415:2011
INTERNATIONAL ISO
STANDARD 18415
First edition
2007-09-01
Corrected version
2007-12-01
Cosmetics — Microbiology — Detection
of specified and non-specified
microorganisms
Cosmétiques — Microbiologie — Détection des micro-organismes
spécifiés et non spécifiés
Reference number
ISO 18415:2007(E)
ISO 2007
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SIST EN ISO 18415:2011
ISO 18415:2007(E)
PDF disclaimer

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COPYRIGHT PROTECTED DOCUMENT
© ISO 2007

All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means,

electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or

ISO's member body in the country of the requester.
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Published in Switzerland
ii © ISO 2007 – All rights reserved
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SIST EN ISO 18415:2011
ISO 18415:2007(E)
Contents Page

Foreword............................................................................................................................................................ iv

Introduction ........................................................................................................................................................ v

1 Scope ..................................................................................................................................................... 1

2 Normative references ........................................................................................................................... 1

3 Terms and definitions........................................................................................................................... 2

4 Principle................................................................................................................................................. 3

5 Diluents and culture media.................................................................................................................. 3

5.1 General................................................................................................................................................... 3

5.2 Diluent for the microbial suspension (tryptone sodium chloride solution) ................................... 3

5.3 Culture media ........................................................................................................................................ 4

6 Apparatus and glassware .................................................................................................................... 5

7 Strains of microorganism .................................................................................................................... 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

9.3 Incubation of the initial suspension ................................................................................................... 7

9.4 Isolation of specified and non-specified microorganisms............................................................... 7

9.5 Procedure for identification of the specified microorganism: Pseudomonas aeruginosa........... 7

9.6 Procedure for identification of the specified microorganism: Escherichia coli ............................ 8

9.7 Procedure for identification of the specified microorganism: Staphylococus aureus ................. 8

9.8 Procedure for the identification of the specified microorganism: Candida albicans.................... 9

9.9 Procedure for the identification of non-specified microorganisms ................................................ 9

10 Expression of the results................................................................................................................... 10

10.1 Detection of specified microorganisms ...........................................................................................10

10.2 Detection of non-specified microorganisms ................................................................................... 10

10.3 Absence of microorganisms ............................................................................................................. 10

11 Neutralization of the antimicrobial properties of the product........................................................ 10

11.1 General................................................................................................................................................. 10

11.2 Preparation of inoculum .................................................................................................................... 10

11.3 Validation of detection method by enrichment ............................................................................... 11

12 Test report ........................................................................................................................................... 12

Annex A (informative) General scheme for identification of microorganisms .......................................... 13

Annex B (informative) Other Media ................................................................................................................ 14

Annex C (informative) Neutralizers of antimicrobial activity of preservatives and rinsing liquids ......... 17

Bibliography ..................................................................................................................................................... 18

© ISO 2007 – All rights reserved iii
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SIST EN ISO 18415:2011
ISO 18415:2007(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.

International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.

The main task of technical committees is to prepare International Standards. Draft International Standards

adopted by the technical committees are circulated to the member bodies for voting. Publication as an

International Standard requires approval by at least 75 % of the member bodies casting a vote.

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.

ISO 18415 was prepared by Technical Committee ISO/TC 217, Cosmetics.
This corrected version of ISO 18415:2007 contains the following corrections:
⎯ p. 2, 3.6.1: modification of definition;
⎯ p. 3, 3.8: correction of term's number;
⎯ p.8, 9.7.1: correction of text in the second paragraph.
iv © ISO 2007 – All rights reserved
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SIST EN ISO 18415:2011
ISO 18415:2007(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:
⎯ potential alteration of cosmetic products;
⎯ pathogenicity of microorganisms;

⎯ site of application of the cosmetic product (hair, skin, eyes, mucous membranes);

⎯ type of user (adults, children, including 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

infection. 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.
© ISO 2007 – All rights reserved v
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SIST EN ISO 18415:2011
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SIST EN ISO 18415:2011
INTERNATIONAL STANDARD ISO 18415:2007(E)
Cosmetics — Microbiology — Detection of specified and non-
specified microorganisms
1 Scope

This International Standard gives general guidelines for the detection and identification of specified

microorganisms in cosmetic products as well as for the detection and identification of other kinds of aerobic

mesophilic non-specified microorganisms in cosmetic products.

