SIST EN ISO 21149:2017

SLOVENSKI STANDARD
oSIST prEN ISO 21149:2017
01-februar-2017
Kozmetika - Mikrobiologija - Ugotavljanje prisotnosti in števila aerobnih mezofilnih
bakterij (ISO/FDIS 21149:2017)
Cosmetics - Microbiology - Enumeration and detection of aerobic mesophilic bacteria
(ISO/FDIS 21149:2017)
Kosmetische Mittel - Mikrobiologie - Zählung und Nachweis von aeroben mesophilen
Bakterien (ISO/FDIS 21149:2017)
Cosmétiques - Microbiologie - Dénombrement et détection des bactéries aérobies
mésophiles (ISO/FDIS 21149:2017)
Ta slovenski standard je istoveten z: prEN ISO 21149
ICS:
07.100.40 Kozmetika - mikrobiologija Cosmetics microbiology
oSIST prEN ISO 21149:2017 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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oSIST prEN ISO 21149:2017

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oSIST prEN ISO 21149:2017
FINAL
INTERNATIONAL ISO/FDIS
DRAFT
STANDARD 21149
ISO/TC 217
Cosmetics — Microbiology —
Secretariat: ISIRI
Enumeration and detection of aerobic
Voting begins on:
2017­01­02 mesophilic bacteria
Voting terminates on:
Cosmétiques — Microbiologie — Dénombrement et détection des
2017­03­27
bactéries aérobies mésophiles
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 21149:2017(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 21149:2017
ISO/FDIS 21149:2017(E)

COPYRIGHT PROTECTED DOCUMENT
© ISO 2017, Published in Switzerland
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form
or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior
written permission. Permission can be requested from either ISO at the address below or ISO’s member body in the country of
the requester.
ISO copyright office
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Tel. +41 22 749 01 11
Fax +41 22 749 09 47
copyright@iso.org
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oSIST prEN ISO 21149:2017
ISO/FDIS 21149:2017(E)

Contents Page
Foreword .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
4.1 General . 2
4.2 Plate count . 2
4.3 Membrane filtration . 2
4.4 Detection of bacteria by enrichment . 2
5 Diluents, neutralizers and culture media . 3
5.1 General . 3
5.2 Neutralizing diluents and diluents . 3
5.3 Diluent for the bacterial suspension (tryptone sodium chloride solution) . 4
5.4 Culture media . 4
6 Apparatus and glassware . 7
7 Strains of microorganisms . 7
8 Handling of cosmetic products and laboratory samples . 7
9 Procedure. 7
9.1 General recommendation . 7
9.2 Preparation of the initial suspension . 7
9.2.1 General. 7
9.2.2 Water­miscible products. 8
9.2.3 Water­immiscible products . 8
9.3 Counting methods . 8
9.3.1 Dilutions for counting methods . 8
9.3.2 Plate­count methods . 8
9.4 Enrichment . 9
9.4.1 General. 9
9.4.2 Incubation of the sample . 9
10 Counting of colonies (plate counts and membrane filtration methods) .9
11 Detection of growth (enrichment method) . 9
12 Expression of results .10
12.1 Method of calculation for plate count .10
12.2 Interpretation .11
12.3 Examples .11
12.4 Detection after enrichment .13
13 Neutralization of the antimicrobial properties of the product .13
13.1 General .13
13.2 Preparation of inoculum .14
13.3 Suitability of counting methods .14
13.3.1 Principle .14
13.3.2 Suitability of the pour-plate method .14
13.3.3 Suitability of the surface spread method .14
13.3.4 Suitability of the membrane filtration method .14
13.4 Suitability of the detection method by enrichment .15
13.4.1 Procedure .15
13.4.2 Interpretation of results .15
13.5 Interpretation of suitability test results .15
14 Test report .16
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Annex A (informative) Other neutralizing diluents .17
Annex B (informative) Other diluents .19
Annex C (informative) Other culture media .20
Annex D (informative) Neutralizers of antimicrobial activity of preservatives and
rinsing liquids .23
Bibliography .24
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oSIST prEN ISO 21149:2017
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Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non­governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation on the meaning of ISO specific terms and expressions related to conformity assessment,
as well as information about ISO’s adherence to the World Trade Organization (WTO) principles in the
Technical Barriers to Trade (TBT) see the following URL: www.iso.org/iso/foreword.html.
The committee responsible for this document is ISO/TC 217, Cosmetics.
This second edition cancels and replaces the first edition (ISO 21149:2006), of which it constitutes a
minor revision with the following changes:
— in 4.1, “validated” has been changed to “demonstrated”;
— in 4.3, “validated” has been changed to “described”;
— in 9.3.2.1, 9.3.2.2 and 9.3.2.3, “validated” has been changed to “described”;
— in 9.3.2.3, “procedure developed during the validation” has been changed to “suitability test
procedure”;
— in 9.4.1, “validation” has been changed to “suitability test”;
— in 12.2.1, “validated according to the chosen method” has been changed to “demonstrated to be
suitable for the chosen method”;
— in 13.3 and 13.4, “validation” has been changed to “suitability”;
— in 13.3.2, 13.3.3 and 13.3.4, “validation” has been changed to “suitability”;
— in 13.3.2, 13.3.3 and 13.3.4, “if the validation count is at least 50 % (0,3 log) of the control count” has
been changed to “if the count is at least 50 % of the control”;
— in 13.4.1, instances of “validation test” have been changed to “suitability test”;
— in 13.4.2, instances of “validation plate” have been changed to “suitability test plate”;
— in 13.5, “validation results” has been changed to “suitability test results” and “validation plates” has
been changed to “suitability test plates”;
— in Clause 14 f), “validation of the method” has been changed to “demonstration of the suitability”;
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— in A.1, B.1 and C.1, “validated” has been changed to “demonstrated to be suitable”.
