ISO 20681:2026

Footwear and footwear components — Test method to assess antimicrobial activity — Agar diffusion test

This document specifies a test method for evaluating the antimicrobial (either antibacterial or antifungal, or both) activity of footwear and footwear components. This document is applicable to all types of footwear and footwear components employing diffusing antimicrobial treatments.

Chaussures et composants de chaussures — Méthode d'essai pour évaluer l'activité antimicrobienne — Essai de diffusion sur gélose

General Information

• Status: Published
• Publication Date: 04-May-2026
• ICS: 61.060 - Footwear
• Technical Committee: ISO/TC 216 - Footwear
• Drafting Committee: ISO/TC 216 - Footwear
• Current Stage: 6060 - International Standard published
• Start Date: 05-May-2026
• Due Date: 26-Apr-2026
• Completion Date: 05-May-2026

Overview

ISO 20681:2026 specifies a standardized agar diffusion test method for assessing the antimicrobial activity (antibacterial and/or antifungal) of footwear and footwear components. This international standard is applicable to all types of footwear products and components that utilize diffusing antimicrobial treatments. By providing a harmonized approach, ISO 20681:2026 supports the reliable evaluation of materials claiming antimicrobial efficacy, helping manufacturers, laboratories, and regulatory bodies verify performance claims and maintain consumer safety.

Key Topics

• Agar Diffusion Test Method: Describes a laboratory procedure for measuring antimicrobial activity by observing the inhibition of microbial growth around a tested footwear sample.
• Antibacterial and Antifungal Assessment: Covers methods for testing both bacteria (e.g., Staphylococcus aureus, Klebsiella pneumoniae) and yeast (Candida albicans), supporting comprehensive antimicrobial evaluation.
• Sample Preparation: Details requirements for obtaining, preparing, and, if needed, sterilizing test and control specimens to ensure consistency and validity.
• Selection of Microorganisms: Specifies standard strains and guidance on using appropriate microbial cultures for robust, repeatable test results.
• Result Interpretation: Instructs how to calculate and report the zone of inhibition, which quantifies the antimicrobial performance of tested products.
• Reporting Requirements: Defines essential elements for inclusion in test reports, ensuring transparency and traceability.

Applications

ISO 20681:2026 is relevant for a range of stakeholders in the footwear industry, including:

• Footwear Manufacturers: Allows manufacturers to scientifically substantiate claims about the antimicrobial properties of shoes, insoles, linings, and other components, supporting product differentiation and compliance with international markets.
• Testing Laboratories: Provides standardized procedures for contract labs and in-house testing facilities, ensuring consistency in testing footwear for antimicrobial efficacy.
• Quality Control and Compliance: Assists quality assurance teams in verifying that supplied materials or finished products meet required antimicrobial specifications.
• Regulatory and Certification Bodies: Offers a recognized benchmark for assessing compliance with health, safety, and consumer protection regulations concerning antimicrobial footwear.

By adopting ISO 20681:2026, organizations can enhance consumer confidence, reduce the risk of product recalls, and support claims for improved hygiene and comfort in footwear products.

Related Standards

For comprehensive antimicrobial testing and terminology in the footwear sector, the following standards are closely associated with ISO 20681:2026:

• ISO 7218: Microbiology of the food chain - General requirements and guidance for microbiological examinations
• ISO 19952: Footwear - Vocabulary
• ISO 16187: Footwear and footwear components - Test method to assess antibacterial activity
• ISO 19574: Footwear and footwear components - Qualitative test method to assess antifungal activity (growth test)
• ISO 20150: Footwear and footwear components - Quantitative challenge test method to assess antifungal activity
• ISO 20645: Textile fabrics - Determination of antibacterial activity - Agar diffusion plate test
• ISO/IEC 17025: General requirements for the competence of testing and calibration laboratories
• EN 12353: Chemical disinfectants and antiseptics - Preservation of test organisms for antimicrobial activity testing

Implementing ISO 20681:2026 alongside these related standards ensures a robust, harmonized system for the evaluation and verification of antimicrobial treatments in footwear manufacturing and supply chains.

Keywords: ISO 20681:2026, footwear antimicrobial test, agar diffusion test, antibacterial activity footwear, antifungal activity footwear, footwear testing standards, antimicrobial footwear assessment, ISO footwear standards, zone of inhibition, test method for footwear components.

