International

Standard

ISO 16187
Footwear and footwear Second edition
components — Test method to 2025-02
assess antibacterial activity
iTeh Standards
Chaussure et composants de chaussure — Méthode d'essai pour
évaluer l'activité antibactérienne
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ISO 16187:2025
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Reference number
ISO 16187:2025(en) © ISO 2025
 ISO 16187:2025(en)

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ISO 16187:2025
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© ISO 2025
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
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 ISO 16187:2025(en)

Contents Page

Foreword..................................................................................................................................................................................................................................................... iv
1 Scope.............................................................................................................................................................................................................................................. 1
2 Normative references.................................................................................................................................................................................................. 1
3 Terms and definitions................................................................................................................................................................................................. 1
4 Principle..................................................................................................................................................................................................................................... 1
5 Safety............................................................................................................................................................................................................................................. 2
6 Apparatus................................................................................................................................................................................................................................. 2
7 Reagents and culture medium........................................................................................................................................................................... 2
7.1 General.........................................................................................................................................................................................................................2
7.2 Water............................................................................................................................................................................................................................. 3
7.3 Nutrient broth (NB)......................................................................................................................................................................................... 3
7.3.1 Composition.......................................................................................................................................................................................... 3
7.3.2 Preparation...........................................................................................................................................................................................3
7.4 Nutrient agar (NA)............................................................................................................................................................................................ 3
7.4.1 Composition.......................................................................................................................................................................................... 3
7.4.2 Preparation...........................................................................................................................................................................................3
7.5 Tryptic soy broth (TSB)................................................................................................................................................................................ 4
7.5.1 Composition.......................................................................................................................................................................................... 4
7.5.2 Preparation...........................................................................................................................................................................................4
7.6 Tryptone soy agar (TSA).............................................................................................................................................................................. 4
iTeh Standards
7.6.1 Composition.......................................................................................................................................................................................... 4
7.6.2 Preparation...........................................................................................................................................................................................4
7.7 (https://standards.iteh.ai)
Soybean casein digest broth with lecithin and polyoxyethylene medium (SCDLP)...............................4
7.7.1 Composition.......................................................................................................................................................................................... 4

7.8
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7.7.2 Preparation...........................................................................................................................................................................................5
Sodium chloride solution (physiological saline).................................................................................................................... 5
7.8.1 Composition.......................................................................................................................................................................................... 5
7.8.2 Preparation...........................................................................................................................................................................................
ISO 16187:2025 5
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Test microorganisms................................................................................................................................................................................................... 5
8.1 Test strains.............................................................................................................................................................................................................. 5
8.2 Storage of strains...............................................................................................................................................................................................5
9 Preparation of test inoculums............................................................................................................................................................................ 6
10 Preparation of test samples.................................................................................................................................................................................. 6
10.1 General.........................................................................................................................................................................................................................6
10.2 Test specimen........................................................................................................................................................................................................ 6
10.3 Pre-treatment of the test specimen................................................................................................................................................... 6
11 Test procedure.................................................................................................................................................................................................................... 6
12 Expression of results.................................................................................................................................................................................................... 7
12.1 Calculation of the number of viable bacteria............................................................................................................................. 7
12.2 Judgement of test effectiveness............................................................................................................................................................. 7
12.3 Calculation of antibacterial activity ratio.................................................................................................................................... 8
13 Test report............................................................................................................................................................................................................................... 8
Annex A (normative) Static challenge test............................................................................................................................................................. 10
Annex B (normative) Film contact method........................................................................................................................................................... 11
Annex C (normative) Dynamic challenge test.................................................................................................................................................... 13
Bibliography.......................................................................................................................................................................................................................................... 14


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 ISO 16187:2025(en)

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 document 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).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed patent
rights in respect thereof. As of the date of publication of this document, ISO had not received notice of (a)
patent(s) which may be required to implement this document. However, implementers are cautioned that
this may not represent the latest information, which may be obtained from the patent database available at
www.iso.org/patents. ISO shall not be held responsible for identifying any or all such patent rights.
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
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Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www.iso.org/iso/foreword.html.