Microorganisms considered as specified in this International Standard might differ from country to country

according to national practices or regulations. Most of them considered as specified microorganisms include

one or more of the following species: Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and

Candida albicans.

In order to ensure product quality and safety for consumers, it is advisable to perform an appropriate

microbiological risk analysis in order to determine the types of cosmetic product to which this International

Standard is applicable. Products considered to present a low microbiological 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 microbial growth in a non-

selective liquid medium (enrichment broth) suitable to detect microbial contamination, followed by isolation of

microorganisms on non-selective agar media. Other methods can be appropriate depending on the level of

detection required.

In this International Standard specific indications are given for identification of Pseudomonas aeruginosa,

Escherichia coli, Staphylococcus aureus and Candida albicans. Other microorganisms that grow under the

conditions described in this International Standard, may be identified by using suitable tests according to a

general scheme (see Annex A). Other standards (e.g., ISO 18416, ISO 21150, ISO 22717, ISO 22718) may

be appropriate.

Because of the large variety of cosmetic products within this field of application, this method might not be

suited in every detail, to some products (e.g. certain water-immiscible products). 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 referenced documents are indispensable for the application 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 21148:2005, Cosmetics — Microbiology — General instructions for microbiological examination

EN 12353, Chemical disinfectants and antiseptics — Preservation of test organisms used for the

determination of bactericidal, mycobactericidal, sporicidal and fungicidal activity

© ISO 2007 – All rights reserved 1
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SIST EN ISO 18415:2011
ISO 18415:2007(E)
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

3.4
sample dilution
dilution of the initial suspension
3.5
aerobic mesophilic microorganisms

mesophilic bacteria or yeast growing aerobically under the conditions specified in this International Standard

NOTE In the described conditions other types of microorganism (e.g. molds) are detectable.

3.6
specified microorganisms

aerobic mesophilic bacteria or yeast undesirable in a cosmetic product and recognised as a skin pathogen

species that may be harmful for human health or as an indication of hygienic failure in the manufacturing

process
3.6.1
Pseudomonas aeruginosa

Gram-negative rod (bacilli), motile, smooth colonies pigmented brown or greenish

NOTE 1 The main characteristics for identification are growth on a selective cetrimide agar medium, oxidase positive,

production of diffusible fluorescent pigments and production of a soluble phenazine pigment (pyocyanin) in suitable media.

NOTE 2 Pseudomonas aeruginosa can be isolated from a wide variety of environmental sources, especially in water

and has a very high potential to spoil many different substrates. It can produce infections of human skin or eye areas. It is

undesirable in cosmetic products for its potential pathogenicity and its capacity to affect the physico-chemical properties of

the cosmetic formula.
3.6.2
Escherichia coli
Gram-negative rod (bacilli), motile, smooth colonies

NOTE 1 The main characteristics are catalase positive, oxidase negative, fermentation of lactose, production of indole,

growth on selective medium containing bile salts with characteristic colonies.

NOTE 2 Escherichia coli can be isolated from the moist environmental sources (air, water, soil) and is a faecal

contamination indicator.
3.6.3
Staphylococus aureus

Gram-positive cocci, mainly aggregated in grape-like clusters, smooth colonies generally pigmented in yellow

NOTE 1 The main characteristics for identification are growth on a specific selective medium, catalase positive,

coagulase positive.
2 © ISO 2007 – All rights reserved
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SIST EN ISO 18415:2011
ISO 18415:2007(E)

NOTE 2 Staphylococcus aureus is an opportunistic pathogen for humans, which often can be also present on the skin

of healthy individuals without causing them any apparent illness. It is a specified microorganism and undesirable in

cosmetic products.
3.6.4
Candida albicans

yeast that forms white to beige, creamy and convex colonies on the surface of a non-selective agar medium

NOTE The main characteristics for identification are production of germ tube and/or pseudomycelium and

chlamydospore when the test is performed following the method specified in this International Standard

3.7
non-specified microorganism

aerobic mesophilic bacteria or yeast found in cosmetic products, not defined in 3.6

3.8
enrichment broth

non-selective liquid medium containing suitable neutralizers and/or dispersing agents and validated 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 following steps (isolation and identification) are performed according to need by using appropriate

conditions of incubation and suitable identification test, as described in this International Standard.