Standards Content (Sample)

International
Standard
ISO 20681
First edition
Footwear and footwear
2026-05
components — Test method to
assess antimicrobial activity — Agar
diffusion test
Chaussures et composants de chaussures — Méthode d'essai pour
évaluer l'activité antimicrobienne — Essai de diffusion sur gélose
Reference number
© ISO 2026
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: [phone]
Email: [e-mail]
Website: www.iso.org
Published in Switzerland
ii
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 media . 3
7.1 General .3
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
7.6.1 Composition .4
7.6.2 Preparation .4
7.7 Malt medium .4
7.7.1 Composition .4
7.7.2 Preparation .4
7.8 Malt extract agar (MEA).5
7.8.1 Composition .5
7.8.2 Preparation .5
7.9 Physiological saline (sodium chloride solution) .5
7.9.1 Composition .5
7.9.2 Preparation .5
7.10 Buffer solution .5
7.10.1 Buffer stock .5
7.10.2 Preparation of buffer stock .5
7.10.3 Preparation of buffer solution .5
8 Test microorganisms . 5
9 Preparation of specimens . 6
9.1 General .6
9.2 Test and control specimens .6
9.3 Pretreatment of specimens .6
10 Test procedure . 7
11 Expression of results . 7
11.1 Judgement of test effectiveness .7
11.2 Calculation of zone of inhibition .7
12 Test report . 8
Annex A (normative) Testing of antibacterial activity . 9
Annex B (normative) Testing of antifungal activity .11
Bibliography .13

iii
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).
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
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 216, Footwear.
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
International Standard ISO 20681:2026(en)
Footwear and footwear components — Test method to assess
antimicrobial activity — Agar diffusion test
CAUTION — Test methods specified in this document require the use of microbes. 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 a test method for evaluating the antimicrobial (either antibacterial or antifungal, or
both) activity of footwear and footwear components.
This document is applicable to all types of footwear and footwear components employing diffusing
antimicrobial 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,
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
ISO 19952, Footwear — Vocabulary
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 19952 and the following apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
3.1
antimicrobial activity
efficacy of a material or finish used to prevent or mitigate the growth of microbes, to reduce the number of
microbes or to kill microbes
3.2
control specimen
specimen with material identical to the test specimen but without antimicrobial treatment
3.3
zone of inhibition
region of no microbial growth near the boundary between the specimen and the agar medium surface
4 Principle
The test specimens and control specimens are placed on agar plates which have been inoculated with a
microbial suspension of a selected test strain specified or claimed in independent tests with one Gram-
positive and one Gram-negative bacterial test organism, and one microfungus (yeast) test organism.

Antimicrobial performance is determined by calculating the zone of inhibition at the contact between the
agar medium and the specimen.
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. 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 CSV, Annex A, and ISO 7218.
6 Apparatus
Usual microbiological laboratory equipment in accordance with ISO 7218 and in particular the following.
Disposable apparatus is an acceptable alternative to reusable glassware and plastic if it has suitable
specifications.
6.1 Biological safety cabinet.
6.2 Microbiological incubator, capable of maintaining temperature at (30 ± 2) °C and (37 ± 2) °C.
6.3 Autoclave, capable of maintaining a temperature of (121 ± 2) °C and a pressure of (103 ± 5) kPa, for
wet sterilization, used in accordance with ISO 7218.
6.4 Ultraviolet lamp, capable of emitting light intensity of 100 000 μWs/cm .
6.5 Vortex mixer.
6.6 Shaking incubator, capable of maintaining temperature at (30 ± 2) °C and (37 ± 2) °C and a rotational
frequency of (120 ± 10) r/min.
6.7 Oven, for dry sterilization.
6.8 pH-meter, having an accuracy of ±0,1 pH units.
6.9 Balance, capable of weighing to ±0,01 g.
6.10 Vernier calliper, having an accuracy of ±0,02 mm.
6.11 Petri dishes, that have been sterilized, made of glass or plastic, in diameter sizes of 90 mm to 100 mm
or 55 mm to 60 mm.
6.12 Pipette, having the most suitable volume for each use.
6.13 Spectrophotometer, capable of measuring at a 500 nm to 700 nm wavelength, or McFarland’s
nephelometer.
6.14 Microscope, at least magnification ×20 (better magnification ×50).
6.15 Water bath, capable of maintaining a temperature of (45 ± 2) °C.

7 Reagents and culture media
7.1 General
Reagents used in tests shall be either of analytical grade or suited for microbiological purposes, or both.
Dehydrated products available commercially can be used for preparing the culture media. The manufacture
user’s instructions for the preparation of these products should be strictly followed.
ISO 11133 or similarly applicable standards should be followed for the preparation, production and
...