EN ISO 11930:2019
(Main)Cosmetics - Microbiology - Evaluation of the antimicrobial protection of a cosmetic product (ISO 11930:2019)
Cosmetics - Microbiology - Evaluation of the antimicrobial protection of a cosmetic product (ISO 11930:2019)
This document specifies a procedure for the interpretation of data generated by the preservation efficacy test or by the microbiological risk assessment, or both, when evaluating the overall antimicrobial protection of a cosmetic product.
It comprises:
— a preservation efficacy test;
— a procedure for evaluating the overall antimicrobial protection of a cosmetic product that is not considered low risk, based on a risk assessment described in ISO 29621.
The preservation efficacy test is a reference method to evaluate the preservation of a cosmetic formulation. It is applicable to cosmetic products in the marketplace.
This test does not apply to those cosmetic products for which the microbiological risk has been determined to be low according to Annex A and ISO 29621.
This test is primarily designed for water-soluble or water-miscible cosmetic products and can be used with modification to test products in which water is the internal (discontinuous) phase.
NOTE This test can be used as a guideline to establish a development method during the development cycle of cosmetic products. In this case, the test can be modified or extended, or both, for example, to make allowance for prior data and different variables (microbial strains, media, incubation conditions exposure time, etc.). Compliance criteria can be adapted to specific objectives. During the development stage of cosmetic products, other methods, where relevant, can be used to determine the preservation efficacy of formulations.
Kosmetische Mittel - Mikrobiologie - Bewertung des antimikrobiellen Schutzes eines kosmetischen Mittels (ISO 11930:2019)
Cosmétiques - Microbiologie - Évaluation de la protection antimicrobienne d'un produit cosmétique (ISO 11930:2019)
Le présent document spécifie un mode opératoire pour l'interprétation des données résultant de l'essai d'efficacité de la protection antimicrobienne et/ou de l'appréciation du risque microbiologique lors de l'évaluation globale de la protection antimicrobienne d'un produit cosmétique.
Il comprend:
— un essai d'efficacité de la protection antimicrobienne; et
— un mode opératoire permettant d'évaluer la protection antimicrobienne globale d'un produit cosmétique qui n'est pas identifié comme étant à faible risque microbiologique d'après l'appréciation du risque décrite dans l'ISO 29621.
L'essai d'efficacité de la protection antimicrobienne est une méthode de référence pour évaluer la protection antimicrobienne d'une formulation cosmétique. Il s'applique aux produits cosmétiques disponibles sur le marché.
Cet essai n'est pas applicable aux produits cosmétiques pour lesquels il a été démontré que le risque microbiologique est faible, conformément à l'Annexe A et à l'ISO 29621.
Cet essai est principalement conçu pour les produits cosmétiques solubles dans l'eau ou miscibles à l'eau, et peut être utilisé avec modification pour soumettre à essai des produits dont la phase interne (discontinue) est aqueuse.
NOTE Cet essai peut servir de ligne directrice pour établir une méthode de développement au cours du cycle de développement d'un produit cosmétique. Dans ce cas, l'essai peut être modifié et/ou élargi, par exemple pour prendre en compte des données antérieures et différents paramètres (souches microbiennes, milieux, conditions et durée d'incubation, etc.). Les critères de conformité peuvent être adaptés à des objectifs spécifiques. Au cours de la phase de développement des produits cosmétiques, d'autres méthodes peuvent être utilisées, le cas échéant, pour déterminer l'efficacité de la protection antimicrobienne des formulations.
Kozmetika - Mikrobiologija - Vrednotenje protimikrobne zaščite kozmetičnih izdelkov (ISO 11930:2019)
Ta dokument določa postopek za interpretacijo podatkov, pridobljenih s preskusom ohranjanja učinkovitosti ali z oceno mikrobiološkega tveganja ali obojega pri vrednotenju skupne protimikrobne zaščite kozmetičnih izdelkov.
Vključuje:
– preskus ohranjanja učinkovitosti;
– postopek za ocenjevanje celotne protimikrobne zaščite kozmetičnih izdelkov, ki na podlagi ocene tveganja, navedene v standardu ISO 29621, ne veljajo za izdelke z nizkim tveganjem.
Preskus ohranjanja učinkovitosti je referenčna metoda za vrednotenje ohranjanja kozmetične formulacije. Uporablja se za kozmetične izdelke na trgu.
Ta preskus se ne uporablja za tiste kozmetične izdelke, za katere je bilo ugotovljeno, da je njihovo mikrobiološko tveganje nizko v skladu z dodatkom A in standardom ISO 29621.