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This document was prepared by Technical Committee ISO/TC 216, Footwear, in collaboration with the
European Committee for Standardization (CEN) Technical Committee CEN/TC 309, Footwear, in accordance
with the Agreement on technical cooperation between ISO and CEN (Vienna Agreement).
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This second edition cancels and replaces the first edition (ISO 16187:2013), which has been technically
revised.
ISO 16187:2025
The main changes are as follows:
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— a new term “neutralizer” and its definition have been added;
— a new Clause 4 has been added;
— AS No. has been revised to CGMCC No.;
— the light intensity of UV lamp has been added;
— the normative references and bibliography have been revised and updated;
— TSA and TSB have been added as alternative culture medium if NA and NB are not available;
— Annex D has been deleted.
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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International Standard ISO 16187:2025(en)

Footwear and footwear components — Test method to assess

antibacterial activity
CAUTION — Test methods specified in this document require the use of bacteria. These tests shall
only be carried out in facilities with containment techniques for handling microorganisms and by
persons with training and experience in the use of microbiological techniques.

1 Scope
This document specifies quantitative test methods to evaluate the antibacterial activity of footwear and
footwear components.
This document is applicable to all types of footwear and footwear components employing non-diffusing
antibacterial treatments.

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,
iTeh Standards
the latest edition of the referenced document (including any amendments) applies.
ISO 7218, Microbiology of the food chain — General requirements and guidance for microbiological examinations
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ISO 19952, Footwear — Vocabulary
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3 Terms and definitions
For the purposes of this document, the terms ISO 16187:2025given in ISO 19952 and the following apply.
and definitions
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ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https://​w ww​.iso​.org/​obp
— IEC Electropedia: available at https://​w ww​.electropedia​.org/​
3.1
antibacterial activity
efficacy of a material or finish used to prevent or mitigate the growth of bacteria, to reduce the number of
bacteria or to kill bacteria
3.2
control sample
material identical to the test material but without antibacterial treatment
3.3
neutralizer
chemical agent used to inactivate, neutralize, or quench the antibacterial properties of antibacterial agents

4 Principle
The test specimens and control specimens are inoculated with a bacterial suspension of a selected test
strain specified or claimed in independent tests with one Gram-positive and one Gram-negative bacterial
test organism.


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 ISO 16187:2025(en)

Three test methods are available to assess antibacterial activity in a challenge test procedure under static
or under dynamic conditions.
Antibacterial performance is quantitatively determined by counting the number of viable cells and
calculating the antibacterial activity ratio.

5 Safety
Handling of microorganisms which are potentially hazardous requires a high degree of technical competence
and can be subject to current national legislation and regulations. Only personnel trained in microbiological
techniques should carry out such tests.
NOTE Refer to country-specific codes of practice for personal hygiene, disinfection and sterilization.

Persons who perform the test should consult IEC 60068-2-10:2005/AMD1:2018, Annex A, and ISO 7218.

6 Apparatus
Disposable apparatus is an acceptable alternative to re-usable glassware and plastic if it has the suitable
specifications.
Apparatus includes usual microbiological laboratory equipment in accordance with ISO 7218 and, in
particular, the following.

6.1 Biological safety cabinet.

6.2
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Incubator, capable of maintaining a temperature of (37 ± 2) °C.
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6.3 Autoclave, capable of maintaining a temperature of (121 ± 2) °C and a pressure of (103 ± 5) kPa, for
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wet sterilization, used in accordance with ISO 7218.

6.4 Humidity chamber, capable of maintaining a temperature of (37 ± 2) °C and a relative humidity of
(85 ± 5) %. ISO 16187:2025
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6.5 Ultraviolet lamp, capable of emitting light intensity of 100 000 μWs/cm2.

6.6 Wide mouth jars, with cap, 100 ml, capable of being used with an autoclave (6.3).

6.7 Cover film, that does not affect bacterial growth or absorb water, which can be made of either
polyethylene, polypropylene or polyester [poly (ethylene terephthalate)]. Film that is 0,05 mm to 0,10 mm
thick should be used. For example, disposal bag suitable for use with an autoclave (6.3).

6.8 Vortex mixer.

6.9 Dimensional shaker, two dimensional or three dimensional, capable of adjusting to 50 r/min.

6.10 Shaking incubator, capable of maintaining a temperature of (37 ± 2) °C and a rotational frequency of
(120 ± 10) r/min.

7 Reagents and culture medium

7.1 General
The preparation and test shall be freshly prepared in order to ensure the culture quality.


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 ISO 16187:2025(en)

This can be done according to ISO 11133, or according to national standards, in case no national
regulations apply.
Dehydrated products available on the commercial market can be used for preparing the culture media. The
manufacture user’s instructions for the preparation of these products should be strictly followed.
Reagents used in tests shall be of analytical grade and/or suited for microbiological purposes.