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oSIST prEN ISO 21149:2017
FINAL DRAFT INTERNATIONAL STANDARD ISO/FDIS 21149:2017(E)
Cosmetics — Microbiology — Enumeration and detection
of aerobic mesophilic bacteria
1 Scope
This document gives general guidelines for enumeration and detection of mesophilic aerobic bacteria
present in cosmetics
— by counting the colonies on agar medium after aerobic incubation, or
— by checking the absence of bacterial growth after enrichment.
Because of the large variety of cosmetic products within this field of application, this method may not be
appropriate for some products in every detail (e.g. certain water immiscible products). Other methods
(e.g. automated) may be substituted for the tests presented here provided that their equivalence has
been demonstrated or the method has been otherwise validated.
If needed, microorganisms enumerated or detected may be identified using suitable identification tests
described in the standards given in the Bibliography.
In order to ensure product quality and safety for consumers, it is advisable to perform an appropriate
microbiological risk analysis, so as to determine the types of cosmetic products to which this document
is applicable. Products considered to present a low microbiological risk include those with low water
activity, hydro-alcoholic products, extreme pH values, etc.
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 (including Legionella), mycobactericidal, sporicidal, fungicidal and virucidal
(including bacteriophages) activity
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— IEC Electropedia: available at http://www.electropedia.org/
— ISO Online browsing platform: available at http://www.iso.org/obp
3.1
aerobic mesophilic bacteria
mesophilic bacteria growing aerobically under the conditions specified in this document
Note 1 to entry: In the described conditions, other types of microorganisms (e.g. yeast, mould) can be detected.
1) To be published.
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3.2
product
portion of an identified cosmetic product received in the laboratory for testing
3.3
sample
portion of the product (at least 1 g or 1 ml) which is used in the test to prepare the initial suspension
3.4
initial suspension
suspension (or solution) of a sample in a defined volume of an appropriate liquid (diluent, neutralizer,
broth or combination of them)
3.5
sample dilution
dilution of the initial suspension
4 Principle
4.1 General
This method involves enumeration of colonies on a non-selective agar medium or by the presence
or absence of bacterial growth after enrichment. The possible inhibition of microbial growth by the
[7]
sample shall be neutralized to allow the detection of viable microorganism . In all cases and whatever
the methodology, the neutralization of the antimicrobial properties of the product shall be checked and
[8][9][10]
demonstrated (see Clause 13) .
4.2 Plate count
Plate count consists of the following steps.
a) Preparation of poured plates or spread plates, using a specified culture medium, and inoculation of
the plates using a defined quantity of the initial suspension or dilution of the product.
b) Aerobic incubation of the plates at 32,5 °C ± 2,5 °C for 72 h ± 6 h.
c) Counting the number of colony forming units (CFU) and calculation of the number of aerobic
mesophilic bacteria per millilitre or per gram of product.
4.3 Membrane filtration
Membrane filtration consists of the following steps.
a) Transfer a suitable amount of the sample prepared as described in Clause 13 in the filtration
apparatus wetted with a small volume of an appropriate sterile diluent, filter immediately and
wash according to the validated procedure (see 13.3.4). Transfer the membrane filter onto the
surface of the specified agar medium as specified in ISO 21148.
b) Aerobic incubation of the membranes at 32,5 °C ± 2,5 °C for 72 h ± 6 h.
c) Counting the number of colony forming units (CFU) and calculation of the number of aerobic
mesophilic bacteria per millilitre or per gram of product.
4.4 Detection of bacteria by enrichment
Detection of bacteria by enrichment consists of the following steps.
a) Incubation at 32,5 °C ± 2,5 °C for at least 20 h of a defined quantity of the initial suspension in a
non­selective liquid medium containing suitable neutralizers and/or dispersing agents.
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b) Transfer of a defined quantity of the previous suspension on non-selective solid agar medium.
c) Aerobic incubation at 32,5 °C ± 2,5 °C for 48 h to 72 h.
d) Detection of growth and expression of results as “presence/absence” of aerobic mesophilic bacteria
per sample S of product.
5 Diluents, neutralizers 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 following diluents, neutralizers and culture media are suitable for enumeration and detection of
aerobic mesophilic bacteria. Other diluents, neutralizers and culture media may be used if they have
been demonstrated to be suitable for use.
