ISO/FDIS 7581

Evaluation of bactericidal activity of a non-porous antimicrobial surface used in a dry environment

This document specifies the test conditions and the levels of activity to determine the bactericidal activity of non-porous surfaces used in a dry environment . It defines a protocol to validate the bactericidal character of a surface and to measure its performance. It is not intended to be used to substantiate cleaning or disinfecting properties. This document is applicable to surfaces claiming to have an activity against vegetative bacteria. The olbigatory test conditions are defined in this document. It does not apply to porous surfaces. It does not refer to methods for testing the toxicological and ecotoxicological properties of the surfaces. This document is used to measure bactericidal action, not bacteriostatic activity of a surface.

Évaluation de l’activité bactéricide d’une surface antimicrobienne non poreuse utilisée dans un environnement sec

General Information

• Status: Not Published
• ICS: 07.100.99 - Other standards related to microbiology
• Technical Committee: ISO/TC 330 - Surfaces with biocidal and antimicrobial properties
• Drafting Committee: ISO/TC 330 - Surfaces with biocidal and antimicrobial properties
• Current Stage: 5020 - FDIS ballot initiated: 2 months. Proof sent to secretariat
• Start Date: 04-Sep-2023
• Completion Date: 04-Sep-2023

Standards Content (Sample)

Pr ISO /FDIS 7581:2022(E)
ISO /TC 330
Secretariat: AFNOR
Date: 2022-06-232023-08-18
Evaluation of bactericidal activity of a non-porous antimicrobial
surface used in a dry environment
FDIS stage

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ISO/DIS 7581:2022(E)
© ISO 20222023
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
EmailE-mail: [e-mail]
Website: www.iso.orgwww.iso.org
Published in Switzerland
ii © ISO 2022 – All rights reserved

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ISO/DIS 7581:2022(E)
Contents
Foreword . 5
Introduction . 6
1 Scope . 1
2 Normative reference . 1
3 Terms and definitions . 1
4 Apparatus, reagents and materials . 3
4.1 Test organisms . 3
4.2 Culture media and reagents . 3
4.3 Reference and test surfaces . 5
4.3.1 General . 5
4.3.2 Reference surfaces . 5
4.3.3 Test surfaces . 5
4.4 Apparatus and materials . 6
5 Preparation of the test organism suspensions . 8
5.1 Stock cultures of the test bacteria . 8
5.2 Working culture of the test bacteria . 8
5.3 Test suspensions . 8
5.4 Quantifying of bacterial test suspensions . 8
5.5 Calculation of the weighted mean bacteria concentration in the test suspensions . 9
6 Evaluation method of the bactericidal activity of a non-porous surface and its efficacy . 10
6.1 Obligatory experimental conditions . 10
6.2 Test procedure . 10
6.2.1 Sampling . 10
6.2.2 Test (T0 and Txh) . 11
6.2.3 Reference surfaces (C and C ) . 12
0 xh
6.2.4 Validation (V ) . 12
n
7 Results. 13
7.1 Determination of the number of viable bacteria . 13
7.2 Verification of the methodology . 14
7.3 Expression of results . 14
7.3.1 Reduction . 14
7.3.2 Conclusion: Bactericidal activity of non-porous surfaces . 15
8 Test report . 15
Annex A (normative) Corresponding obligatory reference strains . 17
Annex B (informative) Standard test report of the measurement of the bactericidal activity of non-
porous surfaces . 18
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ISO/DIS 7581:2022(E)
Annex C (informative) Examples of surface preparation protocols . 21
Annex D (informative) Neutralizers . 22
Annex E (informative) Technical guide to depositing the inoculum . 24
Bibliography . 27