Ta preskus je primarno zasnovan za kozmetične izdelke, ki so topni v vodi ali se mešajo z vodo, in jih je z modifikacijo mogoče uporabiti za preskušanje proizvodov, v katerih je voda notranja (prekinjena) faza.
OPOMBA: Ta preskus je mogoče uporabiti kot smernico za vzpostavitev razvojne metode med razvojnim ciklom kozmetičnih izdelkov. V tem primeru je mogoče preskus spremeniti ali razširiti ali oboje, na primer, da se upoštevajo predhodni podatki in različne spremenljivke (mikrobni sevi, mediji, čas izpostavljenosti v pogojih inkubacije itd.).
Merila skladnosti je mogoče prilagoditi posebnim ciljem. V fazi razvoja kozmetičnih izdelkov je mogoče za določitev učinkovitosti ohranjanja formulacij uporabiti druge metode, kjer je to ustrezno.
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
01-julij-2019
Nadomešča:
SIST EN ISO 11930:2012
Kozmetika - Mikrobiologija - Vrednotenje protimikrobne zaščite kozmetičnih
izdelkov (ISO 11930:2019)
Cosmetics - Microbiology - Evaluation of the antimicrobial protection of a cosmetic
product (ISO 11930:2019)
Kosmetische Mittel - Mikrobiologie - Bewertung des antimikrobiellen Schutzes eines
kosmetischen Produktes (ISO 11930:2019)
Cosmétiques - Microbiologie - Évaluation de la protection antimicrobienne d'un produit
cosmétique (ISO 11930:2019)
Ta slovenski standard je istoveten z: EN ISO 11930:2019
ICS:
07.100.40 Kozmetika - mikrobiologija Cosmetics microbiology
71.100.70 Kozmetika. Toaletni Cosmetics. Toiletries
pripomočki
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
EN ISO 11930
EUROPEAN STANDARD
NORME EUROPÉENNE
February 2019
EUROPÄISCHE NORM
ICS 07.100.40 Supersedes EN ISO 11930:2012
English Version
Cosmetics - Microbiology - Evaluation of the antimicrobial
protection of a cosmetic product (ISO 11930:2019)
Cosmétiques - Microbiologie - Évaluation de la Kosmetische Mittel - Mikrobiologie - Bewertung des
protection antimicrobienne d'un produit cosmétique antimikrobiellen Schutzes eines kosmetischen
(ISO 11930:2019) Produktes (ISO 11930:2019)
This European Standard was approved by CEN on 27 December 2018.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this
European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references
concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN
member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by
translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management
Centre has the same status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2019 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 11930:2019 E
worldwide for CEN national Members.
Contents Page
European foreword . 3
European foreword
This document (EN ISO 11930:2019) has been prepared by Technical Committee ISO/TC 217
"Cosmetics" in collaboration with Technical Committee CEN/TC 392 “Cosmetics” the secretariat of
which is held by AFNOR.
This European Standard shall be given the status of a national standard, either by publication of an
identical text or by endorsement, at the latest by August 2019, and conflicting national standards shall
be withdrawn at the latest by August 2019.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
This document supersedes EN ISO 11930:2012.
This document has been prepared under a mandate given to CEN by the European Commission and the
European Free Trade Association.
According to the CEN-CENELEC Internal Regulations, the national standards organizations of the
following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,
Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia,
France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta,
Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and the United Kingdom.
Endorsement notice
The text of ISO 11930:2019 has been approved by CEN as EN ISO 11930:2019 without any modification.