7.2 Water
Water used in tests shall be free from all toxic or microorganism inhibitory substances and be analytical-
grade water for microbiological media preparation, which is freshly distilled and/or ion-exchanged and/or
ultra-filtered and/or filtered with reverse osmosis (RO).
NOTE Water grade 3 according to ISO 3696 can be used.

7.3 Nutrient broth (NB)

7.3.1 Composition

Beef extract 3,0 g

Peptone 5,0 g

Sodium chloride, NaCl 5,0 g

Water iTeh Standards 1 000 ml

7.3.2 Preparation (https://standards.iteh.ai)

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bath until the components are completely Preview
Stir and adjust pH to (7,2 ± 0,2) (at room temperature). Heat with stirring on a hotplate or in a boiling-water
dissolved. Sterilize with autoclave (6.3) at (121 ± 2) °C for 15 min.

7.4 Nutrient agar (NA) ISO 16187:2025

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7.4.1 Composition

Beef extract 5,0 g

Peptone 10,0 g

Sodium chloride, NaCl 5,0 g

Agar 15,0 g

Water 1 000 ml
NOTE If solidification is insufficient, 15 g to 18 g of agar can be used.

7.4.2 Preparation

Stir and adjust pH to (7,2 ± 0,2) (at room temperature). Heat with stirring on a hotplate or in a boiling-water
bath until the components are completely dissolved. Sterilize with autoclave (6.3) at (121 ± 2) °C for 15 min.
Cool and shake solution well, then pour into the Petri dishes.


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 ISO 16187:2025(en)

7.5 Tryptic soy broth (TSB)

7.5.1 Composition

Tryptone, pancreatic digest of casein 17,0 g

Soya peptone, papain digest of soya 3,0 g

Sodium chloride, NaCl 5,0 g

Glucose 2,5 g

Dipotassium hydrogen phosphate, K 2HPO4 2,5 g

Water 1 000 ml

7.5.2 Preparation

Stir and adjust pH to (7,2 ± 0,2) (at room temperature). Heat with stirring on a hotplate or in a boiling-water
bath until the components are completely dissolved. Sterilize with autoclave (6.3) at (121 ± 2) °C for 15 min.
NOTE TSB can be used as alternative culture medium if NB is not available.

7.6 Tryptone soy agar (TSA)

7.6.1 Composition
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Tryptone, pancreatic digest of casein

Soya peptone, papain digest of soya

15,0 g

5,0 g

Sodium chloride, NaCl

Document5,0 Preview
g

Agar 15,0 g
ISO 16187:2025
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Water 1 000 ml

7.6.2 Preparation

Stir and adjust pH to (7,2 ± 0,2) (at room temperature). Heat with stirring on a hotplate or in a boiling-water
bath until the components are completely dissolved. Sterilize with autoclave (6.3) at (121 ± 2) °C for 15 min.
Cool and shake solution well, then pour into the Petri dishes.
NOTE TSA can be used as alternative culture medium if NA is not available.

7.7 Soybean casein digest broth with lecithin and polyoxyethylene medium (SCDLP)

7.7.1 Composition

Peptone, digest of casein 17,0 g

Peptone, digest of soybean 3,0 g

Sodium chloride, NaCl 5,0 g

Potassium dihydrogen phosphate, KH2PO4 2,5 g

Glucose 2,5 g


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 ISO 16187:2025(en)

Lecithin 1,0 g

Polysorbate 80 7,0 g

Water 1 000 ml
If the neutralizing power is insufficient, the content of polysorbate 80 or lecithin may be adjusted, or another
neutralizing agent may be added. The use of any unspecified neutralizer shall be recorded along with the
name and concentration.
NOTE Information about selection and evaluation of alternative antibacterial neutralizing agents can be found in
ASTM E 1054 and EN 1040.

7.7.2 Preparation

After mixing well, adjust pH to (7,2 ± 0,2) (at room temperature) and sterilize with autoclave (6.3) at
(121 ± 2) °C for 15 min.

7.8 Sodium chloride solution (physiological saline)

7.8.1 Composition

Sodium chloride, NaCl 8,5 g

Water 1 000 ml

7.8.2 Preparation iTeh Standards

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After mixing well, adjust pH to (6,9 ± 0,2) (at room temperature) and sterilize at (121 ± 2) °C for 15 min.

