Cosmetics - Microbiology - Testing of impregnated or coated wipes and masks (ISO 21322:2020)

This document gives guidance for the enumeration and/or detection of microorganisms present in a
cosmetic product that is impregnated or coated onto a substrate (i.e. wipes and masks) where sampling
and microbiological influence of the manufactured product presents particular challenges in terms of
microbiological sampling and testing.
The principle of this document can also be applied to test similar products (e.g. cushion, impregnated
sponge, etc.) or applicators (e.g. brush, puff, sponge, etc.) with modification of the procedure as
appropriate.

Kosmetische Mittel - Mikrobiologie - Prüfung von getränkten oder beschichteten Feuchttüchern und Masken (ISO 21322:2020)

Dieses Dokument enthält eine Anleitung für die Auszählung und/oder zum Nachweis von Mikroorganismen, die in kosmetischen Mitteln vorhanden sind, die sich auf einem damit getränkten oder beschichteten Träger befinden (z. B. Reinigungstücher und Masken), wenn die Probenahme und der mikrobiologische Einfluss des hergestellten Produktes im Hinblick auf die mikrobiologische Probenahme und Prüfung eine besondere Herausforderung darstellen.
Der Grundsatz dieses Dokumentes kann auch auf ähnliche Produkte (z. B. Kissen, getränkte Schwämme usw.) oder Applikatoren (z. B. Pinsel, Quasten, Schwämme usw.) angewendet werden, ggf. mit einer Veränderung des Verfahrens.

Cosmétiques - Microbiologie - Essais sur lingettes et masques imprégnés ou enduits (ISO 21322:2020)

Le présent document fournit des recommandations pour le dénombrement et/ou la détection des micro-organismes présents dans les produits cosmétiques imprégnés ou enduits sur un substrat (lingettes et masques), pour lesquels l'échantillonnage et le contexte microbiologique du produit fabriqué posent des problèmes particuliers lorsqu'il s'agit de réaliser un échantillonnage et des essais microbiologiques.
Le principe du présent document peut également être appliqué à des produits similaires (par exemple, cushion, éponges imprégnées, etc.) ou des applicateurs (par exemple, pinceau, houpette, éponge, etc.), en modifiant le mode opératoire comme approprié.

Kozmetika - Mikrobiologija - Preskušanje impregniranih izdelkov ali izdelkov, obdelanih s premazi - Robčki in maske (ISO 21322:2020)

General Information

Status
Published
Public Enquiry End Date
01-Oct-2021
Publication Date
15-Jun-2022
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
21-Apr-2022
Due Date
26-Jun-2022
Completion Date
16-Jun-2022

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SLOVENSKI STANDARD
SIST EN ISO 21322:2022
01-julij-2022
Kozmetika - Mikrobiologija - Preskušanje impregniranih izdelkov ali izdelkov,
obdelanih s premazi - Robčki in maske (ISO 21322:2020)
Cosmetics - Microbiology - Testing of impregnated or coated wipes and masks (ISO
21322:2020)
Kosmetische Mittel - Mikrobiologie - Prüfung von getränkten oder beschichteten
Feuchttüchern und Masken (ISO 21322:2020)

Cosmétiques - Microbiologie - Essais sur lingettes et masques imprégnés ou enduits

(ISO 21322:2020)
Ta slovenski standard je istoveten z: EN ISO 21322:2021
ICS:
07.100.40 Kozmetika - mikrobiologija Cosmetics microbiology
71.100.70 Kozmetika. Toaletni Cosmetics. Toiletries
pripomočki
SIST EN ISO 21322:2022 en,fr,de

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

---------------------- Page: 1 ----------------------
SIST EN ISO 21322:2022
---------------------- Page: 2 ----------------------
SIST EN ISO 21322:2022
EN ISO 21322
EUROPEAN STANDARD
NORME EUROPÉENNE
December 2021
EUROPÄISCHE NORM
ICS 07.100.40
English Version
Cosmetics - Microbiology - Testing of impregnated or
coated wipes and masks (ISO 21322:2020)

Cosmétiques - Microbiologie - Essais sur lingettes et Kosmetische Mittel - Mikrobiologie - Prüfung von

masques imprégnés ou enduits (ISO 21322:2020) getränkten oder beschichteten Reinigungstüchern und

Masken (ISO 21322:2020)
This European Standard was approved by CEN on 5 December 2021.