Foreword 4
Introduction 5
1 Scope 1
2 Normative reference 1
3 Terms and definitions 1
4 Apparatus, reagents and materials 3
4.1 Test organisms 3
4.2 Culture media and reagents 3
4.3 Reference and test surfaces 4
4.4 Apparatus and materials 6
5 Preparation of the test organism suspensions 7
5.1 Stock cultures of the test bacteria 7
5.2 Working culture of the test bacteria 7
5.3 Test suspensions 7
5.4 CFU Counting from bacterial test suspensions 8
5.5 Calculation of the weighted mean bacteria concentration in the test suspensions 8
6 Evaluation method of the bactericidal activity of a non-porous surface and its efficacy 9
6.1 Obligatory experimental conditions 9
6.2 Test procedure 10
7 Results 12
7.1 Determination of the number of viable bacteria 12
7.2 Verification of the methodology 13
7.3 Expression of results 13
8 Test report 14
Annex A (normative) Corresponding obligatory reference strains 16
Annex B (informative) Standard test report of the measurement of the bactericidal activity of non-porous
surfaces 17
Annex C (informative) Examples of surface preparation protocols 20
Annex D (informative) Neutralizers 21
Annex E (informative) Technical guide to depositing the inoculum 23
Bibliography 25
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ISO/DIS 7581:2022(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 documentsdocument 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 drawnISO draws attention to the possibility that some of the elementsimplementation of this
document may beinvolve the subjectuse 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. 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.htmlwww.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 330, Surfaces with biocidal and antimicrobial
properties.
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.htmlwww.iso.org/members.html.
© ISO 2022 – All rights reserved v

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ISO/DIS 7581:2022(E)
Introduction
The surfaces in our environment can constitute significant reservoirs of bacteria and a risk to health that is
essential to control.
In healthcare establishments, healthcare-associated infections (HAI) are a major cause of mortality and
disability the world over. HAI affects 5 % to 15 % of hospital patients in developed countries and can affect
9 % to 37 % of patients admitted to intensive care units. Each year, about 1 in 25 U.S. hospital patients is
diagnosed with at least one infection related to hospital care alone. At least five million HAI are estimated to
occur in acute care hospitals in Europe, causing 135 000 deaths per year and approximately 25 million
additional days spent in hospital, representing a financial cost of €13 billion to €24 billion.
In public transport, the risks of contamination between travellers are extremely high due to the frequency and
number of contacts points. In establishments receiving the public, all common areas and collective spaces
constitute a risk of the spread of pathogens microorganisms.
In the agri-food sector, microbial hazards are the highest risk amongst those that can impact consumer health,
above malnutrition and chemical contaminants. In addition to the impact on health, the consequences of a case
of contamination can be disastrous for the image and sustainability of a company. For instance,:
— — Anan estimated 600 million – almost 1 in 10 people in the world – fall ill after eating contaminated
food and 420 000 die every year, resulting in the loss of 33 million healthy life years [disability-adjusted
life years ].];
— — US$110 billion is lost each year in productivity and medical expenses resulting from unsafe food in
low- and middle-income countries.;
— — Childrenchildren under 5 years of age carry 40 % of the foodborne disease burden, with 125 000
deaths every year.;
— — Diarrhoealdiarrhoeal diseases are the most common illnesses resulting from the consumption of
contaminated food, causing 550 million people to fall ill and 230 000 deaths every year.
The drive to control microbial risks extends to many other sectors (transport, pharmaceuticals, aeronautics,
cosmetics, the phytosanitary sector, services, etc.), and even to the entire industrial manufacturing sector.
Pathogenic agents can be transmitted in a variety of ways, such as via food and water, via air (aerosols), via
body contact with infected person and via surfaces contaminated by body secretions and fomites. Healthcare-
associated infections, or infections acquired in healthcare settings are the most frequent adverse event in
healthcare delivery worldwide. Hundreds of millions of patients are affected by health carehealthcare-
associated infections worldwide each year, leading to significant mortality and financial losses for health
systems. Of every 100 hospitalized patients at any given time, 7 in developed and 10 in developing countries
will acquire at least one healthcare-associated infection. Such infections annually account for 37 000
attributable deaths in Europe and potentially many more that can be related, and they account for 99 000
deaths in the USA. Amongst the available tools for reducing microbial risks, surfaces with biocidal properties
can help to control cross-contaminations when used in combination with standard cleaning and disinfecting
practice.
This document was written to address the need to demonstrate the biocidal efficacy of a surface under
ambient conditions close to the conditions found in the field. The method described in this document simulates
the contamination of a surface by a microdroplet. For example, this pathway of contamination is
representative of a surface contamination following a sneeze (microdroplet post sneeze might have a mobility
of several meters). The temperatures and humidity required for the test were defined in relation to conditions
that are representative of the atmosphere in the healthcare facilities.
Numerous non-porous surfaces claim to perform a bactericidal function:
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ISO/DIS 7581:2022(E)
— — surfaces of materials that have been treated with or include a biocidal product in order to give the
material bactericidal properties, either temporarily or permanently.
— — surfaces of materials, such as certain metals, that claim to have intrinsic bactericidal properties.
The method prescribes the representative basic strains to be tested. Additional strains may be tested,
depending on the intended use. For a given use, further experiments including in use condition (soiling
substances, ageing, adaption of environmental condition) are needed for demonstrating bactericidal activity
of the tested surface as claimed.
This method will be revised in the near future to include interfering substances and to adapt
strains/efficacy/contact time specifications to the needs of different sectors, in addition to the medical area.
Ageing simulation testing will be produced soon by ISO/TC 330.
Surfaces with bactericidal properties supplement, but do not replace, the regular use of surface-treatment
products, such as detergents, detergent-disinfectants and surface disinfectants which must be demonstrated
to be compatible with maintaining the surface bactericidal activity.

