Nanotechnologies — Evaluation of the antimicrobial performance of textiles containing manufactured nanomaterials

This document specifies the antimicrobial performance assessment method of textiles containing manufactured (metals/metal oxides) nanomaterials (TCMNMs). The textiles in this document include fabric, yarn and fibre in which manufactured nanomaterials are used during production or finishing process. Further, this document also specifies protocols to determine the quantity of nanomaterials released from textile following washing and/or exposure to artificial human body sweat. This document only covers the antibacterial, antifungal, and the anti-odour performance assessment method of TCMNMs. This document does not cover textiles that have therapeutic application as well as environment, health and safety (EHS) issues related to TCMNMs. Further, it does not cover the release of nanomaterials from TCMNMs as a result of aging, dry attrition and abrasion, although it is considered as an effective factor in releasing nanomaterials.

Nanotechnologies — Evaluation de la performance antimicrobienne des textiles contenant des nanomatériaux manufacturés

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Status
Published
Publication Date
06-Oct-2021
Current Stage
6060 - International Standard published
Start Date
07-Oct-2021
Due Date
08-May-2022
Completion Date
07-Oct-2021
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TECHNICAL ISO/TS
SPECIFICATION 23650
First edition
2021-10
Nanotechnologies — Evaluation of
the antimicrobial performance of
textiles containing manufactured
nanomaterials
Nanotechnologies — Evaluation de la performance antimicrobienne
des textiles contenant des nanomatériaux manufacturés
Reference number
ISO/TS 23650:2021(E)
© ISO 2021

---------------------- Page: 1 ----------------------
ISO/TS 23650:2021(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2021
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
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii
  © ISO 2021 – All rights reserved

---------------------- Page: 2 ----------------------
ISO/TS 23650:2021(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Symbols and abbreviated terms.2
5 Characteristics of metal or metal oxide nanomaterials in TCMNMs.3
5.1 General . 3
5.2 Physicochemical characteristics of metal or metal oxide nanomaterials . 3
5.3 Characterization methods. 4
6 Measurement of the released metal or metal oxide nanomaterials .4
6.1 Principle . 4
6.2 Human perspiration solution preparation . 4
6.2.1 General . 4
6.2.2 Measurement method . 4
6.3 Washing procedure . 5
7 Determination of antimicrobial activities of TCMNMs . 5
7.1 Principle . 5
7.2 Antibacterial activity . 5
7.3 Antifungal activity . 5
7.4 Anti-odour property . 6
8 Test report . 6
Annex A (informative) Physical characterization techniques of nanomaterials in TCMNMs .9
Annex B (informative) Chemical characterization of nanomaterials in TCMNMs .10
Annex C (informative) Determination of antibacterial, antifungal, and anti-odour activity
of TCMNMs .11
Bibliography .13
iii
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---------------------- Page: 3 ----------------------
ISO/TS 23650:2021(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 229, Nanotechnologies.
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.
iv
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---------------------- Page: 4 ----------------------
ISO/TS 23650:2021(E)
Introduction
The utilization of nanotechnology in textile industry has presented novel functions such as
antimicrobial activity, stain resistance, flame retardancy, mechanical strength enhancement, UV
resistance, and wrinkle resistance into the conventional textiles without significant loss or change of
[17]
the original properties. According to the nanodatabase website there are already over 400 textiles
containing manufactured nanomaterials (TCMNMs), making them the second largest market among
other nanoproducts.
The rapid and continued growth of TCMNMs is increasing the need to develop international standards
specific for manufactured nanomaterials (MNMs) in textiles and testing processes guidelines. It is a
dual need of industry and consumer.
TCMNMs can be classified into three groups based on how nanomaterials are integrated into the
[1]
textiles including nanofinished, nanocomposite, and nanofibrous textiles :
a) Nanofinished textiles: The textiles that the applied nanoscale property is added after the textile
fabrication through post-manufacture treatments and coatings to create nanostructured surfaces
on fibre media. Most nanotextiles on the consumer market belong to this category.
b) Nanocomposite textiles: The textiles composed of fibres containing one or more nanostructured
or nanoscale components produced by pre-manufacture integration of nanoscale properties into
fibrous components.
c) Nanofibrous textiles: The textiles made of nanofibres which have a nanoscale cross-sectional area
and may or may not have a nanoscale length.
Natural and manufactured textile fibres can be treated with different nanomaterials and chemicals to
provide enhanced antimicrobial properties. The antimicrobial activities of TCMNMs include activities
against bacteria, fungal, viruses, and other microorganisms. Also, the antimicrobial activities can help
to impart anti-odour property as the consequence of the reduced microbial activity. For antimicrobial
TCMNMs, various metals, mainly silver and copper, and metal oxides such as copper oxide (CuO),
titanium dioxide (TiO ) and zinc oxide (ZnO) are normally used.