INTERNATIONAL ISO
STANDARD 11930
Second edition
2019-01
Cosmetics — Microbiology —
Evaluation of the antimicrobial
protection of a cosmetic product
Cosmétiques — Microbiologie — Évaluation de la protection
antimicrobienne d'un produit cosmétique
Reference number
ISO 11930:2019(E)
©
ISO 2019
ISO 11930:2019(E)
© ISO 2019
All rights reserved. Unless otherwise specified, or required in the context of its implementation, 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
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2019 – All rights reserved
ISO 11930:2019(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Preservation efficacy test . 3
5.1 General . 3
5.2 Materials, apparatus, reagents and culture media . 3
5.2.1 General. 3
5.2.2 Materials . 3
5.2.3 Diluents . 3
5.2.4 Neutralizer . 4
5.2.5 Culture media . 5
5.3 Microbial strains . 6
5.4 Preparation and enumeration of inocula . 7
5.4.1 General. 7
5.4.2 Preparation of bacterial and Candida albicans suspensions . 7
5.4.3 Preparation of Aspergillus brasiliensis spore suspension . 8
5.5 Demonstration of the neutralizer efficacy . 9
5.5.1 Principle . 9
5.5.2 Procedure . 9
5.5.3 Calculations . 9
5.5.4 Interpretation of results and conclusion on neutralizer efficacy.10
5.6 Determination of the preservation efficacy of the formulation .10
5.6.1 Procedure .10
5.6.2 Counting of colonies.11
5.6.3 Calculations .11
5.7 Interpretation of test results and conclusions .12
5.7.1 Criteria .12
5.7.2 General case (efficacy of the neutralizer is demonstrated for all strains) .13
5.7.3 Case of formulations for which the efficacy of the neutralizer is not
demonstrated for some strains .13
5.8 Test report .13
6 Overall evaluation of the antimicrobial protection of the cosmetic product .14
6.1 General .14
6.2 Case 1 — Preservation efficacy test has been performed on the formulation.14
6.3 Case 2 — Preservation efficacy test has not been performed on the formulation .15
Annex A (normative) Decision diagram .16
Annex B (normative) Evaluation criteria for the preservation efficacy test .17
Annex C (informative) Examples of neutralizers for the antimicrobial activity of
preservatives and washing liquids .18
Annex D (informative) Packaging characteristics .20
Bibliography .21
ISO 11930:2019(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www .iso .org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO's adherence to the
World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see www .iso
.org/iso/foreword .html.
This document was prepared by Technical Committee ISO/TC 217, Cosmetics.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www .iso .org/members .html.
This second edition cancels and replaces the first edition (ISO 11930:2012), which has been technically
revised. The main changes compared to the previous edition are as follows.
— Two types of diluents, composition 1 and composition 2 can be used as the diluents for bacteria and
Candida albicans on the revised version (5.2.3).
— 5.6.2 Paragraph 2 has been changed to “When counts of surviving microorganisms obtained in
5.6.1.4 c) are less than 30 for bacteria and C. albicans or less than 15 for A. brasiliensis at the dilution
where neutralization has been checked, record the number of colonies on Petri dishes and express
results by multiplying by the dilution factor. If no colonies are observed at the dilution where
neutralization has been checked, note the result as <1 and multiply by the dilution factor.”
iv © ISO 2019 – All rights reserved
ISO 11930:2019(E)
Introduction
This document is designed to be used in the overall evaluation of the antimicrobial protection of a
cosmetic product.
The antimicrobial protection of a product can come from many sources:
— chemical preservation;
— inherent characteristics of the formulation;
— package design;
— manufacturing process.
This document defines a series of steps to be taken when assessing the overall antimicrobial protection
of a cosmetic product. A reference method for a preservation efficacy test (challenge test) along with
evaluation criteria is also described in this document.
The test described in this document involves, for each test microorganism, placing the formulation in
contact with a calibrated inoculum, and then measuring the changes in the microorganism count at set
time intervals for a set period and at a set temperature.
The data generated by the risk assessment (see ISO 29621) or by the preservation efficacy test, or both,
are used to establish the level of antimicrobial protection required to minimize user risk.
INTERNATIONAL STANDARD ISO 11930:2019(E)
Cosmetics — Microbiology — Evaluation of the
antimicrobial protection of a cosmetic product
1 Scope
This document specifies a procedure for the interpretation of data generated by the preservation
efficacy test or by the microbiological risk assessment, or both, when evaluating the overall
antimicrobial protection of a cosmetic product.
It comprises:
— a preservation efficacy test;
— a procedure for evaluating the overall antimicrobial protection of a cosmetic product that is not
considered low risk, based on a risk assessment described in ISO 29621.
The preservation efficacy test is a reference method to evaluate the preservation of a cosmetic
formulation. It is applicable to cosmetic products in the marketplace.
This test does not apply to those cosmetic products for which the microbiological risk has been
determined to be low according to Annex A and ISO 29621.
This test is primarily designed for water-soluble or water-miscible cosmetic products and can be used
with modification to test products in which water is the internal (discontinuous) phase.
NOTE This test can be used as a guideline to establish a development method during the development cycle
of cosmetic products. In this case, the test can be modified or extended, or both, for example, to make allowance
for prior data and different variables (microbial strains, media, incubation conditions exposure time, etc.).