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, Republic of North Macedonia, 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

© 2021 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 21322:2021 E

worldwide for CEN national Members.
---------------------- Page: 3 ----------------------
SIST EN ISO 21322:2022
EN ISO 21322:2021 (E)
Contents Page

European foreword ....................................................................................................................................................... 3

---------------------- Page: 4 ----------------------
SIST EN ISO 21322:2022
EN ISO 21322:2021 (E)
European foreword

The text of ISO 21322:2020 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 21322:2021 by

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

This European Standard shall be given the status of a national standard, either by publication of an

identical text or by endorsement, at the latest by June 2022, and conflicting national standards shall be

withdrawn at the latest by June 2022.

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.

Any feedback and questions on this document should be directed to the users’ national standards body.

A complete listing of these bodies can be found on the CEN website.

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, Republic of

North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the

United Kingdom.
Endorsement notice

The text of ISO 21322:2020 has been approved by CEN as EN ISO 21322:2021 without any modification.

---------------------- Page: 5 ----------------------
SIST EN ISO 21322:2022
---------------------- Page: 6 ----------------------
SIST EN ISO 21322:2022
INTERNATIONAL ISO
STANDARD 21322
First edition
2020-06
Cosmetics — Microbiology — Testing
of impregnated or coated wipes and
masks
Cosmétiques — Microbiologie — Essais sur lingettes et masques
imprégnés ou enduits
Reference number
ISO 21322:2020(E)
ISO 2020
---------------------- Page: 7 ----------------------
SIST EN ISO 21322:2022
ISO 21322:2020(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2020

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 2020 – All rights reserved
---------------------- Page: 8 ----------------------
SIST EN ISO 21322:2022
ISO 21322:2020(E)
Contents Page

Foreword ..........................................................................................................................................................................................................................................v

Introduction ................................................................................................................................................................................................................................vi

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

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

3 Terms and definitions ..................................................................................................................................................................................... 1

4 Principle ........................................................................................................................................................................................................................ 2

4.1 General information ........................................................................................................................................................................... 2

4.2 Selection of the test sample ......................................................................................................................................................... 2

4.3 Selection of the method ................................................................................................................................................................... 3

4.4 Recovery of microorganisms from the test sample ................................................................................................. 3

4.5 Enumeration of aerobic mesophilic microorganisms ........................................................................................... 3

4.5.1 General...................................................................................................................................................................................... 3

4.5.2 Plate count method overview ............................................................................................................................... 3

4.5.3 Membrane filtration method overview ........................................................................................................ 3

4.6 Detection of specified microorganisms by enrichment method .................................................................. 4

5 Diluents, neutralizers and culture media ................................................................................................................................... 4

5.1 General ........................................................................................................................................................................................................... 4

5.2 Diluents and neutralizers .............................................................................................................................................................. 4

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

5.3.1 Media for enumeration and detection ........................................................................................................... 4

5.3.2 Media for preparation of spores of Bacillus subtilis ...................................................................... 4

6 Apparatus and glassware ............................................................................................................................................................................ 4

7 Strains of microorganisms ......................................................................................................................................................................... 4

8 Handling of cosmetic products and laboratory samples ............................................................................................ 5

9 Procedure..................................................................................................................................................................................................................... 5

9.1 General recommendation .............................................................................................................................................................. 5

9.2 Selection and preparation of the test sample ............................................................................................................... 5

9.2.1 Selection of the test sample .................................................................................................................................... 5

9.2.2 Preparation of the initial suspension ............................................................................................................. 5

9.3 Recovery of microorganisms ...................................................................................................................................................... 6

9.3.1 General...................................................................................................................................................................................... 6

9.3.2 Stomaching ........................................................................................................................................................................... 6

9.3.3 Shaking/Stirring ...................................................................... ......................................................................................... 6

9.4 Enumeration of aerobic mesophilic microorganisms ........................................................................................... 6

9.4.1 General...................................................................................................................................................................................... 6

9.4.2 Pour plate method .......................................................................................................................................................... 6

9.4.3 Surface spread method ............................................................................................................................................... 7

9.4.4 Membrane filtration method ................................................................................................................................. 7

9.4.5 Incubation .............................................................................................................................................................................. 7

9.4.6 Counting of colonies...................................................................................................................................................... 7

9.5 Detection of specified microorganisms by enrichment method .................................................................. 8

9.5.1 General...................................................................................................................................................................................... 8

9.5.2 Test for specified microorganisms ................................................................................................................... 8

10 Expression of results ........................................................................................................................................................................................ 9