2
Several characteristics of MNMs have great impacts on their antimicrobial performance including
size, shape, surface area, chemical composition, surface chemistry and surface charge. The size and
shape of MNMs have important impacts on their antimicrobial property due to their association to
their surface area. Generally, the antibacterial properties of nanoparticles are size-dependent. Smaller
particles with higher surface area to volume ratio have more contact with either bacteria or fungi cells,
[2]
or both, leading to improve either the bactericidal or fungicidal effectiveness, or both . Therefore,
when they incorporate in textiles even at low concentrations they show noticeable antimicrobial
[3]-[5]
activity compared to their micro-and macro scale counterparts. The shape of MNMs remarkably
influences the rate of interaction and uptake by microbial cells. For instance, spherical-shaped of
[7]
gold nanoparticles demonstrated higher cellular uptake than nanorod shaped particles . Surface
charge of MNMs is another important characteristic that can be measured by Zeta potential method.
The antimicrobial effect of MNMs is triggered by the electrostatic interaction between the positively
charged MNMs and the negatively charged microbial cell membranes ultimately leading to cell damage
and inhibition of their growth and reproduction. Surface chemistry of MNMs has an important effect
on their antimicrobial activity. The presence of functional groups, capping agents or biomolecules
on the surface of nanomaterials has also potential influence in their antibacterial activities. Surface
functionalization of antimicrobial nanoparticles such as silver nanoparticles with bioactive molecules
[8]
exhibited enhanced antibacterial activity compared to the bare ones . The above-mentioned inter-
relationship highlights the important effect of physiochemical characteristics on antimicrobial
performance of TCMNMs.
Currently, there are various antimicrobial TCMNMs products in the market such as underwear,
shirts, socks, and bed sheets/covers. The antimicrobial mechanism of action of nanomaterials can
generally be described as one of three models: oxidative stress induction, metal ion release, or non-
[1]
oxidative mechanisms, which can occur simultaneously as well . The antimicrobial activity of
v
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ISO/TS 23650:2021(E)
TCMNMs can decline significantly after several washing cycles and exposure to body sweat due to the
possible release of incorporated nanomaterials and also the chemical action of sweat and laundering
solution on the nanocompounds. Currently, there is no ISO document specific to TCMNM products.
Therefore, the development of a standard to determine antimicrobial performance of TCMNMs
subjected to washing process and body sweating can facilitate the trade and growth of market. It is
worth mentioning that already published ISO standards are related to the assessment of antimicrobial
properties of conventional textiles. Moreover, there is an ASTM standard document for detection and
[9]
characterization of silver nanomaterials in textiles . However, these documents do not address the
potential release of nanomaterials/nanostructure from TCMNNs following washing or sweating and
their possible consequence on the antimicrobial activity of these textiles.
This document does not address nano-safety and environmental impact of the release of nanomaterials
from TCMNMs into the air, water and to landfill. Data related to the release of nanomaterials from the
fabrics under different conditions such as sweating, mechanical stresses (repetitive abrasion) during
washing process in a laundry machine, are considered as essential information for understanding the
potential releases to the environment.
Artificial sweat solution is an appropriate candidate to use as a material to resemble the human skin
sweat to determine the amount of release of nanomaterials from TCMNMs to human body. For many
TCMNMs applications, such as human clothes, there is a high possibility of skin contact and interaction
[10]
with incorporated nanomaterials . In such condition, the involved interaction and release of the
nanomaterial can also affect the antibacterial performance of TCMNMs.
Considering the effect of the release of nanomaterials from TCMNMs by washing process and
human sweat, this document specifies the measurement methods of the released nanomaterials, the
antimicrobial performance and the assessment method of TCMNMs. Further, from TCMNMs subjected
to washing process and exposed to artificial human body sweat solution are specified.
vi
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TECHNICAL SPECIFICATION ISO/TS 23650:2021(E)
Nanotechnologies — Evaluation of the antimicrobial
performance of textiles containing manufactured
nanomaterials
1 Scope
This document specifies the antimicrobial performance assessment method of textiles containing
manufactured (metals/metal oxides) nanomaterials (TCMNMs). The textiles in this document include
fabric, yarn and fibre in which manufactured nanomaterials are used during production or finishing
process. Further, this document also specifies protocols to determine the quantity of nanomaterials
released from textile following washing and/or exposure to artificial human body sweat.
This document only covers the antibacterial, antifungal, and the anti-odour performance assessment
method of TCMNMs.
This document does not cover textiles that have therapeutic application as well as environment, health
and safety (EHS) issues related to TCMNMs. Further, it does not cover the release of nanomaterials
from TCMNMs as a result of aging, dry attrition and abrasion, although it is considered as an effective
factor in releasing nanomaterials.
2 Normative references
The following referenced documents are indispensable for the application of this document. For dated
references, only the edition cited applies. For undated references, the latest edition of the referenced
document (including any amendments) applies.