5.2 Neutralizing diluents and diluents
5.2.1 General
The diluent is used to disperse the sample. It may contain neutralizers if the specimen to be tested
has antimicrobial properties. The efficacy of the neutralization shall be demonstrated before the
determination of the count (see Clause 13). Information relative to suitable neutralizers is given in
Annex D.
5.2.2 Neutralizing diluents
5.2.2.1 Fluid casein digest–soy lecithin–polysorbate 20 medium (SCDLP 20 broth)
5.2.2.1.1 Composition
Pancreatic digest of casein 20,0 g
Soy lecithin 5,0 g
Polysorbate 20 40,0 ml
Water 960,0 ml
5.2.2.1.2 Preparation
Dissolve the polysorbate 20 in 960 ml of water by mixing while heating in a water bath at 49 °C ± 2 °C.
Add pancreatic digest of casein and soy lecithin. Heat for about 30 min to obtain solution. Mix and
dispense the medium into suitable containers. Sterilize in the autoclave at 121 °C for 15 min. After
sterilization, the pH shall be equivalent to 7,3 ± 0,2 when measured at room temperature.
5.2.2.2 Other neutralizing diluents
Other neutralizing diluents may be used as appropriate (see Annex A and Annex D).
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5.2.3 Diluent
5.2.3.1 Fluid A
5.2.3.1.1 Composition
Peptic digest of animal tissue 1,0 g
Water 1 000 ml
5.2.3.1.2 Preparation
Dissolve 1 g of peptone in water to make 1 l. Heat with frequent agitation. Dispense into suitable
containers. Sterilize in the autoclave at 121 °C for 15 min. After sterilization, the pH shall be equivalent
to 7,1 ± 0,2 when measured at room temperature.
5.2.3.2 Other diluents
Other diluents may be used as appropriate (see Annex B).
5.3 Diluent for the bacterial suspension (tryptone sodium chloride solution)
5.3.1 Composition
Tryptone, pancreatic digest of casein 1,0 g
Sodium chloride 8,5 g
Water 1 000 ml
5.3.2 Preparation
Dissolve the components in the water by mixing while heating. Dispense into suitable containers.
Sterilize in the autoclave at 121 °C for 15 min. After sterilization, the pH shall be equivalent to 7,0 ± 0,2
when measured at room temperature.
5.4 Culture media
5.4.1 General
Culture media may be prepared as follows or from dehydrated culture media according to the
instructions of the manufacturer. Ready-to-use media may be used when their composition and/or
growth yields are comparable to those of the formulas given herein.
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5.4.2 Culture media for counting
5.4.2.1 Soybean–casein digest agar medium (SCDA) or tryptic soy agar (TSA)
5.4.2.1.1 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
5.4.2.1.2 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.
5.4.2.2 Other media for counting
Other media may be used as appropriate (see Annex C).
5.4.3 Culture media for detection
5.4.3.1 General
When chosen, an enrichment broth and an agar medium shall be used for bacterial detection.
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.
5.4.3.2 Enrichment broth: Eugon LT 100 broth
5.4.3.2.1 General
This medium contains ingredients
— which neutralize inhibitory substances present in the sample: lecithin and polysorbate 80, and
— dispersing agent: octoxynol 9.
5.4.3.2.2 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
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Glucose 5,5 g
Egg lecithin 1,0 g
Polysorbate 80 5,0 g
Octoxynol 9 1,0 g
Water 1 000 ml
5.4.3.2.3 Preparation
Dissolve successively polysorbate 80, octoxynol 9 and egg lecithin 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, the pH shall
be equivalent to 7,0 ± 0,2 when measured at room temperature.
5.4.3.3 Agar media for detection
5.4.3.3.1 Eugon LT 100 agar medium
5.4.3.3.1.1 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
Agar 15,0 g
Water 1 000 ml
5.4.3.3.1.2 Preparation
Dissolve successively polysorbate 80, octoxynol 9 and egg lecithin into boiling water until their complete
dissolution. Dissolve the other components by mixing while heating. Mix gently to avoid foam. 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.
5.4.3.3.2 Other agar media for detection
Other media may be used as appropriate (see Annex C).
5.4.4 Agar medium for cultivation of reference strains
Use soybean-casein digest agar medium (SCDA) or tryptic soy agar (TSA) (5.4.2.1).
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6 Apparatus and glassware
The laboratory equipment, apparatus and glassware are described in ISO 21148.
7 Strains of microorganisms
For testing the efficacy of neutralizers, two strains representative of both Gram negative and Gram
[8][11]
positive microorganisms , respectively, are used:
2) 3) 4) 5)
— Pseudomonas aeruginosa ATCC 9027 (equivalent strain: CIP 82.118 or NCIMB 8626 or NBRC
6)
13275 or KCTC 2513 or other equivalent national collection strain);
— Staphylococcus aureus ATCC 6538 (equivalent strain: CIP 4.83 or NCIMB 9518 or NBRC 13276 or
KCTC 1916 or other equivalent national collection strain).
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).
The culture should be reconstituted according to the procedures provided by the supplier of
reference strain.
The strains may 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 (3.2) and samples (3.3) before or after analysis.
Sampling of cosmetic products to be analysed should be carried out, as described in ISO 21148. Analyse
samples as specified in ISO 21148 and in accordance
...