Compliance criteria can be adapted to specific objectives. During the development stage of cosmetic products,
other methods, where relevant, can be used to determine the preservation efficacy of formulations.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
ISO 16212, Cosmetics — Microbiology — Enumeration of yeast and mould
ISO 18415, Cosmetics — Microbiology — Detection of specified and non-specified microorganisms
ISO 21148:2017, Cosmetics — Microbiology — General instructions for microbiological examination
ISO 21149, Cosmetics — Microbiology — Enumeration and detection of aerobic mesophilic bacteria
ISO 29621, Cosmetics — Microbiology — Guidelines for the risk assessment and identification of
microbiologically low-risk products
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 21148 and the following apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https: //www .iso .org/obp
ISO 11930:2019(E)
— IEC Electropedia: available at https: //www .electropedia .org/
3.1
cosmetic formulation
preparation of raw materials with a qualitatively and quantitatively defined composition
3.2
cosmetic product
cosmetic formulation (3.1) that has undergone all stages of production, including packaging in its final
container
3.3
antimicrobial protection of a cosmetic product
ability of a cosmetic product (3.2) to overcome microbial contamination that might present a potential
risk to the user or to the aesthetic and functional integrity of the product, during intended use
Note 1 to entry: The overall antimicrobial protection includes preservation of the formulation, the specific
manufacturing process and protective packaging.
3.4
preservation of a cosmetic formulation
set of means used to avoid microbial proliferation in a cosmetic formulation (3.1)
EXAMPLE Preservatives, multifunctional compounds, hostile raw materials, extreme pH, low water-
activity values.
3.5
reference method
method applied by interested parties to assess a product on the market and in case of dispute
3.6
development method
in-house method
method used during the development stage of a product before the product is put on the market
3.7
consumer
end user of a cosmetic product (3.2)
4 Principle
The evaluation of the antimicrobial protection of a cosmetic product combines the following elements
(see Annex A).
a) The characteristics of its formulation (see ISO 29621) or the results of the preservation efficacy
test (if performed), or both.
The preservation efficacy test is described in Clause 5.
b) The characteristics of the cosmetic product in conjunction with the production conditions
(see ISO 22716 and ISO 29621), the packaging materials and, if justified, recommendations for use
of the product (see ISO 29621) and, when relevant, the area of application and the targeted user
population (see Annex D).
This document describes a procedure for the interpretation of data generated by the preservation
efficacy test (if appropriate) and by the microbiological risk assessment.
2 © ISO 2019 – All rights reserved
ISO 11930:2019(E)
5 Preservation efficacy test
5.1 General
The evaluation of the preservation of a cosmetic formulation is based on inoculation of the formulation
with calibrated inocula (prepared from relevant strains of microorganisms). The number of surviving
microorganisms is measured at defined intervals during a period of 28 days. For each time and
each strain, the log reduction value is calculated and compared to the minimum values required for
evaluation criteria A or B (see Annex B).
When used as a reference method, procedures shall be strictly followed in order to avoid variability
in results. To determine the preservation efficacy of a formulation during product development, other
suitable development methods may be used.
Prior to the test, neutralizer efficacy shall be established (see 5.5), and the microbiological quality of
the product shall be determined (in accordance with ISO 21149 and ISO 16212, or with ISO 18415)
to ensure that any microorganisms present in the test sample do not interfere with recovery of test
organisms.
5.2 Materials, apparatus, reagents and culture media
5.2.1 General
When water is used in diluents, neutralizers or culture media preparation, use distilled water or
purified water as specified in ISO 21148:2017, 8.2.
5.2.2 Materials
In addition to the microbiology laboratory equipment described in ISO 21148, the following materials
should be used
5.2.2.1 Glass beads, 3 mm to 4 mm in diameter.
5.2.2.2 Sintered glass filter, of porosity 2 (40 µm to 100 µm).
5.2.2.3 Sterile glass containers with closures, of suitable volumes.
5.2.2.4 Centrifuge, capable of a centrifugal force of 2 000g.
5.2.3 Diluents
5.2.3.1 General
Unless otherwise specified, all reagents shall be equilibrated at ambient temperature before use. When
available, ready-to-use reagents and media may be used.
5.2.3.2 Diluents for bacteria and Candida albicans
5.2.3.2.1 Composition 1
Sodium chloride 8,5 g
Water 1 000 ml
ISO 11930:2019(E)
5.2.3.2.2 Preparation
Dissolve sodium chloride in the water by mixing. Dispense into suitable containers. Sterilize in the
autoclave at 121 °C for 15 min.
5.2.3.2.3 Composition 2
Tryptone pancreatic digest of casein 1,0 g
Sodium chloride 8,5 g
Water 1 000 ml
5.2.3.2.4 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.