The possible inhibition of microbial growth by the sample shall be neutralized to allow the detection of viable

[2]

microorganisms . In all cases and whatever the methodology, the neutralization of the antimicrobial

[2],[3],[4]
properties of the product shall be checked and validated .
5 Diluents and culture media
5.1 General

General specifications are given in ISO 21148. When water is used in a formula, 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 C.

The enrichment broth (5.3.2.1) or any of the ones listed in Annex B is suitable for checking the presence of

specified and non-specified microorganisms in accordance with this International Standard provided that they

are validated in accordance with Clause 11.

Other diluents and culture media may be used if it has been demonstrated that they are suitable for use.

5.2 Diluent for the microbial suspension (tryptone sodium chloride solution)
5.2.1 General

The diluent is used for the preparation of bacteria and yeast suspensions used for the validation procedure

(see Clause 11).
© ISO 2007 – All rights reserved 3
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SIST EN ISO 18415:2011
ISO 18415:2007(E)
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, 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 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.
5.3.2 Enrichment broth
5.3.2.1 Eugon LT100 broth
5.3.2.1.1 General

This medium contains ingredients which neutralize inhibitory substances present in the sample: lecithin and

polysorbate 80, and dispersing agent: octoxynol 9.
5.3.2.1.2 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
4 © ISO 2007 – All rights reserved
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SIST EN ISO 18415:2011
ISO 18415:2007(E)
5.3.2.1.3 Preparation

Dissolve the components polysorbate 80, octoxynol 9 and egg lecithin, one after another in boiling water to

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, the pH shall be

equivalent to 7,0 ± 0,2 when measured at room temperature.
5.3.2.2 Other enrichment broths
Other enrichment broths may be used as appropriate (see Annex B).
5.3.3 Non-selective agar medium
5.3.3.1 General

This medium is used for the isolation and detection of specified and non-specified microorganisms present in

the initial suspension after enrichment and for the preparation of inoculum used in the validation procedure.

5.3.3.2 Soybean-casein digest agar medium (SCDA) or tryptic soy agar (TSA)
5.3.3.2.1 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
5.3.3.2.2 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.

5.3.3.3 Other non-selective agar medium
Other non-selective, non-neutralizing agar media may be used (see Annex B).
6 Apparatus and glassware
The laboratory equipment, apparatus and glassware are described in ISO 21148.
7 Strains of microorganism

For testing the recovery efficiency of the test conditions, three specified microorganisms are used: two strains

representative of both Gram-negative and Gram-positive bacteria and a strain of yeast.

⎯ Pseudomonas aeruginosa ATCC 9027 (equivalent strain: CIP 82.118 or NCIMB 8626 or NBRC 13275 or

KCTC 2513 or other equivalent national collection strain).
© ISO 2007 – All rights reserved 5
---------------------- Page: 17 ----------------------
SIST EN ISO 18415:2011
ISO 18415:2007(E)

An alternative to the Gram-negative strain may be: Escherichia coli ATCC 8739 (equivalent strain: CIP 53.126

or NCIMB 8545 or NBRC 3972 or KCTC 2571 or other equivalent national collection strain).

1) 2) 3) 4)

⎯ Staphylococcus aureus ATCC 6538 (equivalent strain: CIP 4.83 or NCIMB 9518 or NBRC 13276 or

KCTC 1916 or other equivalent national collection strain);
6) 7)

⎯ Candida albicans ATCC 10231 (equivalent strain: IP 48.72 or NCPF 3179 or NBRC 1594 or

KCTC 17205 or other equivalent national collection strain).