ISO 105-E04, Textiles — Tests for colour fastness — Part E04: Colour fastness to perspiration
ISO 6330, Textiles — Domestic washing and drying procedures for textile testing
ISO 20743:2021, Textiles — Determination of antibacterial activity of textile products
ISO 13629-1, Textiles — Determination of antifungal activity of textile products — Part 1: Luminescence
method
ISO/TS 80004-1, Nanotechnologies — Vocabulary — Part 1: Core terms
EN 16711-1, Textiles — Determination of metal content — Part 1: Determination of metals using microwave
digestion
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 6330, ISO/TS 80004-1 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
— IEC Electropedia: available at http:// www .electropedia .org/
1
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ISO/TS 23650:2021(E)
3.1
nanomaterial
material with any external dimension in the nanoscale or having internal structure or surface structure
in the nanoscale
Note 1 to entry: This generic term is inclusive of nano-object and nanostructured material.
[SOURCE: ISO/TS 80004-1:2015, 2.4, modified — Note 2 to entry has been deleted.]
3.2
textile
woven fabric, knitted fabric, etc., formed by the interlocking of fibres and yarns having certain cohesion
and which is generally intended for clothing or furniture applications
Note 1 to entry: Textiles often include certain types of non-woven fabrics.
[SOURCE: ISO 16373-3:2014, 2.4]
3.3
antimicrobial activity
ability to kill/destroy/inactivate microorganisms, prevent their proliferation and/or prevent their
pathogenic action
[SOURCE: ISO 18369-1:2017, 3.1.11.12]
3.4
antibacterial activity
activity of an antibacterial finish used to prevent or mitigate the growth of bacteria, to reduce the
number of bacteria or to kill bacteria
[SOURCE: ISO 20743:2021, 3.4]
3.5
antifungal activity
activity to prevent or mitigate the growth of fungus, expressed as the difference of growth value in
logarithm of ATP (3.6) between the control and test sample
[SOURCE: ISO 13629-1:2012, 3.6]
3.6
ATP
adenosine triphosphate, a multifunctional nucleotide present in living fungi
[SOURCE: ISO 13629-1:2012, 3.5]
3.7
washing procedure
cycle of the washing action including water supplying, washing, and repeated rinsing, spinning and
water supplying and ended by spinning as predetermined on the washing machine
[SOURCE: ISO 6330:2012, 3.7]
4 Symbols and abbreviated terms
AAS Atomic absorption spectroscopy
AFM Atomic force microscopy
AES Auger electron spectroscopy
2
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ISO/TS 23650:2021(E)
ATP Adenosine triphosphate
BET Brunauer, Emmett, and Teller
ELS Electrophoretic light scattering
FESEM Field emission scanning electron microscopy
ICP-AES Inductively coupled plasma-atomic emission spectroscopy
ICP-MS Inductively coupled plasma-mass spectrometry
MNM Manufactured nanomaterial
TCMNM Textiles containing manufactured nanomaterial
TEM Transmission electron microscopy
SAED Selected area (electron) diffraction
XPS X-ray photoelectron spectroscopy
5 Characteristics of metal or metal oxide nanomaterials in TCMNMs
5.1 General
As was mentioned earlier in the introduction section, knowledge about the physicochemical
characteristics of nanomaterials used in TCMNMs is important, considering their noticeable effects
on their antimicrobial performance. Subject to the stakeholder agreement and the specific application,
these characteristics as shown in Table 1 should be measured and reported.
A wide variety of analytical techniques are available for detection and characterization of nanomaterials
in textiles. The selection of the appropriate techniques depends on capabilities, advantages and
limitations of the techniques. Also, the cost and availability of the instrument need to be taken into
account. There are no single techniques to both detect and characterize MNMs in textiles.
5.2 Physicochemical characteristics of metal or metal oxide nanomaterials
The commercially available techniques to measure the physiochemical characteristics of nanomaterials
and definitions relevant to the characterization of them are available in ISO/TR 18196 and ISO/TS
80004-6, respectively. Also, the characteristics and measurement methods for powder or colloidal
forms of silver nanoparticles applied as antibacterial agents are available in ISO/TS 20660.
These physicochemical characteristics include shape, size, surface charge, chemical composition, and
surface chemistry of MNMs. Table 1 summarizes the list of physicochemical characteristics and their
measurement methods for TCMNMs.
Table 1 — List of physicochemical characteristics of metallic or metal oxides nanomaterials
used in TCMNMs
Characteristic/property Measurement methods
Particle size, shape, and size distribution FESEM, TEM, SEM, AFM
Zeta potential ELS
Surface area BET
Surface chemistry XPS, AES
Chemical composition AAS, ICP-AES, ICP-MS
Phase identification TEM/SAED
3
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ISO/TS 23650:2021(E)
As mentioned before, nanomaterials utilized in textiles are either incorporated in the main fibre
texture or applied as a coating onto the textiles by different methods. On the other hand, such fibres and
textile fabrics made out of them can be further processed for different purposes. In some cases, there
can be complexities for the characterization and detection of the nanomaterials used for antibacterial
properties. This includes the possible elemental and chemical similarities of different chemical agents
for various purposes (e.g. dyeing, printing) with those of the used nanomaterials. Therefore, to
identify the latter from the former, care should be taken to choose a set of appropriate measurement
techniques, since for such cases normally no single technique can be suitable to resolve the issue. In this
[15]
respect, ASTM E3025-16 also explores some of the physicochemical characteristics measurement
methods and the relevant detection challenges of textiles containing silver nanomaterials which can be
[9]
considered .