5.2.3.3 Diluent for preparation of Aspergillus brasiliensis: polysorbate solution
Prepare a solution of polysorbate 80 (0,5 g/l). Dissolve by mixing while heating until complete
dissolution is achieved. Dispense the solution into suitable containers. Sterilize in the autoclave at
121 °C for 15 min.
5.2.4 Neutralizer
5.2.4.1 General
The suitability and effectiveness of the neutralizing agent with respect to the test strains used and to
the tested formulation shall be demonstrated as specified in 5.5.
The neutralizer described in 5.2.4.2 is frequently used. Examples of other suitable neutralizers are
given in Annex C (see Table C.1).
5.2.4.2 Eugon LT 100 liquid broth
5.2.4.2.1 General
This medium contains ingredients that neutralize inhibitory substances present in the sample (lecithin
1) ®
and polysorbate 80) and dispersing agent octoxynol 9 (Triton X100 ). It may be prepared as described
in 5.2.4.2.2, or from dehydrated culture medium, according to the manufacturer’s instructions. A ready-
to-use medium may also be used.
5.2.4.2.2 Composition
Pancreatic digest of casein 15 g
Papaic digest of soybean meal 5 g
Sodium chloride 4 g
L-cystine 0,7 g
1) Triton X100® is an example of a suitable product available commercially. This information is given for the
convenience of users of this document and does not constitute an endorsement by ISO of this product.
4 © ISO 2019 – All rights reserved
ISO 11930:2019(E)
Sodium sulphite 0,2 g
Glucose 5,5 g
Egg lecithin 1 g
Polysorbate 80 5 g
Octoxynol 9 1 g
Water 1 000 ml
5.2.4.2.3 Preparation
Dissolve successively into boiling water polysorbate 80, octoxynol 9 and egg lecithin until they are
completely dissolved. Dissolve the other components by mixing while heating. Dispense the medium
into suitable containers. Sterilize in the autoclave at 121 °C for 15 min. Mix well after sterilization while
the liquid is still hot to redissolve settled substances. After sterilization, the pH shall be equivalent to
7,0 ± 0,2 when measured at room temperature.
5.2.5 Culture media
5.2.5.1 General
Culture media may be prepared as in 5.2.5.2, 5.2.5.3 and 5.2.5.4, 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 in 5.2.5.2.1, 5.2.5.3.1 and 5.2.5.4.1.
5.2.5.2 Culture medium for bacteria: tryptic soy agar (TSA) or soybean casein digest agar
medium
5.2.5.2.1 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
5.2.5.2.2 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. Mix well
after sterilization while the liquid is still hot to redissolve settled substances. After sterilization and
cooling down, the pH shall be equivalent to 7,3 ± 0,2 when measured at room temperature.
ISO 11930:2019(E)
5.2.5.3 Culture medium for C. albicans: Sabouraud dextrose agar medium (SDA)
5.2.5.3.1 Composition
Dextrose 40,0 g
Peptic digest of animal tissue 5,0 g
Pancreatic digest of casein 5,0 g
Agar 15,0 g
Water 1 000 ml
5.2.5.3.2 Preparation
Dissolve the components or the dehydrated complete medium in the water by mixing while heating.
Dispense the medium into suitable containers. Sterilize in an autoclave at 121 °C for 15 min. After
sterilization, the pH shall be equivalent to 5,6 ± 0,2 when measured at room temperature.
5.2.5.4 Culture medium for A. brasiliensis: potato dextrose agar (PDA)
5.2.5.4.1 Composition
Potato infusion 200,0 g
Dextrose 20,0 g
Agar (see 5.2.5.4.2, Note 1) 20,0 g
Water 1 000 ml
5.2.5.4.2 Preparation
Dissolve the components or the dehydrated complete medium in the water by heating. Dispense the
medium into suitable containers. Sterilize in an autoclave at 121 °C for 15 min. After sterilization, the
pH shall be equivalent to 5,6 ± 0,2 when measured at room temperature.
NOTE Commercially available dehydrated medium powders that contain less than 20 g/l of agar can be
supplemented with extra agar to the final concentration of 20 g/l if necessary.