10.1 Enumeration of aerobic mesophilic microorganisms ........................................................................................... 9

10.2 Detection of specified microorganisms ............................................................................................................................. 9

11 Suitability test ......................................................................................................................................................................................................... 9

12 Test report ................................................................................................................................................................................................................... 9

© ISO 2020 – All rights reserved iii
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SIST EN ISO 21322:2022
ISO 21322:2020(E)

Annex A (normative) Guidance on methods for microbiological testing of impregnated or

coated products — Wipes and masks ..........................................................................................................................................10

Annex B (informative) Expression and interpretation of results ........................................................................................14

Annex C (normative) Suitability test method ...........................................................................................................................................21

Bibliography .............................................................................................................................................................................................................................26

iv © ISO 2020 – All rights reserved
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SIST EN ISO 21322:2022
ISO 21322:2020(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.
© ISO 2020 – All rights reserved v
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SIST EN ISO 21322:2022
ISO 21322:2020(E)
Introduction

For technical reasons, current standards in cosmetics microbiology may not be applicable to

impregnated or coated cosmetic products, such as wipes and masks, in which there is no direct access

to the formulation.

Based on their product form or delivery there is a need to adapt these standards to assess the

microbiological quality of these products.
vi © ISO 2020 – All rights reserved
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SIST EN ISO 21322:2022
INTERNATIONAL STANDARD ISO 21322:2020(E)
Cosmetics — Microbiology — Testing of impregnated or
coated wipes and masks
1 Scope

This document gives guidance for the enumeration and/or detection of microorganisms present in a

cosmetic product that is impregnated or coated onto a substrate (i.e. wipes and masks) where sampling

and microbiological influence of the manufactured product presents particular challenges in terms of

microbiological sampling and testing.

The principle of this document can also be applied to test similar products (e.g. cushion, impregnated

sponge, etc.) or applicators (e.g. brush, puff, sponge, etc.) with modification of the procedure as

appropriate.
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 11930, Cosmetics — Microbiology — Evaluation of the antimicrobial protection of a cosmetic product

ISO 16212, Cosmetics — Microbiology — Enumeration of yeast and mould
ISO 18416, Cosmetics — Microbiology — Detection of Candida albicans

ISO 21148, Cosmetics — Microbiology — General instructions for microbiological examination

ISO 21149, Cosmetics — Microbiology — Enumeration and detection of aerobic mesophilic bacteria

ISO 21150, Cosmetics — Microbiology — Detection of Escherichia coli
ISO 22717, Cosmetics — Microbiology — Detection of Pseudomonas aeruginosa
ISO 22718, Cosmetics — Microbiology — Detection of Staphylococcus aureus
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:

— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// 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, for shipment
© ISO 2020 – All rights reserved 1
---------------------- Page: 13 ----------------------
SIST EN ISO 21322:2022
ISO 21322:2020(E)
3.3
impregnated product
product absorbed on the support
3.4
coated product
product adsorbed on the support
3.5
test sample
representative unit of the entire cosmetic product (3.2) for testing
4 Principle
4.1 General information

The method determines the population of viable microorganisms by enumeration of colonies on a

non-selective agar medium and by the presence or absence of specified microorganisms growth after

enrichment.
The method involves the following steps:
— selection of the test sample;
— selection of an appropriate method;
— recovery of microorganisms;

— enumeration of the population of viable microorganisms by filtration or plate count method;

— tests for specified microorganisms by enrichment method.

The experimental conditions shall be evaluated to ensure that the method should not affect the viability

of microorganisms and the recovery of bioburden from the sample and should include the verification

of the efficacy of the neutralization (see Clause 11).

In order to ensure product quality and safety for the consumer, an appropriate microbiological risk

assessment should be performed to determine the types of cosmetic products to which this document

is applicable (see ISO 29621:2017, Table 2).

Other methods may be substituted provided that their equivalence has been demonstrated.

4.2 Selection of the test sample

— Whenever practical, the entire unit should be used for testing with a minimum weight of 1 g. If for

technical reasons the entire unit cannot be tested, a defined Unit Item Portion (UIP) is used for

testing. A “UIP” is a microbiologically-representative subunit of the test sample and is referenced

throughout the document.

— If the unit is < 1 g per unit then the appropriate number of units should be sampled to achieve the

appropriate weight or volume.

— The weight of the test sample shall be recorded even if the results are expressed by unit.