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ISO/DISFDIS 7581:20222023(E)
WARNING — Persons using this document shall have technical microbiological knowledge.
viii © ISO 2022 – All rights reserved
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DRAFT INTERNATIONAL STANDARD ISO/DIS 7581:2022(E)

Evaluation of bactericidal activity of a non-porous antimicrobial
surface used in a dry environment
WARNING — Persons using this document shall have technical microbiological knowledge.
1 Scope
This document specifies the test conditions and the levels of activity to determine the bactericidal activity
of non-porous surfaces used in a dry environment . It defines a protocol to validate the bactericidal
character of a surface and to measure its performance. It is not intended to be used to substantiate
cleaning or disinfecting properties.
This document is applicable to surfaces claiming to have an activity against vegetative bacteria. The
olbigatoryobligatory test conditions are defined in this document. It does not apply to porous surfaces.
It does not refer to methods for testing the toxicological and ecotoxicological properties of the surfaces.
This document is used to measure bactericidal action, not bacteriostatic activity of a surface.
2 Normative reference
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.

EN 12353, Chemical disinfectants and antiseptics — Preservation of test organisms used for the
determination of bactericidal (including Legionella), mycobactericidal, sporicidal, fungicidal and virucidal
(including bacteriophages) activity
3 Terms and definitions
For the purposes of this document, the following terms and definitions 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/obphttps://www.iso.org/obp
— — IEC Electropedia: available at https://www.electropedia.org/https://www.electropedia.org/
3.1
bactericidal activity
capability of a surface to produce a reduction in the number of viable bacterial cells of representative test
organisms (3.6)(3.6) under defined conditions without soil load, nor ageing test nor test conditions
adaptation to a specific sector.
Note 1 to entry: Note to entry: At the moment this document does not include soiling or ageing condition.
3.2
bacteriostatic activity
capability of a surface to inhibit the growth of viable bacterial cells of representative test organisms
(3.6)(3.6) under defined conditions
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ISO/DISFDIS 7581:20222023(E)
Note 1 to entry: The term "bacteriostatic activity" cannot be used for claims according to this document.
3.3
additional condition
test conditions that are optional and not obligatory, that can be used for additional claims of bactericidal
activity (3.1) regarding a surface
3.4
cleaning
all operations that achieve a level of cleanliness, appearance, comfort and hygiene
Note 1 to entry:, : Such operations use, to varying degrees, the following combined factors: chemical action,
mechanical action, temperature, duration of action
3.5
neutralizer
chemical agent or formulation that suppresses the residual microbicidal activity of a product or active
substance from the surface to be tested for a specific test but does not inactivate or inhibit the test
organism (3.6)(3.6)
3.6
test organism
strain of a microorganism selected to evaluate the antimicrobial activity of a surface for a standardized
test
3.7
bactericidal surface
surface that irreversibly kill vegetative bacteria under defined conditions
Note 1 to entry: The adjective “bactericidal” corresponds to the noun “bactericide”.
3.8
reference surface
surface without any bactericidal activity (3.1) or properties, that is used to evaluate the quantity of
culturable bacteria present at the moment when the bactericidal activity of the test surface (3.9)(3.9) is
evaluated
3.9
test surface
surface claiming bactericidal activity (3.1)
Note 1 to entry: This method is suitable for testing any type of non-porous surfaces (Metalssuch as metals, plastic,
glass, coated surfaces…), etc.) as long as recovery of bacteria from the test surface shall not lose more than 1 log at
T=0.
Note 2 to entry: Very hydrophobic surfaces, for which a drying time greater than 10 min (see 6.2.2)6.2.2) is
necessary, cannot be tested according this method.
3.10
ambient light
light corresponding to a maximum value of 2000 2 000 lux
Note 1 to entry: Any specific light or light spectrum (e.g. strong light, UV, etc.) is not considered as
ambient.
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ISO/DISFDIS 7581:20222023(E)
3.11
porous surface
surface permeable to water, air, or other fluids
4 Apparatus, reagents and materials
4.1 Test organisms
Bactericidal activity shall be evaluated using the following four strains:
— — Pseudomonas aeruginosa ATCC 15442 = CIP 103-467;
— — Staphylococcus aureus ATCC 6538 = CIP 4.83;
— — Enterococcus hirae ATCC 10541 = CIP 5855;
— — Escherichia coli ATCC 10536 = CIP 54127.
The reference strain numbers in other culture collections shall be in accordance with the strains specified
in Annex A.".Annex A.
The activity data can be completed with other strains using the experimental design described in this
document, varying the conditions to meet the needs of the intended -practice application(s). If additional
strains are used, they shall be incubated under optimum growth conditions (temperature, time,
atmosphere) to be recorded in the test report.
Their suitability for supplying the inoculum and controls with a sufficient concentration shall be verified.
If these additional test strains are not classified at a culture collection centre, their identification
characteristics shall be stated. In addition, they shall be held by the testing laboratory or national culture
collection under a reference for a five-year period.
NOTE ISO/TC 330 is currently adapting the method in terms of strains/efficacy levels/contact
times/interfering substances according to different sectors’ needs and to include not only the medical area.