The culture should be reconstituted according to the procedures provided by the supplier of reference strain.

The strains can 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

(3.1) and samples (3.2) 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 given in Clause 9.

9 Procedure
9.1 General recommendations

Use sterile material, equipment and aseptic techniques to prepare the sample, initial suspension and dilutions.

In the case of the preparation of an initial suspension in an appropriate diluent before transfer in the

enrichment broth, the time that elapses between the end of the preparation and the moment the initial

suspension and/or sample dilutions come 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 in the enrichment broth
9.2.1 General

The enrichment is prepared from a sample 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 mass 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).
1) American Type Culture Collection.
2) Collection de l’Institut Pasteur.
3) National Collection of Industrial and Marine Bacteria.
4) National Biological Resource Center.
5) Korean Collection for Type Culture.
6) Institut Pasteur.
7) National Collection of Pathogenic Fungi.
6 © ISO 2007 – All rights reserved
---------------------- Page: 18 ----------------------
SIST EN ISO 18415:2011
ISO 18415:2007(E)

NOTE In some cases, and when possible, filtration of the cosmetic product through a membrane which is afterwards

immersed in the enrichment broth facilitates the neutralization of the antimicrobial properties of the product (see 11.3.3).

9.2.2 Water-miscible products

Transfer the sample, S, of product to a suitable container containing an appropriate volume (at least 9 ml) of

broth.
9.2.3 Water-immiscible products

Transfer the sample, S, of product to a suitable container containing a suitable quantity of dispersing agent

(e.g. polysorbate 80).

Disperse the sample within the solubilizing agent and add an appropriate volume (at least 9 ml) of broth.

9.2.4 Filterable products
Use a membrane filter having a nominal pore size no greater than 0,45 µm.
...

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.LKKosmetik - Mikrobiologie - Nachweis von spezifizierten und nichtspezifizierten Mikroorganismen (ISO 18415:2007)Cosmétiques - Microbiologie - Détection des micro-organismes spécifiés et non spécifiés (ISO 18415:2007)Cosmetics - Microbiology - Detection of specified and non-specified microorganisms (ISO 18415:2007)71.100.70SULSRPRþNLCosmetics. Toiletries07.100.99Drugi standardi v zvezi z mikrobiologijoOther standards related to microbiologyICS:Ta slovenski standard je istoveten z:FprEN ISO 18415kSIST FprEN ISO 18415:2011en,fr,de01-januar-2011kSIST FprEN ISO 18415:2011SLOVENSKI

STANDARD
kSIST FprEN ISO 18415:2011
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
FINAL DRAFT
FprEN ISO 18415
November 2010 ICS 07.100.99; 71.100.70 English Version

Cosmetics - Microbiology - Detection of specified and non-specified microorganisms (ISO 18415:2007)

Cosmétiques - Microbiologie - Détection des micro-organismes spécifiés et non spécifiés (ISO 18415:2007)

Kosmetik - Mikrobiologie - Nachweis von spezifizierten und nichtspezifizierten Mikroorganismen (ISO 18415:2007) This draft European Standard is submitted to CEN members for unique acceptance procedure. It has been drawn up by the Technical Committee CEN/TC 392.

If this draft becomes a European Standard, 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.

This draft European Standard was established by CEN 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, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.

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 supporting documentation.

Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without notice and shall not be referred to as a European Standard.

EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre:
Avenue Marnix 17,

B-1000 Brussels © 2010 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. FprEN ISO 18415:2010: EkSIST FprEN ISO 18415:2011

FprEN ISO 18415:2010 (E) 2 Contents Page Foreword ..............................................................................................................................................................3 kSIST FprEN ISO 18415:2011

FprEN ISO 18415:2010 (E) 3 Foreword The text of ISO 18415:2007 has been prepared by Technical Committee ISO/TC 217 “Cosmetics” of the International Organization for Standardization (ISO) and has been taken over as FprEN ISO 18415:2010 by Technical Committee CEN/TC 392 “Cosmetics” the secretariat of which is held by NEN. This document is currently submitted to the Unique Acceptance Procedure. Endorsement notice The text of ISO 18415:2007 has been approved by CEN as a FprEN ISO 18415:2010 without any modification.