Selection of the test sample shall be according to A.1.
2 © ISO 2020 – All rights reserved
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SIST EN ISO 21322:2022
ISO 21322:2020(E)
4.3 Selection of the method

The method should be conducted according to an appropriate procedure based on the specifics of the

product (size, volume, single unit/multi–unit package, level of bioburden. etc.) and should ensure that a

representative sample is evaluated.
Selection of the method shall be according to A.1 and A.2.
4.4 Recovery of microorganisms from the test sample

The degree to which microorganisms adhere to the test sample surface is dependent on the wipe or

mask in which the liquid portion of the formulation has been either impregnated or coated. Preliminary

treatments may be necessary to separate microorganisms from the test sample.

Regardless of the treatment, the verification of recovery method should be performed in order to

demonstrate that the method can release microorganisms from the test sample without having an

adverse effect on their viability (see Clause 11).
4.5 Enumeration of aerobic mesophilic microorganisms
4.5.1 General

The enumeration of aerobic mesophilic microorganisms includes bacteria, yeasts and moulds.

Based on the nature of the test sample, the volume of diluent used to immerse the test sample and the

expected level of bioburden, two types of counting methods may be used:
— plate count method;
— membrane filtration method.
4.5.2 Plate count method overview
Plate count method consists of either using a pour plate or spread plate method.
Each method consists of the following steps.

— Prepare the agar plates and diluent using a non-selective agar medium for plating the diluent in

which the sample was immersed.
— Incubate the plates for enumeration and/or detection.

— Count the number of colonies forming units (CFU) based on the number of aerobic mesophilic

microorganisms recovered per unit or g.
4.5.3 Membrane filtration method overview
Membrane filtration consists of the following steps.

— Transfer the diluent or a defined quantity of diluent in which the test sample was immersed to a

filtration apparatus wetted with a small volume of an appropriate sterile diluent.

— After filtration and rinsing, transfer the membrane filter onto the surface of plates with non-

selective agar medium.
— Aerobic incubation of the plates.

— Count the number of colony forming units (CFU) and calculate of the number of aerobic mesophilic

microorganisms per g or unit.
© ISO 2020 – All rights reserved 3
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SIST EN ISO 21322:2022
ISO 21322:2020(E)
4.6 Detection of specified microorganisms by enrichment method

The objective of the enrichment method is to incubate a test sample in a non-selective broth medium to

increase the number of microorganisms that are present in a test sample.

— The first step of an enrichment method is to incubate the test sample in a non-selective broth

medium to increase the number of microorganisms present in the test sample.

— The second step of an enrichment method is to isolate specified microorganisms that may be present

on a test sample through the use of selective agar media followed by confirmatory identification

tests for characteristic colonies. See ISO 18416, ISO 21150, ISO 22717 and ISO 22718.

5 Diluents, neutralizers and culture media
5.1 General

The diluents, neutralizers and culture media suitable for enumeration and detection of aerobic

mesophilic microorganisms are described in ISO 11930, ISO 16212 and ISO 21149. Other diluents,

neutralizers and culture media may be used if they have been demonstrated to be suitable for use.

Use the general instructions given in ISO 21148. When water is mentioned in this document, use

distilled water or purified water as specified in ISO 21148.
5.2 Diluents and neutralizers

The diluent is used to disperse the sample. It is required that it contain neutralizers if the sample to be

tested has antimicrobial properties or contains a preservative. The efficacy of the neutralization shall

be demonstrated before the determination of the count (see Clause 11). Diluents and neutralizers shall

be in accordance with ISO 11930, ISO 16212, ISO 18416, ISO 21149, ISO 21150, ISO 22717 and ISO 22718.

5.3 Culture media
5.3.1 Media for enumeration and detection

Culture media for enumeration and/or detection shall be in accordance with ISO 11930, ISO 16212,

ISO 18416, ISO 21149, ISO 21150, ISO 22717 and ISO 22718.
5.3.2 Media for preparation of spores of Bacillus subtilis
See C.1.3.1.
6 Apparatus and glassware
The laboratory equipment, apparatus and glassware are described in ISO 21148.
7 Strains of microorganisms

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

reference strain. The strains may be stored in the laboratory conforming to EN 12353 or according to

another suitable method.