5.3 Microbial strains
2)
The test shall be run using the following strains as test microorganisms :
® TM3) ® TM4) ® TM5)
— Pseudomonas aeruginosa ATCC 9027 (equivalent strain: CIP 82.118 or NCIMB 8626
® TM6) ® TM7)
or NBRC 13275 or KCTC 2513 or other equivalent national collection strain);
2) These are examples of suitable products available commercially. This information is given for the convenience
of users of this document and does not constitute an endorsement by ISO of these products. ®
3) ATCC : American Type Culture Collection ®
4) CIP : Collection de l’Institut Pasteur ®
5) NCIMB : National Collection of Industrial Marine Bacteria ®
6) NBRC : NITE Biological Resource Center, JP ®
7) KCTC : Korean Collection for Type Cultures
6 © ISO 2019 – All rights reserved
ISO 11930:2019(E)
® TM ® TM ® TM
— Staphylococcus aureus ATCC 6538 (equivalent strain: CIP 4.83 or NCIMB 9518 or
8)
® TM ® TM ® TM
NBRC 13276 or KCTC 3881 or NCTC 10788 or other equivalent national collection
strain);
® TM ® TM ® TM
— Escherichia coli ATCC 8739 (equivalent strain: CIP 53.126 or NCIMB 8545 or
® TM ® TM ® TM
NBRC 3972 or KCTC 2571 or NCTC 12923 or other equivalent national collection strain);
9) 10)
® TM TM ® TM
— Candida albicans ATCC 10231 (equivalent strain: IP 48.72 or NCPF 3179 or
® TM ® TM
NBRC 1594 or KCTC 7965 or other equivalent national collection strain);
® TM ® TM11)
— Aspergillus brasiliensis ATCC 16404 (equivalent strain: IP 1431 or IMI 149007 or
® TM ® TM
NBRC 9455 or KCTC 6196 or other equivalent national collection strain).
The culture can be acquired frozen, freeze-dried, on slants or in ready-to-use formats and should be
prepared according to the procedures provided by the supplier of the reference strain. The strains
should be stored in a laboratory conforming to EN 12353 or according to another suitable method.
5.4 Preparation and enumeration of inocula
5.4.1 General
To perform the tests, use the strains stored in the laboratory (see 5.3) to obtain the stock cultures and
the working cultures.
The stock culture is a confluent culture obtained by streaking slant tubes or plates with the stored
strain (single-use vial or bead). After incubation, the stock culture can be kept between 2 °C and 8 °C for
two months and is used to obtain the working cultures.
The working culture, prepared when needed to perform a test, is used to obtain the calibrated
suspension (inoculum).
The same growth conditions (agar media and incubation) are used for both stock cultures and working
cultures (see 5.4.2 and 5.4.3).
NOTE 1 A limited number of serial subcultures and the use of confluent cultures instead of isolated colonies
lower the risk of change in the susceptibility of strains. The standardization of growth conditions and of inoculum
preparation improves the reproducibility of the test.
NOTE 2 Avoid thawing when multi-dose containers are used (for example, containers with several beads
brought out of the freezer to take one bead, then replaced in the freezer).
5.4.2 Preparation of bacterial and Candida albicans suspensions
5.4.2.1 To prepare the working culture of the test microorganism, prepare a subculture from the
stock culture by streaking slant tubes or plates (TSA for bacteria, SDA for C.albicans) in order to obtain a
confluent culture. Incubate at (32,5 ± 2,5) °C for 18 h to 24 h.
Prepare in the same way a second subculture, starting from the first subculture, and incubate at
(32,5 ± 2,5) °C for 18 h to 24 h. A third subculture can be grown in the same way, starting from the
second. The second subculture and the third one (if it was carried out) form the working cultures.
If the second subculture cannot be carried out in a timely manner, then the first subculture can be kept
for up to 48 h in the incubator (32,5 ± 2,5) °C and used to prepare the second subculture. In this case,
prepare the third 18 h to 24 h subculture and use this in the test. ®
8) NCTC : National Collection of Type Cultures
9) IP: Institut Pasteur ®
10) NCPF : National Collection of Pathogenic Fungi
11) IMI: International Mycological Institute, UK
ISO 11930:2019(E)
It is recommended that a fourth subculture not be prepared from the initial stock culture.
5.4.2.2 Take 10 ml of diluent (5.2.3.2) and place in a suitable sterile container with approximately 5 g
of sterile glass beads. Transfer loopfuls of the cells harvested from the agar medium into the diluent; the
cells should be suspended in the diluent by rubbing the loop in a small amount of the diluent against the
side of the container to dislodge the cells.
5.4.2.3 Shake the container manually or mechanically, for a maximum of 3 min, to homogenize the
suspension. Aspirate the upper part of the suspension (avoiding any contact with the glass beads) and
transfer the obtained suspension to a sterile container.