8 Test microorganisms Document Preview

8.1 Test strains ISO 16187:2025

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The following strains shall be used in all antibacterial activity tests.
a) Staphylococcus aureus CGMCC1) 1.89 or ATCC® 6538TM2) or WDCM 00032.
b) Klebsiella pneumoniae CGMCC 1.1736 or ATCC® 4352TM2) or WDCM 00192.
If required, other strains or other species can be used. However, the selected organisms shall contain at least
one Gram-positive and one Gram-negative organism as the antibacterial agents can have different activities.
Test strains shall be obtained from agencies of the World Federation of Culture Collection (WFCC).
The bacteria strains and their supply sources shall be included in the test report.

8.2 Storage of strains

Inoculate the strains to the nutrient agar (NA) (7.4) or tryptone soy agar (TSA) (7.6) and incubate at
(37 ± 2) °C for 24 h. Store at (5 ± 3) °C (maximum one month) and keep it as stock culture of the strains.
Transfer and incubate one time each month.
Strains can be preserved in accordance with the supplier’s direction or EN 12353.

1) CGMCC refers to the China General Microbiological Culture Collection Centre, ATCC® is the American Type Culture
Collection, WDCM is World Data Centre for Microorganisms (refer to WDCM and website: http://​refs​.wdcm​.org).
2) ATCC® 6538TM and ATCC® 4352TM are examples of suitable products available commercially. This information is
given for the convenience of users of this document and does not constitute an endorsement by ISO of these products.


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 ISO 16187:2025(en)

9 Preparation of test inoculums

Using a sterile inoculating loop, transfer one colony (8.2) into 20 ml of nutrient broth (NB) (7.3) or tryptic
soy broth (TSB) (7.5) and incubate in the shaking incubator (6.10) at (37 ± 2) °C for approximately 16 h
(overnight culture). Estimate the number of bacteria with microscopic observation or other methods.
Prepare physiological saline (7.8) with 1 % nutrient broth (NB) (7.3) or tryptic soy broth (TSB) (7.5). Use
this media to prepare a suspension with a bacterial concentration of 2,5 × 105 CFU/ml to 10 × 105 CFU/ml as
test inoculum.
If necessary, store the test inoculum on ice and use it within 4 h.
If required, the inoculum may be > 1,0 × 106 CFU/ml.

10 Preparation of test samples

10.1 General
Test only the components or material which are claimed to be antibacterial. If the whole footwear is claimed
as antibacterial, major components, including upper, lining, insole, insock and outsole, shall be tested
separately.
In the case where only one material of a component is claimed to be antibacterial, it shall be tested separately,
if possible. Otherwise, the whole component shall be tested.
Each test sample shall be at least 80 % of the surface area of the component or material. If a single material
accounts for less than 80 %, take two main materials used in the composition of the component.
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The test samples can be obtained directly from the footwear raw materials.

10.2 Test specimen

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The area of test specimen should be Preview
approximately 500 mm 2
. For the static challenge test method described
in Annex A, the area of test specimen shall have a thickness of less than 2,0 mm. The area and the weight
shall be reported in the test report. If a larger ISO
test specimen is used, then the volume of bacterial suspension
16187:2025
should be increased proportionally.
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If it is impossible to lower the thickness of the test specimen (for example, if components are thicker and
cannot be separated or cut without changing critical properties such as surface morphology which can
affect how the bacteria interact with the surface), the thickness shall be indicated in the test report.
At least six test specimens shall be taken for each material or component and for each test strain.

10.3 Pre-treatment of the test specimen

Pre-treatment of the test specimen is optional and should only be conducted if necessary due to high
bioburden (contamination, etc.).
If sterilization methods are applied, they shall be reported in detail, and shall not affect the antibacterial
properties or the material itself.
NOTE The test and control sample can be sterilized with the autoclave (6.3) at (121 ± 2) °C and 103 kPa for 15 min,
or with ultraviolet rays [ultraviolet lamp (6.5), 100 000 μWs/cm2, 300 mm away from the sample, each side for one
hour respectively] or by immersion in 70 % ethanol (analytical grade) for 10 minutes and drying overnight in the
workbench, or other suitable sterilizing methods.

11 Test procedure
Table 1 lists the circumstances under which each test method should be applied.


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