kSIST FprEN ISO 18415:2011
kSIST FprEN ISO 18415:2011
Reference numberISO 18415:2007(E)© ISO 2007

INTERNATIONAL STANDARD ISO18415First edition2007-09-01Corrected version2007-12-01 Cosmetics — Microbiology — Detection of specified and non-specified microorganisms Cosmétiques — Microbiologie — Détection des micro-organismes spécifiés et non spécifiés

kSIST FprEN ISO 18415:2011

ISO 18415:2007(E) PDF disclaimer This PDF file may contain embedded typefaces. In accordance with Adobe's licensing policy, this file may be printed or viewed but shall not be edited unless the typefaces which are embedded are licensed to and installed on the computer performing the editing. In downloading this file, parties accept therein the responsibility of not infringing Adobe's licensing policy. The ISO Central Secretariat accepts no liability in this area. Adobe is a trademark of Adobe Systems Incorporated. Details of the software products used to create this PDF file can be found in the General Info relative to the file; the PDF-creation parameters were optimized for printing. Every care has been taken to ensure that the file is suitable for use by ISO member bodies. In the unlikely event that a problem relating to it is found, please inform the Central Secretariat at the address given below.

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ISO 2007 All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or ISO's member body in the country of the requester. ISO copyright office Case postale 56 • CH-1211 Geneva 20 Tel.

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ii © ISO 2007 – All rights reserved
kSIST FprEN ISO 18415:2011

ISO 18415:2007(E) © ISO 2007 – All rights reserved iiiContents Page Foreword............................................................................................................................................................iv Introduction........................................................................................................................................................v 1 Scope.....................................................................................................................................................1 2 Normative references...........................................................................................................................1 3 Terms and definitions...........................................................................................................................2 4 Principle.................................................................................................................................................3 5 Diluents and culture media..................................................................................................................3 5.1 General...................................................................................................................................................3 5.2 Diluent for the microbial suspension (tryptone sodium chloride solution)...................................3 5.3 Culture media........................................................................................................................................4 6 Apparatus and glassware....................................................................................................................5 7 Strains of microorganism....................................................................................................................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 9.3 Incubation of the initial suspension...................................................................................................7 9.4 Isolation of specified and non-specified microorganisms...............................................................7 9.5 Procedure for identification of the specified microorganism: Pseudomonas aeruginosa...........7 9.6 Procedure for identification of the specified microorganism: Escherichia coli............................8 9.7 Procedure for identification of the specified microorganism: Staphylococus aureus.................8 9.8 Procedure for the identification of the specified microorganism: Candida albicans....................9 9.9 Procedure for the identification of non-specified microorganisms................................................9 10 Expression of the results...................................................................................................................10 10.1 Detection of specified microorganisms...........................................................................................10 10.2 Detection of non-specified microorganisms...................................................................................10 10.3 Absence of microorganisms.............................................................................................................10 11 Neutralization of the antimicrobial properties of the product........................................................10 11.1 General.................................................................................................................................................10 11.2 Preparation of inoculum....................................................................................................................10 11.3 Validation of detection method by enrichment...............................................................................11 12 Test report...........................................................................................................................................12 Annex A (informative)

General scheme for identification of microorganisms..........................................13 Annex B (informative)

Other Media................................................................................................................14 Annex C (informative)

Neutralizers of antimicrobial activity of preservatives and rinsing liquids.........17 Bibliography.....................................................................................................................................................18

kSIST FprEN ISO 18415:2011

ISO 18415:2007(E) iv © ISO 2007 – All rights reserved 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. International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2. The main task of technical committees is to prepare International Standards. Draft International Standards adopted by the technical committees are circulated to the member bodies for voting. Publication as an International Standard requires approval by at least 75 % of the member bodies casting a vote. 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. ISO 18415 was prepared by Technical Committee ISO/TC 217, Cosmetics. This corrected version of ISO 18415:2007 contains the following corrections: ⎯ p. 2, 3.6.1: modification of definition; ⎯ p. 3, 3.8: correction of term's number; ⎯ p.8, 9.7.1: correction of text in the second paragraph. kSIST FprEN ISO 18415:2011