5.3 Characterization methods
The brief description of the mentioned characterization methods of TCMNMs is given in Annex A
and Annex B. For chemical composition analysis, the sample shall be digested according to one of the
procedures of acid digestion or microwave-assisted acid digestion presented in Clause B.3. The goal of
digestion is to completely decompose the solid matrix of TCMNMs to transfer the nanomaterials into
the solution for the further determination step. The choice of the digestion method depends on the
instrument availability and agreement between the concerned parties.
6 Measurement of the released metal or metal oxide nanomaterials
6.1 Principle
The nanomaterial released from textiles is measured during exposure to human perspiration and
washing procedure as described in 6.2 and 6.3, respectively.
6.2 Human perspiration solution preparation
6.2.1 General
Artificial perspiration solution shall be used to simulate human perspiration. Since perspiration
varies widely from one person to another, it is not possible to design a method with universal validity.
Generally, fresh human perspiration is weakly acidic. However, micro-organisms cause the pH to
become weakly alkaline (pH 7,5 to pH 8,5). Therefore, two different artificial alkaline (pH 8) and acidic
sweat solutions (pH 5,5) as specified in ISO 105-E04 shall be utilized as the natural perspiration source.
The preparation of artificial alkaline and acidic sweat solutions shall be made according to ISO 105-E04.
6.2.2 Measurement method
The amount of nanomaterials released from textiles is determined by measuring the concentration
difference before and after they are being exposed to as-prepared artificial body sweat solution using
the formula:
AA−
01
X = ×100 (1)
A
0
4
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ISO/TS 23650:2021(E)
where
X is the amount of nanomaterials released from the textile sample;
A is the measured amount of nanomaterials (µg/l) in the textile sample solution before it is exposed
0
to the artificial sweat solution;
A is the amount of nanomaterials (µg/l) in the textile sample after it has been exposed to the ar-
1
tificial sweat solution (to be reported as a percentage).
A and A shall be measured after subjecting the samples to acid digestion or microwave-assisted acid
0 1
digestion as explained in Annex B.
It should be mentioned that the measured release can be also due to the possible utilization of
chemicals and or nano-sized particles as dyes. In such case, care must be taken to consider only the
release of the nanomaterial(s) showing antimicrobial properties due to the limited available MNMs for
such applications. Further, it is necessary that the producer or manufacturer should declare the type of
MNMs used in textiles.
6.3 Washing procedure
In this method, the released nanomaterials utilized in TCMNMs are measured after several washing
cycles by regular domestic washing procedure. Considering the wide varieties of textiles covered by
this document, the selection of specific procedure of washing, drying and type of detergent provided
in ISO 6330 shall be done based on the instructions provided by textile manufacturer. The released
amount of nanomaterials during the washing procedure is calculated similarly according to the
Formula (1). However, here, A and A are referred to the number of nanomaterials in textile sample
0 1
pre- and post-washing process, respectively. A and A shall be measured after subjecting the samples
0 1
to microwave or acid digestion as explained in Annex B.
7 Determination of antimicrobial activities of TCMNMs
7.1 Principle
Antimicrobial activities of TCMNMs samples including antibacterial, antifungal and anti-odour shall be
carried out on specimens, pre- and post-washing process and those before and after exposure to human
sweat solution.
7.2 Antibacterial activity
The antibacterial activity of the TCMNMs shall be determined according to the ISO 20743:2021 standard
method using Gram-positive Staphylococcus aureus and Gram-negative bacterium Klebsiella pneumoniae
(AATCC 4352). A brief description of the method is given in Clause C.1.
NOTE 1 Other bacteria can be used after appropriate validation.
[30]
NOTE 2 Refer to World Data Centre for Microorganisms (WDCM): http:// refs .wdcm .org/ search .htm .
7.3 Antifungal activity
The antifungal activity of TCMNM samples shall be determined according to ISO 13629-1. The test
method determines the antifungal activity by measuring the intensity of luminescence produced by an
enzymatic reaction (ATP method). A brief description of the method is given Clause C.2.
The reference fungi to be used shall be selected from the following list:
— Aspergillus niger;
— Penicillium citrinum;
5
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ISO/TS 23650:2021(E)
— Cladosporium cladosporioides;
— Trichophyton mentagrophytes.
NOTE 1 Other fungi can be used after appropriate validation.
[30]
NOTE 2 Refer to WDCM and its website: http:// refs .wdcm .org/ search .htm .
7.4 Anti-odour property
Sweat secreted by axillary glands is odourless. The human axillary malodour is mainly produced by
[11]
bacteria flora found on axillary skin dominated by genus of Gram-positive Corynebacteria . The
antibacterial TCMNMs can reduce malodour by decreasing the number of Corynebacteria on axillary
skin area. If the manufacturer claims that the product has the anti-odour property, this property of
textile samples shall be determined according to ISO 20743 and carry out the test by using any strains
of Corynebacterium such as Corynebacterium xerosis. A more detailed description is given in Clause C.3.