7 8
5.4.2.4 Adjust the number of cells in the suspension to 1 × 10 cfu/ml to 1 × 10 cfu/ml (bacteria) or
6 7
1 × 10 cfu/ml to 1 × 10 cfu/ml (C. albicans) using the diluent (5.2.3.2) and in accordance with calibration
data produced in the laboratory (e.g. using a spectrophotometer, see ISO 21148:2017, Annex C).
Use this calibrated inoculum within 2 h.
5.4.2.5 At the time of the test, check the initial capacity of the suspension, N. Make successive tenfold
dilutions of the calibrated suspension in the diluent (5.2.3.2). Perform the enumeration by duplicating
1 ml of the suitable dilutions (see 5.6.2) into TSA for bacteria and into SDA for C. albicans. Incubate the
dishes at (32,5 ± 2,5) °C for 24 h to 48 h.
5.4.3 Preparation of Aspergillus brasiliensis spore suspension
5.4.3.1 To obtain the working culture of the test microorganism, use a stock culture (on PDA) aged
not more than 2 months and prepare a suspension in the diluent (5.2.3.3). Inoculate by flooding the
surface of PDA (use an appropriate number of Petri dishes), so as to obtain a confluent culture. Incubate
at (22,5 ± 2,5) °C for 7 days to 11 days.
5.4.3.2 After incubation, transfer 10 ml of the polysorbate solution (5.2.3.3) to the surface of PDA.
Gently detach the spores from the culture surface, for example, using a spatula or glass beads.
Transfer the suspension to an appropriate flask and stir gently for about 1 min in the presence of glass
beads. Filter the suspension through a sintered glass filter of porosity 2 (i.e. 40 µm to 100 µm).
5.4.3.3 Carry out a microscopic examination (magnification × 400) to detect the presence of germinated
spores or mycelium fragments.
— If germinated spores are present, the suspension shall be discarded.
— If mycelium is present in more than one field out of 10, wash the filtered suspension by centrifuging
at 2 000g for 20 min. Wash the spores at least twice by resuspending them in the polysorbate
solution (5.2.3.3) and centrifuging.
6 7
5.4.3.4 Adjust the number of spores in the suspension to a value of about 1 × 10 spores/ml to 1 × 10
spores/ml using the diluent (5.2.3.3) and any appropriate means.
The use of a cell enumeration device (e.g. a haemocytometer) is recommended to adjust the number of
spores. If an appropriate cell count chamber is used, follow the instructions accurately.
The suspension should be used during the same working day. It can be used on the following day if
stored between 2 °C and 8 °C, but, at the time of the test, the absence of germinated spores shall be
checked.
5.4.3.5 At the time of the test, check the initial number of microorganisms, N. Make successive tenfold
dilutions of the calibrated suspension in the diluent (5.2.3.3). Perform the enumeration by duplicating
8 © ISO 2019 – All rights reserved
ISO 11930:2019(E)
1 ml of the suitable dilutions (see 5.6.2) into PDA plates (using an appropriate number of Petri dishes).
Incubate the dishes at (22,5 ± 2,5) °C for 3 days to 5 days.
5.5 Demonstration of the neutralizer efficacy
5.5.1 Principle
Neutralization efficacy is the verification that the test method protocol sufficiently neutralizes the
antimicrobial aspects of a formulation, ensuring that microorganisms can be detected in the product
matrix, without inhibiting the test microorganisms.
A calibrated suspension of microorganisms (about 10 cfu/ml) is inoculated in the neutralizer in the
presence (test) and in the absence (control) of the formulation. The neutralizer efficacy is demonstrated
if the counts performed on the inoculum, N , and on the control, N (mixture of the neutralizer and
v vn
diluent), are equivalent and if the count in the test, N (mixture of the neutralizer and the formulation),
vf
is at least 50 % of N (see 5.5.4).
vn
5.5.2 Procedure
Run the test separately for each strain.
a) Prepare a dilution of the calibrated suspension of microorganisms [N is between 1 × 10 cfu/ml
8 6 7
and 1 × 10 cfu/ml for bacteria, and between 1 × 10 cfu/ml and 1 × 10 cfu/ml for C. albicans and
A. brasiliensis (see 5.4.2 and 5.4.3)] in order to obtain a suspension containing about 10 cfu/ml
(inoculum).
b) Transfer 1 g or 1 ml of the formulation to be tested into 9 ml of neutralizer (5.2.4). Shake to disperse
the formulation. If the initial dilution conditions/neutralizer prove insufficient for neutralization
efficacy, repeat the procedure following guidance in 5.5.4, paragraph 4. Other test conditions are
acceptable provided that at least 1 g or 1 ml of formulation is used and a minimal tenfold dilution is
performed.
