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
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ISO/TS 23650:2021(E)
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© ISO 2021

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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

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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.
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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.

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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ISO/TS 23650:2021(E)

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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ISO/TS 23650:2021(E)
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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ISO/TS 23650:2021(E)
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

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−
X = ×100 (1)
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

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-

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

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.

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
...

ISO/PRF TS 23650

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

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

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

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