8 Test report
The manufacturer or provider shall report the relevant general information and the measurement
results of the fundamental characteristics of the TCMNMs. The test report shall contain the following
information:
a) a reference to this document (i.e. ISO/TS 23650:2021);
b) general information of TCMNMs products such as product name, product application, batch number,
lot number, manufacturing method, type of TCMNM, lab. name and address;
c) details of the measurement results of nanomaterial used such as size, size distribution, zeta
potential, surface area, phase identification, chemical composition, and surface chemistry subject
to the relevant stakeholders agreement;
d) all details of antibacterial characterization test procedure such as name of the test bacteria, strain
number, inoculation method, concentration inoculum, quantitative measurement method and
antibacterial activity va
...

TECHNICAL ISO/TS
SPECIFICATION 23650
First edition
Nanotechnologies — Evaluation of
the antimicrobial performance of
textiles containing manufactured
nanomaterials
Nanotechnologies — Evaluation de la performance antimicrobienne
des textiles contenant des nanomatériaux manufacturés
PROOF/ÉPREUVE
Reference number
ISO/TS 23650:2021(E)
©
ISO 2021

---------------------- Page: 1 ----------------------
ISO/TS 23650:2021(E)

COPYRIGHT PROTECTED DOCUMENT
© ISO 2021
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
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii PROOF/ÉPREUVE © ISO 2021 – All rights reserved

---------------------- Page: 2 ----------------------
ISO/TS 23650:2021(E)

Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Symbols and abbreviated terms . 2
5 Characteristics of metal or metal oxide nanomaterials in TCMNMs .3
5.1 General . 3
5.2 Physicochemical characteristics of metal/metal oxide nanomaterials . 3
5.3 Characterization methods . 4
6 Measurement of the released metal or metal oxide nanomaterials .4
6.1 Principle . 4
6.2 Human perspiration solution preparation . 4
6.2.1 General. 4
6.2.2 Measurement method . 4
6.3 Washing procedure . 5
7 Determination of antimicrobial activities of TCMNMs . 5
7.1 Principle . 5
7.2 Antibacterial activity . 5
7.3 Antifungal activity . 5
7.4 Anti-odour property . 5
8 Test report . 6
Annex A (innormative)Physical characterization techniques of nanomaterials in TCMNMs .10
Annex B (informative) Chemical characterization of nanomaterials in TCMNMs .12
Annex C (informative) Determination of antibacterial, antifungal, and anti-odour activity of
TCMNMs .13
Bibliography .15
© ISO 2021 – All rights reserved PROOF/ÉPREUVE iii

---------------------- Page: 3 ----------------------
ISO/TS 23650:2021(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 229, Nanotechnologies.
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.
iv PROOF/ÉPREUVE © ISO 2021 – All rights reserved

---------------------- Page: 4 ----------------------
ISO/TS 23650:2021(E)

Introduction
The utilization of nanotechnology in textile industry has presented novel functions such as
antimicrobial activity, stain resistance, flame retardancy, mechanical strength enhancement, UV
resistance, and wrinkle resistance into the conventional textiles without significant loss or change of
[1]
the original properties. According to the nanodatabase website there are already over 400 Textiles
Containing Manufactured Nanomaterials (TCMNMs), making them the second largest market among
other nanoproducts.
The rapid and continued growth of TCMNMs is increasing the need to develop international standards
specific for manufactured nanomaterials (MNMs) in textiles and testing processes guidelines. It is a
dual need of industry and consumer.
TCMNMs can be classified into three groups based on how nanomaterials are integrated into the
[2]
textiles including nanofinished, nanocomposite, and nanofibrous textiles :
a) Nanofinished textiles: The textiles that the applied nanoscale property is added after the textile
fabrication through post-manufacture treatments and coatings to create nanostructured surfaces
on fibre media. Most nanotextiles on the consumer market belong to this category.
b) Nanocomposite textiles: The textiles composed of fibres containing one or more nanostructured
or nanoscale components produced by pre-manufacture integration of nanoscale properties into
fibrous components.
c) Nanofibrous textiles: The textiles made of nanofibres which have a nanoscale cross-sectional area
and may or may not have a nanoscale length.
Natural and manufactured textile fibres can be treated with different nanomaterials and chemicals to
provide enhanced antimicrobial properties. The antimicrobial activities of TCMNMs include activities
against bacteria, fungal, viruses, and other microorganisms. Also, the antimicrobial activities can help
to impart anti-odour property as the consequence of the reduced microbial activity. For antimicrobial
TCMNMs, various metals, mainly silver and copper, and metal oxides such as copper oxide (CuO),
titanium dioxide (TiO ) and zinc oxide (ZnO) are normally used.