For testing the recovery of microorganisms on the test samples, spores of Bacillus subtilis ATCC 6633

(equivalent strain CIP 52.62 or NCIMB 8054 or NBRC 3134 or other equivalent national collection

strain) are used.
4 © ISO 2020 – All rights reserved
---------------------- Page: 16 ----------------------
SIST EN ISO 21322:2022
ISO 21322:2020(E)

For testing the efficacy of neutralizers, two strains representative of both Gram negative and Gram

positive bacteria and a yeast are used:

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

KCTC 1916 or other equivalent national collection strain);

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

or KCTC 2513 or other equivalent national collection strain).

An alternative 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).

— 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 strains may be kept in the laboratory according to the EN 12353.
8 Handling of cosmetic products and laboratory samples

If storage is necessary, keep the products to be tested at room temperature. Do not incubate, refrigerate

or freeze products and samples before or after analysis. Sampling and test procedures should follow

the guidelines specified in ISO 21148 and in accordance with the procedure outlined in Clause 9.

9 Procedure
9.1 General recommendation
Use sterile equipment and aseptic technique whenever preparing the test sample
...

SLOVENSKI STANDARD
oSIST prEN ISO 21322:2021
01-september-2021
Kozmetika - Mikrobiologija - Preskušanje impregniranih izdelkov ali izdelkov,
obdelanih s premazi - Robčki in maske (ISO 21322:2020)
Cosmetics - Microbiology - Testing of impregnated or coated wipes and masks (ISO
21322:2020)
Kosmetische Mittel - Mikrobiologie - Prüfung von getränkten oder beschichteten
Feuchttüchern und Masken (ISO 21322:2020)

Cosmétiques - Microbiologie - Essais sur lingettes et masques imprégnés ou enduits

(ISO 21322:2020)
Ta slovenski standard je istoveten z: prEN ISO 21322
ICS:
07.100.40 Kozmetika - mikrobiologija Cosmetics microbiology
71.100.70 Kozmetika. Toaletni Cosmetics. Toiletries
pripomočki
oSIST prEN ISO 21322:2021 en,fr,de

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

---------------------- Page: 1 ----------------------
oSIST prEN ISO 21322:2021
---------------------- Page: 2 ----------------------
oSIST prEN ISO 21322:2021
INTERNATIONAL ISO
STANDARD 21322
First edition
2020-06
Cosmetics — Microbiology — Testing
of impregnated or coated wipes and
masks
Cosmétiques — Microbiologie — Essais sur lingettes et masques
imprégnés ou enduits
Reference number
ISO 21322:2020(E)
ISO 2020
---------------------- Page: 3 ----------------------
oSIST prEN ISO 21322:2021
ISO 21322:2020(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2020

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 2020 – All rights reserved
---------------------- Page: 4 ----------------------
oSIST prEN ISO 21322:2021
ISO 21322:2020(E)
Contents Page

Foreword ..........................................................................................................................................................................................................................................v

Introduction ................................................................................................................................................................................................................................vi

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

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

3 Terms and definitions ..................................................................................................................................................................................... 1

4 Principle ........................................................................................................................................................................................................................ 2

4.1 General information ........................................................................................................................................................................... 2

4.2 Selection of the test sample ......................................................................................................................................................... 2

4.3 Selection of the method ................................................................................................................................................................... 3

4.4 Recovery of microorganisms from the test sample ................................................................................................. 3

4.5 Enumeration of aerobic mesophilic microorganisms ........................................................................................... 3

4.5.1 General...................................................................................................................................................................................... 3

4.5.2 Plate count method overview ............................................................................................................................... 3

4.5.3 Membrane filtration method overview ........................................................................................................ 3

4.6 Detection of specified microorganisms by enrichment method .................................................................. 4

5 Diluents, neutralizers and culture media ................................................................................................................................... 4

5.1 General ........................................................................................................................................................................................................... 4

5.2 Diluents and neutralizers .............................................................................................................................................................. 4

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

5.3.1 Media for enumeration and detection ........................................................................................................... 4

5.3.2 Media for preparation of spores of Bacillus subtilis ...................................................................... 4

6 Apparatus and glassware ............................................................................................................................................................................ 4

7 Strains of microorganisms ......................................................................................................................................................................... 4

8 Handling of cosmetic products and laboratory samples ............................................................................................ 5

9 Procedure..................................................................................................................................................................................................................... 5

9.1 General recommendation .............................................................................................................................................................. 5

9.2 Selection and preparation of the test sample ............................................................................................................... 5

9.2.1 Selection of the test sample .................................................................................................................................... 5

9.2.2 Preparation of the initial suspension ............................................................................................................. 5

9.3 Recovery of microorganisms ...................................................................................................................................................... 6

