SIST EN ISO 18415:2017

SLOVENSKI STANDARD
oSIST prEN ISO 18415:2017
01-februar-2017
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Cosmetics - Microbiology - Detection of specified and non-specified microorganisms
(ISO/FDIS 18415:2017)
Kosmetische Mittel - Mikrobiologie - Nachweis von spezifizierten und nichtspezifizierten
Mikroorganismen (ISO/FDIS 18415:2017)
Cosmétiques - Microbiologie - Détection des micro-organismes spécifiés et non spécifiés
(ISO/FDIS 18415:2017)
Ta slovenski standard je istoveten z: prEN ISO 18415
ICS:
07.100.40 Kozmetika - mikrobiologija Cosmetics microbiology
oSIST prEN ISO 18415:2017 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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oSIST prEN ISO 18415:2017

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oSIST prEN ISO 18415:2017
FINAL
INTERNATIONAL ISO/FDIS
DRAFT
STANDARD 18415
ISO/TC 217
Cosmetics — Microbiology —
Secretariat: ISIRI
Detection of specified and non-
Voting begins on:
2017­01­02 specified microorganisms
Voting terminates on:
Cosmétiques — Microbiologie — Détection des micro-organismes
2017­03­27
spécifiés et non spécifiés
ISO/CEN PARALLEL PROCESSING
RECIPIENTS OF THIS DRAFT ARE INVITED TO
SUBMIT, WITH THEIR COMMENTS, NOTIFICATION
OF ANY RELEVANT PATENT RIGHTS OF WHICH
THEY ARE AWARE AND TO PROVIDE SUPPOR TING
DOCUMENTATION.
IN ADDITION TO THEIR EVALUATION AS
Reference number
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO­
ISO/FDIS 18415:2017(E)
LOGICAL, COMMERCIAL AND USER PURPOSES,
DRAFT INTERNATIONAL STANDARDS MAY ON
OCCASION HAVE TO BE CONSIDERED IN THE
LIGHT OF THEIR POTENTIAL TO BECOME STAN­
DARDS TO WHICH REFERENCE MAY BE MADE IN
©
NATIONAL REGULATIONS. ISO 2017

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oSIST prEN ISO 18415:2017
ISO/FDIS 18415:2017(E)

COPYRIGHT PROTECTED DOCUMENT
© ISO 2017, Published in Switzerland
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form
or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior
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copyright@iso.org
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ii © ISO 2017 – All rights reserved

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oSIST prEN ISO 18415:2017
ISO/FDIS 18415:2017(E)

Contents Page
Foreword .v
Introduction .vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
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
5.2.1 General. 3
5.2.2 Composition . 4
5.2.3 Preparation . 4
5.3 Culture media . 4
5.3.1 General. 4
5.3.2 Enrichment broth . 4
5.3.3 Non­selective agar medium . 5
6 Apparatus and glassware . 5
7 Strains of microorganism . 6
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. 7
9.2.3 Water­immiscible products . 7
9.2.4 Filterable products . 7
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
9.5.1 Gram staining . 7
9.5.2 Oxidase test . 7
9.5.3 Identification test . 8
9.6 Procedure for identification of the specified microorganism: Escherichia coli . 8
9.6.1 Gram staining . 8
9.6.2 Oxidase test . 8
9.6.3 Identification test . 8
9.7 Procedure for identification of the specified microorganism: Staphylococcus aureus . 8
9.7.1 Gram staining . 8
9.7.2 Catalase test . 8
9.7.3 Identification test . 8
9.8 Procedure for the identification of the specified microorganism: Candida albicans . 9
9.8.1 Gram staining . 9
9.8.2 Identification test . 9
9.9 Procedure for the identification of non-specified microorganisms . 9
9.9.1 Gram staining . 9
9.9.2 Oxidase test . 9
9.9.3 Catalase test . 9
9.9.4 Identification test . 9
10 Expression of the results .10
10.1 Detection of specified microorganisms .10
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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 Suitability of detection method by enrichment .10
11.3.1 Principle .10
11.3.2 Procedure .11
11.3.3 Interpretation of suitability test results .11
12 Test report .11
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 .19
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oSIST prEN ISO 18415:2017
ISO/FDIS 18415:2017(E)

Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non­governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation on the meaning of ISO specific terms and expressions related to conformity assessment,
as well as information about ISO’s adherence to the World Trade Organization (WTO) principles in the
Technical Barriers to Trade (TBT) see the following URL: www.iso.org/iso/foreword.html.
The committee responsible for this document is ISO/TC 217, Cosmetics.
This second edition cancels and replaces the first edition (ISO 18415:2007), of which it constitutes a
minor revision with the following changes:
— the term “validated” in 3.8 has been changed to “demonstrated to be suitable”;
— the term “validated” in Clause 4 has been changed to “demonstrated”;
— the phrase “are validated” in 5.1 has been changed to “have been demonstrated to be suitable”;
— instances of the term “validation” in 5.2.1, 5.3.3.1, 11.3.1, 11.3.2 and in the heading title of 11.3.3
have been changed to “suitability test”;
— the term “validation” in the heading title of 11.3 has been changed to “suitability”;
— instances of “validated” in 11.3.3 have been changed to “satisfactory”;
— the term “validation” in Clause 12 f) has been changed to “demonstration of the suitability”.
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oSIST prEN ISO 18415:2017
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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.
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oSIST prEN ISO 18415:2017
FINAL DRAFT INTERNATIONAL STANDARD ISO/FDIS 18415:2017(E)
Cosmetics — Microbiology — Detection of specified and
non-specified microorganisms
1 Scope
This document 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 document 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
document 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 document 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 non­selective agar media. Other methods can be appropriate depending on the level
of detection required.
In this document 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 document 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 documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
1)
ISO 21148:— , Cosmetics — Microbiology — General instructions for microbiological examination
EN 12353, Chemical disinfectants and antiseptics — Preservation of test organisms used for the
determination of bactericidal, mycobactericidal, sporicidal and fungicidal activity
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— IEC Electropedia: available at http://www.electropedia.org/
1) To be published.
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— ISO Online browsing platform: available at http://www.iso.org/obp
3.1
product
portion of an identified cosmetic product received in the laboratory for testing
3.2
sample
portion of the product (3.1) (at least 1 g or 1 ml) that is used in the test to prepare the initial
suspension (3.3)
3.3
initial suspension
suspension (or solution) of the sample (3.2) in a defined volume of an appropriate enrichment broth (3.8)
3.4
sample dilution
dilution of the initial suspension (3.3)
3.5
aerobic mesophilic microorganisms
mesophilic bacteria or yeast growing aerobically under the conditions specified in this document
Note 1 to entry: 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 recognized as a skin
pathogen species that may be harmful for human health or as an indication of hygienic failure in the
manufacturing process
3.6.1
Pseudomonas aeruginosa
Gram­negative rod (bacilli), motile, smooth colonies pigmented brown or greenish
Note 1 to entry: 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 to entry: 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
Gram­negative rod (bacilli), motile, smooth colonies
Note 1 to entry: 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 to entry: 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 to entry: The main characteristics for identification are growth on a specific selective medium, catalase
positive, coagulase positive.
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Note 2 to entry: 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 1 to entry: 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 document.
3.7
non-specified microorganism
aerobic mesophilic bacteria or yeast found in cosmetic products, not defined in 3.6
3.8
enrichment broth
non­selective liquid medium containing suitable neutralizers and/or dispersing agents and
demonstrated to be suitable for the product (3.1) 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 document.
The possible inhibition of microbial growth by the sample shall be neutralized to allow the detection
[9]
of viable microorganisms . In all cases and whatever the methodology, the neutralization of the
[9][10][11]
antimicrobial properties of the product shall be checked and demonstrated .
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 (see 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 document 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.
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 suitability test
procedure (see Clause 11).
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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 with
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
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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
suitability test 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 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 App
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