2
Several characteristics of MNMs have great impacts on their antimicrobial performance including size,
shape, surface area, chemical composition, surface chemistry and surface charge. The size and shape of
MNMs have important impacts on their antimicrobial property due to their association to their surface
area. Generally, the antibacterial properties of nanoparticles are size-dependent. Smaller particles
with higher surface area to volume ratio have more contact with bacteria and/or fungi cells leading to
[3]
improve the bactericidal and/or fungicidal effectiveness . Therefore, when they incorporate in textiles
even at low concentrations they show noticeable antimicrobial activity compared to their micro-and
[4]-[6]
macro scale counterparts. The shape of MNMs remarkably influences the rate of interaction and
uptake by microbial cells. For instance, spherical-shaped of gold nanoparticles demonstrated higher
[7]
cellular uptake than nanorod shaped particles . Surface charge of MNMs is another important
characteristic that can be measured by Zeta potential method. The antimicrobial effect of MNMs is
triggered by the electrostatic interaction between the positively charged MNMs and the negatively
charged microbial cell membranes ultimately leading to cell damage and inhibition of their growth
and reproduction. Surface chemistry of MNMs has an important effect on their antimicrobial activity.
The presence of functional groups, capping agents or biomolecules on the surface of nanomaterials
has also potential influence in their antibacterial activities. Surface functionalization of antimicrobial
nanoparticles such as silver nanoparticles with bioactive molecules exhibited enhanced antibacterial
[8]
activity compared to the bare ones . The above mentioned inter-relationship highlights the important
effect of physiochemical characteristics on antimicrobial performance of TCMNMs.
Currently, there are various antimicrobial TCMNMs products in the market such as underwear, shirts,
socks, bed sheets/covers etc. The antimicrobial mechanism of action of nanomaterials can generally
be described as one of three models: oxidative stress induction, metal ion release, or non-oxidative
[2]
mechanisms, which can occur simultaneously as well . The antimicrobial activity of TCMNMs can
decline significantly after several washing cycles and exposure to body sweat due to the possible
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release of incorporated nanomaterials and also the chemical action of sweat and laundering solution
on the nanocompounds. Currently, there is no ISO standard specific to TCMNM products. Therefore,
the development of a standard to determine antimicrobial performance of TCMNMs subjected
to washing process and body sweating can facilitate the trade and growth of market. It is worth
mentioning that already published ISO standards are related to the assessment of antimicrobial
properties of conventional textiles. Moreover, there is an ASTM standard document for detection and
[9]
characterization of silver nanomaterials in textiles . However, these documents do not address the
potential release of nanomaterials/nanostructure from TCMNNs following washing or sweating and
their possible consequence on the antimicrobial activity of these textiles.
This document does not address nano-safety and environmental impact of the release of nanomaterials
from TCMNMs into the air, water and to landfill. Data related to the release of nanomaterials from the
fabrics under different conditions such as sweating, mechanical stresses (repetitive abrasion) during
washing process in a laundry machine, are considered as essential information for understanding the
potential releases to the environment.
Artificial sweat solution is an appropriate candidate to use as a material to resemble the human skin
sweat to determine the amount of release of nanomaterials from TCMNMs to human body. For many
TCMNMs applications, such as human clothes, there is a high possibility of skin contact and interaction
[10]
with incorporated nanomaterials . In such condition, the involved interaction and release of the
nanomaterial can also affect the antibacterial performance of TCMNMs.
Considering the effect of the release of nanomaterials from TCMNMs by washing process and
human sweat, this document specifies the measurement methods of the released nanomaterials, the
antimicrobial performance and the assessment method of TCMNMs. Further, from TCMNMs subjected
to washing process and exposed to artificial human body sweat solution are specified.
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TECHNICAL SPECIFICATION ISO/TS 23650:2021(E)
Nanotechnologies — Evaluation of the antimicrobial
performance of textiles containing manufactured
nanomaterials
1 Scope
This document specifies the antimicrobial performance assessment method of textiles containing
manufactured (metals/metal oxides) nanomaterials (TCMNMs). The textiles in this document include
fabric, yarn and fibre in which manufactured nanomaterials are used during production or finishing
process. Further, this document also specifies protocols to determine the quantity of nanomaterials
released from textile following washing and/or exposure to artificial human body sweat.
This document only covers the antibacterial, antifungal, and the anti-odour performance assessment
method of TCMNMs.
This document does not cover textiles that have therapeutic application as well as environment, health
and safety (EHS) issues related to TCMNMs. Further, it does not cover the release of nanomaterials
from TCMNMs as a result of aging, dry attrition and abrasion, although it is considered as an effective
factor in releasing nanomaterials.
2 Normative references
The following referenced documents are indispensable for the application of this document. For dated
references, only the edition cited applies. For undated references, the latest edition of the referenced
document (including any amendments) applies.
ISO 6330, Textiles — Domestic washing and drying procedures for textile testing
ISO 20743:2013, Textiles — Determination of antibacterial activity of textile products
ISO 13629-2, Textiles — Determination of antifungal activity of textile products — Part 2: Plate count
method
ISO/TS 80004-1, Nanotechnologies — Vocabulary — Part 1: Core terms
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 6330, ISO/TS 80004-1 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
— IEC Electropedia: available at http:// www .electropedia .org/
3.1
nanomaterial
material with any external dimension in the nanoscale or having internal structure or surface structure
in the nanoscale
Note 1 to entry: This generic term is inclusive of nano-object and nanostructured material.