9.3.1 General...................................................................................................................................................................................... 6

9.3.2 Stomaching ........................................................................................................................................................................... 6

9.3.3 Shaking/Stirring ...................................................................... ......................................................................................... 6

9.4 Enumeration of aerobic mesophilic microorganisms ........................................................................................... 6

9.4.1 General...................................................................................................................................................................................... 6

9.4.2 Pour plate method .......................................................................................................................................................... 6

9.4.3 Surface spread method ............................................................................................................................................... 7

9.4.4 Membrane filtration method ................................................................................................................................. 7

9.4.5 Incubation .............................................................................................................................................................................. 7

9.4.6 Counting of colonies...................................................................................................................................................... 7

9.5 Detection of specified microorganisms by enrichment method .................................................................. 8

9.5.1 General...................................................................................................................................................................................... 8

9.5.2 Test for specified microorganisms ................................................................................................................... 8

10 Expression of results ........................................................................................................................................................................................ 9

10.1 Enumeration of aerobic mesophilic microorganisms ........................................................................................... 9

10.2 Detection of specified microorganisms ............................................................................................................................. 9

11 Suitability test ......................................................................................................................................................................................................... 9

12 Test report ................................................................................................................................................................................................................... 9

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ISO 21322:2020(E)

Annex A (normative) Guidance on methods for microbiological testing of impregnated or

coated products — Wipes and masks ..........................................................................................................................................10

Annex B (informative) Expression and interpretation of results ........................................................................................14

Annex C (normative) Suitability test method ...........................................................................................................................................21

Bibliography .............................................................................................................................................................................................................................26

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oSIST prEN ISO 21322:2021
ISO 21322:2020(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.
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oSIST prEN ISO 21322:2021
ISO 21322:2020(E)
Introduction

For technical reasons, current standards in cosmetics microbiology may not be applicable to

impregnated or coated cosmetic products, such as wipes and masks, in which there is no direct access

to the formulation.

Based on their product form or delivery there is a need to adapt these standards to assess the

microbiological quality of these products.
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oSIST prEN ISO 21322:2021
INTERNATIONAL STANDARD ISO 21322:2020(E)
Cosmetics — Microbiology — Testing of impregnated or
coated wipes and masks
1 Scope

This document gives guidance for the enumeration and/or detection of microorganisms present in a

cosmetic product that is impregnated or coated onto a substrate (i.e. wipes and masks) where sampling

and microbiological influence of the manufactured product presents particular challenges in terms of

microbiological sampling and testing.

The principle of this document can also be applied to test similar products (e.g. cushion, impregnated

sponge, etc.) or applicators (e.g. brush, puff, sponge, etc.) with modification of the procedure as

appropriate.
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 11930, Cosmetics — Microbiology — Evaluation of the antimicrobial protection of a cosmetic product

ISO 16212, Cosmetics — Microbiology — Enumeration of yeast and mould
ISO 18416, Cosmetics — Microbiology — Detection of Candida albicans

ISO 21148, Cosmetics — Microbiology — General instructions for microbiological examination

ISO 21149, Cosmetics — Microbiology — Enumeration and detection of aerobic mesophilic bacteria

ISO 21150, Cosmetics — Microbiology — Detection of Escherichia coli
ISO 22717, Cosmetics — Microbiology — Detection of Pseudomonas aeruginosa
ISO 22718, Cosmetics — Microbiology — Detection of Staphylococcus aureus
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:

— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// 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, for shipment
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3.3
impregnated product
product absorbed on the support
3.4
coated product
product adsorbed on the support
3.5
test sample
representative unit of the entire cosmetic product (3.2) for testing
4 Principle
4.1 General information

The method determines the population of viable microorganisms by enumeration of colonies on a

non-selective agar medium and by the presence or absence of specified microorganisms growth after

enrichment.
The method involves the following steps:
— selection of the test sample;
— selection of an appropriate method;
— recovery of microorganisms;

— enumeration of the population of viable microorganisms by filtration or plate count method;

— tests for specified microorganisms by enrichment method.

The experimental conditions shall be evaluated to ensure that the method should not affect the viability

of microorganisms and the recovery of bioburden from the sample and should include the verification

of the efficacy of the neutralization (see Clause 11).

In order to ensure product quality and safety for the consumer, an appropriate microbiological risk

assessment should be performed to determine the types of cosmetic products to which this document

is applicable (see ISO 29621:2017, Table 2).