c) Leave the “test” tubes for (30 ± 15) min at room temperature. Run a control in parallel with the
same neutralizer, replacing the tested formulation with 1 ml of diluent (5.2.3).
d) Inoculate the “test” tubes [tenfold and, if necessary, the additional dilution in 5.5.2 b)] and “control”
tubes with 1 ml of inoculum [5.5.2 a)] (the final volume is 11 ml). Mix.
e) Prepare the “inoculum control”. Add 1 ml of the inoculum [5.5.2 a)] to 10 ml of diluent (the final
volume is 11 ml). Mix.
f) Enumerate in duplicate by inclusion of 1 ml of each mixture (“test”, “control” and “inoculum
control”) into appropriate agar medium (TSA for bacteria, SDA for C. albicans and PDA for A.
brasiliensis).
The use of a 1 ml volume of the calibrated suspension is recommended to improve the precision of
the counts (“test”, “control” and “inoculum control” mixtures).
g) Incubate at (32,5 ± 2,5) °C for 48 h to 72 h for the bacteria and C. albicans and at (22,5 ± 2,5) °C for
3 days to 5 days for A. brasiliensis.
5.5.3 Calculations
Calculate the number, N , of microorganisms, in colony-forming units per millilitre, present in the
v
inoculum control [see 5.5.2 e)].
N is the mean number of colonies counted in duplicate over the plates in a 1 ml sample.
N shall be about 100.
v
ISO 11930:2019(E)
Calculate the number of microorganisms, in colony-forming units per millilitre, present in the “test”
mixture with the neutralizer in the presence of the formulation, N , and in the “control” mixture with
vf
the neutralizer in the absence of the formulation, N .
vn
N or N is the mean number of colonies counted in duplicate over the plates in a 1 ml sample of “test”
vf vn
or “control” mixture.
5.5.4 Interpretation of results and conclusion on neutralizer efficacy
The efficacy of the neutralizer is demonstrated if N ≥ 0,5N and if N is close to N . If N is not close
vf vn vn v vn
to N , the neutralizer is considered toxic for microorganisms.
v
The inherent variability in enumeration on agar plates shall be taken into account. Two counts are
usually considered different only if their difference exceeds 50 %.
Take note of the test conditions (neutralizer, volume, etc.) and in particular the dilution of the
formulation (1/10, 1/100 or other) for which the efficacy of the neutralizer was demonstrated.
If the results do not comply with the requirements, it is necessary to
— either modify the neutralizer (see Annex C) or make a further dilution of the sample,
— or carry out a membrane filtration, if possible.
If the results still do not comply with the requirements, it is unlikely that the formulation can be
contaminated by the strain concerned. It is possible, even in this case, to issue a test report [see 5.7 and
5.8 f)].
5.6 Determination of the preservation efficacy of the formulation
5.6.1 Procedure
Run the test separately for each strain.
5.6.1.1 Sampling of test product
For each strain, dispense 20 g or 20 ml of the test formulation into a sterile container (5.2.2.3).
5.6.1.2 Inoculation of test microorganisms
Add to each container 0,2 ml of calibrated inoculum (see 5.4.2 and 5.4.3) to obtain between 1 × 10 cfu/
6 4 5
ml and 1 × 10 cfu/ml or g for bacteria, and between 1 × 10 cfu/ml and 1 × 10 cfu/ml or g for C. albicans
and A. brasiliensis in the formulation (final concentration). Mix thoroughly to ensure a homogeneous
distribution of the inoculum.
The initial concentration of microorganisms present in the inoculated product, N , is calculated using
the results of the enumeration of the calibrated inoculum, N [see 5.6.3.2 b)].
5.6.1.3 Incubation of the inoculated formulation
Store the containers holding the inoculated formulation at (22,5 ± 2, 5) °C.
5.6.1.4 Sampling and enumeration
a) At each specified sampling interval: 7 days (T7), 14 days (T14) and 28 days (T28), according to
the test strain (see Annex B), sample 1 g of inoculated formulation and prepare the dilution for
10 © ISO 2019 – All rights reserved
ISO 11930:2019(E)
which neutralization efficacy has been demonstrated (5.5). Ensure the correct dilution factor is
used when calculating N (see 5.6.3.3).
x
Leave in contact for (30 ± 15) min at room temperature.
b) Starting from dilution with demonstrated neutralization efficacy, make successive tenfold dilutions
in the diluent (see 5.2.3).
c) Car
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