[SOURCE: ISO/TS 80004-1:2015, 2.4, modified — Note 2 to entry has been deleted.]
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3.2
textile
woven fabric, knitted fabric, etc., formed by the interlocking of fibres and yarns having certain cohesion
and which is generally intended for clothing or furniture applications
Note 1 to entry: Textiles often include certain types of non-woven fabrics.
[SOURCE: ISO 16373-3:2014, 2.4]
3.3
antimicrobial activity
ability to kill/destroy/inactivate microorganisms, prevent their proliferation and/or prevent their
pathogenic action
[SOURCE: ISO 18369-1:2017, 3.1.11.12]
3.4
antibacterial activity
activity of an antibacterial finish used to prevent or mitigate the growth of bacteria, to reduce the
number of bacteria or to kill bacteria
[SOURCE: ISO 20743:2013, 3.4]
3.5
antifungal activity
activity to prevent or mitigate the growth of fungus, expressed as the difference of growth value in
logarithm of ATP (3.6) between the control and test sample
[SOURCE: ISO 13629-1:2012, 3.6]
3.6
ATP
adenosine triphosphate, a multifunctional nucleotide present in living fungi
[SOURCE: ISO 13629-1:2012, 3.5]
3.7
washing procedure
cycle of the washing action including water supplying, washing, and repeated rinsing, spinning and
water supplying and ended by spinning as predetermined on the washing machine
[SOURCE: ISO 6330:2012, 3.7]
4 Symbols and abbreviated terms
AAS Atomic absorption spectroscopy
AFM Atomic force microscopy
AES Auger electron spectroscopy
ATP Adenosine triphosphate
BET Brunauer, Emmett, and Teller
ELS Electrophoretic light scattering
FESEM Field emission scanning electron microscopy
ICP-AES Inductively coupled plasma-atomic emission spectroscopy
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ICP-MS Inductively coupled plasma-mass spectrometry
MNM Manufactured nanomaterial
TCMNM Textiles containing manufactured nanomaterial
TEM Transmission electron microscopy
SAED Selected area (electron) diffraction
XPS X-ray photoelectron spectroscopy
5 Characteristics of metal or metal oxide nanomaterials in TCMNMs
5.1 General
As was mentioned earlier in the introduction section, knowledge about the physicochemical
characteristics of nanomaterials used in TCMNMs is important, considering their noticeable effects
on their antimicrobial performance. Subject to the stakeholder agreement and the specific application,
these characteristics as shown in Table 1 should be measured and reported.
A wide variety of analytical techniques are available for detection and characterization of nanomaterials
in textiles. The selection of the appropriate techniques depends on capabilities, advantages and
limitations of the techniques. Also, the cost and availability of the instrument need to be taken into
account. There are no single techniques to both detect and characterize MNMs in textiles.
5.2 Physicochemical characteristics of metal/metal oxide nanomaterials
The commercially available techniques to measure the physiochemical characteristics of
nanomaterials and definitions relevant to the characterization of them are available in ISO/TR 18196
and ISO/TS 80004-6, respectively. Also, the characteristics and measurement methods for powder or
colloidal forms of silver nanoparticles applied as antibacterial agents are available in ISO/TS 20660.
These physicochemical characteristics include shape, size, surface charge, chemical composition, and
surface chemistry of MNMs. Table 1 summarizes the list of physicochemical characteristics and their
measurement methods for TCMNMs.
Table 1 — List of physicochemical characteristics of metallic or metal oxides nanomaterials
used in TCMNMs
Characteristic/property Measurement methods
Particle size, shape, and size distribution FESEM, TEM, SEM, AFM
Zeta potential ELS
Surface area BET
Surface Chemistry XPS, AES
Chemical Composition AAS, ICP-AES, ICP-MS
Phase Identification TEM/SAED
As mentioned before, nanomaterials utilized in textiles are either incorporated in the main fibre texture
or applied as a coating onto the textiles by different methods. On the other hand, such fibres and textile
fabrics made out of them can be further processed for different purposes. In some cases, there may
be complexities for the characterization and detection of the nanomaterials used for antibacterial
properties. This includes the possible elemental and chemical similarities of different chemical agents
for various purposes (e.g. dyeing, printing) with those of the used nanomaterials. Therefore, to
identify the latter from the former, care should be taken to choose a set of appropriate measurement
techniques, since for such cases normally no single technique can be suitable to resolve the issue. In this
[15]
respect, ASTM E3025-16 also explores some of the physicochemical characteristics measurement
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methods and the relevant detection challenges of textiles containing silver nanomaterials which can be
[9]
considered .
5.3 Characterization methods
The brief description of the mentioned characterization methods of TCMNMs is presented in Annex A
and Annex B. For chemical composition analysis, the sample shall be digested according to one of
the procedures of acid digestion or microwave-assisted acid digestion presented in B.3. The goal of
digestion is to completely decompose the solid matrix of TCMNMs to transfer the nanomaterials into
the solution for the further determination step. The choice of the digestion method depends on the
instrument availability and agreement between the concerned parties.