Other methods may be substituted provided that their equivalence has been demonstrated.

4.2 Selection of the test sample

— Whenever practical, the entire unit should be used for testing with a minimum weight of 1 g. If for

technical reasons the entire unit cannot be tested, a defined Unit Item Portion (UIP) is used for

testing. A “UIP” is a microbiologically-representative subunit of the test sample and is referenced

throughout the document.

— If the unit is < 1 g per unit then the appropriate number of units should be sampled to achieve the

appropriate weight or volume.

— The weight of the test sample shall be recorded even if the results are expressed by unit.

Selection of the test sample shall be according to A.1.
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4.3 Selection of the method

The method should be conducted according to an appropriate procedure based on the specifics of the

product (size, volume, single unit/multi–unit package, level of bioburden. etc.) and should ensure that a

representative sample is evaluated.
Selection of the method shall be according to A.1 and A.2.
4.4 Recovery of microorganisms from the test sample

The degree to which microorganisms adhere to the test sample surface is dependent on the wipe or

mask in which the liquid portion of the formulation has been either impregnated or coated. Preliminary

treatments may be necessary to separate microorganisms from the test sample.

Regardless of the treatment, the verification of recovery method should be performed in order to

demonstrate that the method can release microorganisms from the test sample without having an

adverse effect on their viability (see Clause 11).
4.5 Enumeration of aerobic mesophilic microorganisms
4.5.1 General

The enumeration of aerobic mesophilic microorganisms includes bacteria, yeasts and moulds.

Based on the nature of the test sample, the volume of diluent used to immerse the test sample and the

expected level of bioburden, two types of counting methods may be used:
— plate count method;
— membrane filtration method.
4.5.2 Plate count method overview
Plate count method consists of either using a pour plate or spread plate method.
Each method consists of the following steps.

— Prepare the agar plates and diluent using a non-selective agar medium for plating the diluent in

which the sample was immersed.
— Incubate the plates for enumeration and/or detection.

— Count the number of colonies forming units (CFU) based on the number of aerobic mesophilic

microorganisms recovered per unit or g.
4.5.3 Membrane filtration method overview
Membrane filtration consists of the following steps.

— Transfer the diluent or a defined quantity of diluent in which the test sample was immersed to a

filtration apparatus wetted with a small volume of an appropriate sterile diluent.

— After filtration and rinsing, transfer the membrane filter onto the surface of plates with non-

selective agar medium.
— Aerobic incubation of the plates.

— Count the number of colony forming units (CFU) and calculate of the number of aerobic mesophilic

microorganisms per g or unit.
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4.6 Detection of specified microorganisms by enrichment method

The objective of the enrichment method is to incubate a test sample in a non-selective broth medium to

increase the number of microorganisms that are present in a test sample.

— The first step of an enrichment method is to incubate the test sample in a non-selective broth

medium to increase the number of microorganisms present in the test sample.

— The second step of an enrichment method is to isolate specified microorganisms that may be present

on a test sample through the use of selective agar media followed by confirmatory identification

tests for characteristic colonies. See ISO 18416, ISO 21150, ISO 22717 and ISO 22718.

5 Diluents, neutralizers and culture media
5.1 General

The diluents, neutralizers and culture media suitable for enumeration and detection of aerobic

mesophilic microorganisms are described in ISO 11930, ISO 16212 and ISO 21149. Other diluents,

neutralizers and culture media may be used if they have been demonstrated to be suitable for use.

Use the general instructions given in ISO 21148. When water is mentioned in this document, use

distilled water or purified water as specified in ISO 21148.
5.2 Diluents and neutralizers

The diluent is used to disperse the sample. It is required that it contain neutralizers if the sample to be

tested has antimicrobial properties or contains a preservative. The efficacy of the neutralization shall

be demonstrated before the determination of the count (see Clause 11). Diluents and neutralizers shall

be in accordance with ISO 11930, ISO 16212, ISO 18416, ISO 21149, ISO 21150, ISO 22717 and ISO 22718.

5.3 Culture media
5.3.1 Media for enumeration and detection

Culture media for enumeration and/or detection shall be in accordance with ISO 11930, ISO 16212,

ISO 18416, ISO 21149, ISO 21150, ISO 22717 and ISO 22718.
5.3.2 Media for preparation of spores of Bacillus subtilis
See C.1.3.1.
6 Apparatus and glassware
The laboratory equipment, apparatus and glassware are described in ISO 21148.
7 Strains of microorganisms

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

reference strain. The strains may be stored in the laboratory conforming to EN 12353 or according to

another suitable method.