ISO 18415:2007(E) © ISO 2007 – All rights reserved vIntroduction 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: ⎯ potential alteration of cosmetic products; ⎯ pathogenicity of microorganisms; ⎯ site of application of the cosmetic product (hair, skin, eyes, mucous membranes); ⎯ type of user (adults, children, including 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 infection. 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.

kSIST FprEN ISO 18415:2011
kSIST FprEN ISO 18415:2011

INTERNATIONAL STANDARD ISO 18415:2007(E) © ISO 2007 – All rights reserved 1Cosmetics — Microbiology — Detection of specified and non-specified microorganisms 1 Scope This International Standard gives general guidelines for the detection and identification of specified microorganisms in cosmetic products as well as for the detection and identification of other kinds of aerobic mesophilic non-specified microorganisms in cosmetic products. Microorganisms considered as specified in this International Standard might differ from country to country according to national practices or regulations. Most of them considered as specified microorganisms include one or more of the following species: Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Candida albicans. In order to ensure product quality and safety for consumers, it is advisable to perform an appropriate microbiological risk analysis in order to determine the types of cosmetic product to which this International Standard is applicable. Products considered to present a low microbiological 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 microbial growth in a non-selective liquid medium (enrichment broth) suitable to detect microbial contamination, followed by isolation of microorganisms on non-selective agar media. Other methods can be appropriate depending on the level of detection required. In this International Standard specific indications are given for identification of Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Candida albicans. Other microorganisms that grow under the conditions described in this International Standard, may be identified by using suitable tests according to a general scheme (see Annex A). Other standards (e.g., ISO 18416, ISO 21150, ISO 22717, ISO 22718) may be appropriate. Because of the large variety of cosmetic products within this field of application, this method might not be suited in every detail, to some products (e.g. certain water-immiscible products). 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 referenced documents are indispensable for the application 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 21148:2005, Cosmetics — Microbiology — General instructions for microbiological examination EN 12353, Chemical disinfectants and antiseptics — Preservation of test organisms used for the determination of bactericidal, mycobactericidal, sporicidal and fungicidal activity kSIST FprEN ISO 18415:2011

ISO 18415:2007(E) 2 © ISO 2007 – All rights reserved 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 3.4 sample dilution dilution of the initial suspension 3.5 aerobic mesophilic microorganisms mesophilic bacteria or yeast growing aerobically under the conditions specified in this International Standard NOTE In the described conditions other types of microorganism (e.g. molds) are detectable. 3.6 specified microorganisms aerobic mesophilic bacteria or yeast undesirable in a cosmetic product and recognised as a skin pathogen species that may be harmful for human health or as an indication of hygienic failure in the manufacturing process 3.6.1 Pseudomonas aeruginosa Gram-negative rod (bacilli), motile, smooth colonies pigmented

brown or greenish NOTE 1 The main characteristics for identification are growth on a selective cetrimide agar medium, oxidase positive, production of diffusible fluorescent pigments and production of a soluble phenazine pigment (pyocyanin) in suitable media. NOTE 2 Pseudomonas aeruginosa can be isolated from a wide variety of environmental sources, especially in water and has a very high potential to spoil many different substrates. It can produce infections of human skin or eye areas. It is undesirable in cosmetic products for its potential pathogenicity and its capacity to affect the physico-chemical properties of the cosmetic formula. 3.6.2 Escherichia coli Gram-negative rod (bacilli), motile, smooth colonies NOTE 1 The main characteristics are catalase positive, oxidase negative, fermentation of lactose, production of indole, growth on selective medium containing bile salts with characteristic colonies. NOTE 2 Escherichia coli can be isolated from the moist environmental sources (air, water, soil) and is a faecal contamination indicator. 3.6.3 Staphylococus aureus Gram-positive cocci, mainly aggregated in grape-like clusters, smooth colonies generally pigmented in yellow NOTE 1 The main characteristics for identification are growth on a specific selective medium, catalase positive, coagulase positive. kSIST FprEN ISO 18415:2011