6 Measurement of the released metal or metal oxide nanomaterials
6.1 Principle
The nanomaterial released from textiles is measured during exposure to human perspiration and
washing procedure as described in 6.2 and 6.3, respectively.
6.2 Human perspiration solution preparation
6.2.1 General
Artificial perspiration solution shall be used to simulate human perspiration. Since perspiration
varies widely from one person to another, it is not possible to design a method with universal validity.
Generally, fresh human perspiration is weakly acidic. However, micro-organisms cause the pH to
become weakly alkaline (pH 7,5 to pH 8,5). Therefore, two different artificial alkaline (pH 8) and acidic
sweat solutions (pH 5,5) as specified in ISO 105-E04 shall be utilized as the natural perspiration source.
The preparation of artificial alkaline and acidic sweat solutions shall be made according to ISO 105-E04.
6.2.2 Measurement method
The amounts of nanomaterials released from textiles are determined by measuring the concentration
difference before and after they are being exposed to as-prepared artificial body sweat solution. The
following formula can be applied to the calculation:
AA−
01
X = ×100 (1)
A
0
where
X is the amount of nanomaterials released from the textile sample;
A is the measured amount of nanomaterials (µg/l) in the textile sample solution before it is exposed
0
to the artificial sweat solution;
A is the amount of nanomaterials (µg/l) in the textile sample after it has been exposed to the ar-
1
tificial sweat solution (to be reported as a percentage).
A and A shall be measured after subjecting the samples to acid digestion or microwave-assisted acid
0 1
digestion as explained in Annex B.
It should be mentioned that the measured release can be also due to the possible utilization of chemicals
and or nano-sized particles as dyes. In such case, care must be taken to consider only the release of
the nanomaterial(s) showing antimicrobial properties due to the limited available MNMs for such
applications. Further, it is necessary that producer or manufacturer should declare the type of MNMs
used in textiles.
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6.3 Washing procedure
In this method, the released nanomaterials utilized in TCMNMs are measured after several washing
cycles by regular domestic washing procedure. Considering the wide varieties of textiles covered by
this document, the selection of specific procedure of washing, drying and type of detergent provided
in ISO 6330 shall be done based on the instructions provided by textile manufacturer. The released
amount of nanomaterials during the washing procedure is calculated similarly according to the
Formula (1). However, here, A and A are referred to the number of nanomaterials in textile sample
0 1
pre- and post-washing process, respectively. A and A shall be measured after subjecting the samples
0 1
to microwave or acid digestion as explained in Annex B.
7 Determination of antimicrobial activities of TCMNMs
7.1 Principle
Antimicrobial activities of TCMNMs samples including antibacterial, antifungal and anti-odour shall be
carried out on specimens, pre- and post-washing process and those before and after exposure to human
sweat solution.
7.2 Antibacterial activity
The antibacterial activity of the TCMNMs shall be determined according to the ISO 20743:2013 standard
method using Gram-positive Staphylococcus aureus and Gram-negative bacterium Klebsiella pneumoniae
(AATCC 4352). A brief description of the method is given in C.1.
NOTE 1 Other bacteria can be used after appropriate validation.
NOTE 2 Refer to World Data Centre for Microorganisms (WDCM) and its website: http:// refs .wdcm .org/
search .htm.
7.3 Antifungal activity
The antifungal activity of TCMNM samples shall be determined according to ISO 13629-2. The test
method determines the antifungal activity by measuring the intensity of luminescence produced by an
enzymatic reaction (ATP method). A brief description of the method is given C.2.
The reference fungi to be used shall be selected from the following list:
— Aspergillus niger
— Penicillium citrinum
— Cladosporium cladosporioides
— Trichophyton mentagrophytes
NOTE 1 Other fungi can be used after appropriate validation.
NOTE 2 Refer to WDCM and its website: http:// refs .wdcm .org/ search .htm.
7.4 Anti-odour property
Sweat secreted by axillary glands is odourless. The human axillary malodour is mainly produced by
[11]
bacteria flora found on axillary skin dominated by genus of Gram-positive Corynebacteria . The
antibacterial TCMNMs can reduce malodour by decreasing the number of Corynebacteria on axillary
skin area. If the manufacturer claims that the product has the anti-odour property, this property of
textile samples shall be determined according to ISO 20743 and carry out the test by using any strains
of Corynebacterium such as Corynebacterium xerosis. A more detailed description is given in C.3.
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8 Test report
The manufacturer or provider shall report the relevant general information and the measurement
results of the fundamental characteristics of the TCMNMs. The test report shall contain the following
information:
a) a reference to this document (i.e. ISO/TS 23650:2021);
b) general information of TCMNMs products such as product name, product application, batch number,
lot number, manufacturing method, type of TCMNM, lab. name and address;
c) details of the measurement results of nanomaterial used such as size, size distribution, zeta
potential, surface area, phase identification, chemical composition, and surface chemistry subject
to the relevant stakeholders agreement;
d) all details of antibacterial characterization test procedure such as name of the test bacteria, strain
number, inoculation method, concentration inoculum, quantitative measurement method and
antibacterial activity value (A), and measuring metho
...

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