For testing the recovery of microorganisms on the test samples, spores of Bacillus subtilis ATCC 6633

(equivalent strain CIP 52.62 or NCIMB 8054 or NBRC 3134 or other equivalent national collection

strain) are used.
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For testing the efficacy of neutralizers, two strains representative of both Gram negative and Gram

positive bacteria and a yeast are used:

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

KCTC 1916 or other equivalent national collection strain);

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

or KCTC 2513 or other equivalent national collection strain).

An alternative 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).

— 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 strains may be kept in the laboratory according to the EN 12353.
8 Handling of cosmetic products and laboratory samples

If storage is necessary, keep the products to be tested at room temperature. Do not incubate, refrigerate

or freeze products and samples before or after analysis. Sampling and test procedures should follow

the guidelines specified in ISO 21148 and in accordance with the procedure outlined in Clause 9.

9 Procedure
9.1 General recommendation

Use sterile equipment and aseptic technique whenever preparing the test sample and diluent.

For the preparation of an initial suspension, the time which elapses between the end of the preparation

of the test sample and the moment the diluent of the initial suspension comes into contact with the

culture medium shall not exceed (30 ± 15) min, unless specifically mentioned in the established

protocols or documents.

The method should follow the procedure developed during the suitability test, to ensure neutralization

of potential inhibitory effects (see Clause 11) and to guarantee the recovery of microorganisms.

9.2 Selection and preparation of the test sample
9.2.1 Selection of the test sample
The test sample shall weigh at least 1 g.

The test sample can be either the entire unit, or multiple units if the weight of one unit is less than 1 g,

or the UIP (see A.1).
Record the exact weight of the test sample, S, and, the number of units, n.
If a UIP is used for testing, record the UIP value of the test sample (see 4.2).
9.2.2 Preparation of the initial suspension

Place the test sample (see 9.2.1) into an appropriate container, with a known volume of diluent, defined

in the suitability test (see Clause 11). The test sample should be completely immersed in the diluent.

Record the value for “V”, the volume of diluent used.
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9.3 Recovery of microorganisms
9.3.1 General

After immersion, the following treatments may be used to remove microorganisms from the test sample:

— stomaching;
— shaking/stirring;
— vortexing (see A.3).

NOTE If necessary, record the volume of the diluent after stomaching or shaking of test sample.

9.3.2 Stomaching

Prepare the initial suspension utilizing a sterile stomacher bag and then place it into the stomacher.

Proceed according to the parameters (time and speed) outlined in the suitability test (see Clause 11).

Record the time and the speed at which the stomaching took place.
9.3.3 Shaking/Stirring

Prepare the initial suspension in the appropriate closed container and mix according to the parameters

(duration and frequency) applied in the suitability test (see Clause 11).

Sterile glass beads may be added to enhance product mixing and organism recovery.

Record the time, frequency and the speed of shaking/stirring (if applicable) and whether glass beads

are added or not.
9.4 Enumeration of aerobic mesophilic microorganisms
9.4.1 General

The enumeration of aerobic mesophilic microorganisms includes bacteria, yeasts and moulds.

The choice of the method depends on the volume of diluent used for the preparation of the initial

suspension (see A.2).

Based on the test sample size, the level of bioburden and the sensitivity of the method, all of the diluent

in which the test sample is immersed (V) or a fraction of V (Vd) is used for enumeration.

Usually, the volume V or Vd of the initial suspension diluent is the dilution used for enumeration. No

further diluting of the initial preparation is required.

The minimal volume of diluent shall be equivalent to at least 1 g of the test sample.

9.4.2 Pour plate method

Use the appropriate number of Petri dishes to properly evaluate the volume of diluent needed to

properly immerse and transfer the product to be plated (V or Vd).

The diluent (V) is divided into two work streams: one half (V/2) is for the enumeration of bacteria and

the other half (V/2) for enumeration of yeasts and moulds.

If the enumeration is conducted on a fraction of the total diluent (Vd), two equal fractions (Vd/2) shall

be plated: one for bacteria and the other for yeasts and moulds.
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If Petri dishes, 85 mm to 100 mm in diameter are used, add 1 ml of the diluent and pour 15 ml to

20 ml of the melted agar medium (as specified in ISO 21149 and ISO 16212) kept in a water bath not to

exceed 48 °C.
If larger Petri dishes (140
...

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