ISO 18415:2007(E) © ISO 2007 – All rights reserved 3NOTE 2 Staphylococcus aureus is an opportunistic pathogen for humans, which often can be also present on the skin of healthy individuals without causing them any apparent illness. It is a specified microorganism and undesirable in cosmetic products. 3.6.4 Candida albicans yeast that forms white to beige, creamy and convex colonies on the surface of a non-selective agar medium NOTE The main characteristics for identification are production of germ tube and/or pseudomycelium and chlamydospore when the test is performed following the method specified in this International Standard 3.7 non-specified microorganism aerobic mesophilic bacteria or yeast found in cosmetic products, not defined in 3.6 3.8 enrichment broth non-selective liquid medium containing suitable neutralizers and/or dispersing agents and validated 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 following steps (isolation and identification) are performed according to need by using appropriate conditions of incubation and suitable identification test, as described in this International Standard. The possible inhibition of microbial growth by the sample shall be neutralized to allow the detection of viable microorganisms [2]. In all cases and whatever the methodology, the neutralization of the antimicrobial properties of the product shall be checked and validated [2],[3],[4]. 5 Diluents and culture media 5.1 General General specifications are given in ISO 21148. When water is used in a formula, 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 C. The enrichment broth (5.3.2.1) or any of the ones listed in Annex B is suitable for checking the presence of specified and non-specified microorganisms in accordance with this International Standard provided that they are validated in accordance with Clause 11. Other diluents and culture media may be used if it has been demonstrated that they are suitable for use. 5.2 Diluent for the microbial suspension (tryptone sodium chloride solution) 5.2.1 General The diluent is used for the preparation of bacteria and yeast suspensions used for the validation procedure (see Clause 11). kSIST FprEN ISO 18415:2011

ISO 18415:2007(E) 4 © ISO 2007 – All rights reserved 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, 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 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. 5.3.2 Enrichment broth 5.3.2.1 Eugon LT100 broth 5.3.2.1.1 General This medium contains ingredients which neutralize inhibitory substances present in the sample: lecithin and polysorbate 80, and dispersing agent: octoxynol 9. 5.3.2.1.2 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 kSIST FprEN ISO 18415:2011

ISO 18415:2007(E) © ISO 2007 – All rights reserved 55.3.2.1.3 Preparation Dissolve the components polysorbate 80, octoxynol 9 and egg lecithin, one after another in boiling water to 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, the pH shall be equivalent to 7,0 ± 0,2 when measured at room temperature. 5.3.2.2 Other enrichment broths Other enrichment broths may be used as appropriate (see Annex B). 5.3.3 Non-selective agar medium 5.3.3.1 General This medium is used for the isolation and detection of specified and non-specified microorganisms present in the initial suspension after enrichment and for the preparation of inoculum used in the validation procedure. 5.3.3.2 Soybean-casein digest agar medium (SCDA) or tryptic soy agar (TSA) 5.3.3.2.1 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 5.3.3.2.2 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. 5.3.3.3 Other non-selective agar medium Other non-selective, non-neutralizing agar media may be used (see Annex B). 6 Apparatus and glassware The laboratory equipment, apparatus and glassware are described in ISO 21148. 7 Strains of microorganism For testing the recovery efficiency of the test conditions, three specified microorganisms are used: two strains representative of both Gram-negative and Gram-positive bacteria and a strain of yeast. ⎯ Pseudomonas aeruginosa ATCC 9027 (equivalent strain: CIP 82.118 or NCIMB 8626 or NBRC 13275 or KCTC 2513 or other equivalent national collection strain). kSIST FprEN ISO 18415:2011

ISO 18415:2007(E) 6 © ISO 2007 – All rights reserved An alternative to the Gram-negative strain may be: Escherichia coli ATCC 8739 (equivalent strain: CIP 53.126 or NCIMB 8545 or NBRC 3972 or KCTC 2571 or other equivalent national collection strain). ⎯ Staphylococcus aureus ATCC1) 6538 (equivalent strain: CIP2) 4.83 or NCIMB3) 9518 or NBRC4) 13276 or KCTC5) 1916 or other equivalent national collection strain); ⎯ Candida albicans ATCC 10231 (equivalent strain: IP6) 48.72 or NCPF7) 3179 or NBRC 1594 or KCTC 17205 or other equivalent national collection strain). The culture should be reconstituted according to the procedures provided by the supplier of reference strain. The strains can 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 (3.1) and samples (3.2) 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 given in Clause 9. 9 Procedure 9.1 General recommendations Use sterile material, equipment and aseptic techniques to prepare the sample, initial suspension and dilutions. In the case of the preparation of an initial suspension in an appropriate diluent before transfer in the enrichment broth, the time that elapses between the end of the preparation and the moment the initial suspension and/or sample dilutions come 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 in the enrichment broth 9.2.1 General The enrichment is prepared from a sample 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 mass 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).

1) American Type Culture Collection. 2) Collection de l’Institut Pasteur. 3) National Collection of Industrial and Marine Bacteria. 4) National Biological Resource Center. 5) Korean Collection for Type Culture. 6) Institut Pasteur. 7) National Collection of Pathogenic Fungi. kSIST FprEN ISO 18415:2011

ISO 18415:2007(E) © ISO 2007 – All rights reserved 7NOTE In some cases, and when possible, filtration of the cosmetic product through a membrane which is afterwards immersed in the enrichment broth facilitates the neutralization of the antimicrobial properties of the product (see 11.3.3). 9.2.2 Water-miscible products Transfer the sample, S, of product to a suitable container containing an appropriate volume (at least 9 ml) of broth. 9.2.3 Water-immiscible products Transfer the sample, S, of product to a suitable container containing a suitable quantity of dispersing agent (e.g. polysorbate 80). Disperse the sample within the solubilizing agent and add an appropriate volume (at least 9 ml) of broth. 9.2.4 Filterable products Use a membrane filter having a nominal pore size no greater than 0,45 µm. Transfer the sample, S, on to the membrane in a filtration apparatus (see ISO 21148). Filter immediately and wash the membrane using defined volumes of water and/or diluent. Transfer the membrane and immerse into a tube or flask of suitable size containing an appropriate volume (at least 9 ml) of enrichment broth. This preparation is equivalent to an initial suspension. 9.3 Incubation of the initial suspension Incubate the initial suspension prepared in broth (see 9.2) at 32,5 °C ± 2,5 °C for at least 20 h. 9.4 Isolation of specified and non-specified microorganisms Using a sterile loop, streak an aliquot of the incubated enrichment broth on to the surface of a Petri dish (diameter 85 mm to 100 mm) containing approximately 15 ml to 20 ml of non-selective agar medium. If larger Petri dishes are used, the volume of the agar is increased accordingly. Invert the Petri dish and incubate at 32,5 °C ± 2,5 °C for 48 h to 72 h. The procedure for identification of colonies is described below for Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Candida albicans and for others microorganisms described in 9.9. 9.5 Procedure for identification of the specified microorganism: Pseudomonas aeruginosa 9.5.1 Gram staining Proceed to a Gram staining as described in ISO 21148 on a part of suspect8) colony well isolated from a surface of soybean-casein digest agar. The microscopic observation of the Gram stain shall reveal Gram-negative rods. 9.5.2 Oxidase test Follow the procedure specified in ISO 21148. Check for oxidase positive test.

8) Suspect for Pseudomonas aeruginosa means smooth colonies generally pigmented greenish to yellowish. kSIST FprEN ISO 18415:2011

ISO 18415:2007(E) 8 © ISO 2007 – All rights reserved 9.5.3 Identification test Use a suitable identification protocol for non-fermenting Gram-negative rod (i.e. standardized and/or miniaturized biochemical system) with a dedicated database (or equivalent like a catalogue) including the typical characteristics of Pseudomonas aeruginosa. When using an identification kit, follow the instructions given

...

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