4.2 Culture media and reagents

The reagents shall be of analytical grade and/or appropriate for microbiological purposes. They shall be
free from substances that are toxic or inhibitory to the test organisms.
4.2.1 Water, which shall be free from any substances that are toxic or that inhibit the bacteria. It shall
be freshly glass-distilled water or water for injection or possibly deionized or demineralized water.
Sterilize in the autoclave (4.4.1).(4.4.1).
NOTE If the water is sterilized during the sterilization of the reagents, this is not necessary.
4.2.2 Microbial suspension diluents
Tryptone-salt solution:
Tryptone, pancreatic digest of casein  .  1,0 g
Sodium chloride    .                        8,5 g
Water (see 4.2.1)   4.2.1)                    1,000 ml
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ISO/DISFDIS 7581:20222023(E)
Sterilize in the autoclave (see 4.4.1).4.4.1). After sterilization, the pH of the medium shall be equivalent
to 7,0 ± 0,2 when measured at 20 °C.
4.2.3 Liquid for bacteria recovery and for membrane rinsing
4.2.3.1 Composition:
Tryptone, pancreatic digest of casein  1.                   1,0 g
Sodium chloride
8.,5 g
Polysorbate 80
5.,0 g
Water (see 4.2.1)     1 000 ml

Water (see 4.2.1)                                          1 000 ml
4.2.3.2 Preparation:
a) a) Dissolve the sodium chloride and tryptone in the water (see 4.2.2).4.2.2). Add the Polysorbate 80,
mix and complete up to 1 000 ml with water
b) b) distribute into smaller adapted flasks. Sterilize in the autoclave.
c) c) If necessary, add a neutralizer to the recovery liquid
The use of ana validated neutralizer of the respective antimicrobial is compulsory to warrant a correct
test time for bacteria recovery and for membrane rinsing (see 4.2.3):4.2.3): The functionality of the
respective neutralizer shall be validated beforehand. The neutralizer:
— — shall be validated for the tested surface, as per 6.2.46.2.4 and it shall be sterile:;
— — shall be mentioned in the test report.
Examples of neutralizers are given in Annex D.Annex D.
4.2.4 Agar for bacteria maintenance and counting ([tryptone soya agar: (TSA))]
Use agar to preserve the bacterial strains and count the viable bacteria.
Tryptone, pancreatic digest of casein           .  15,0 g
Soya peptone, papaic digest of soybean meal   .
5,0 g
Sodium chloride                  5,0 g
Agar                  15,0 g
Water (see 4.2.1)    1 4.2.1)              1 000 ml
Sterilize in the autoclave. After sterilization, the pH of the medium shall be equivalent to 7,2 ± 0,2 when
measured at 20 °C. Pre pored plate TSA are acceptable if they meet the composition and pH value
describesdescribed in 4.2.4.4.2.4.

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ISO/DISFDIS 7581:20222023(E)
4.3 Reference and test surfaces
4.3.1 General
The surfaces shall be used only once.
4.3.2 Reference surfaces
4.3.2.1 Description
Inox 304 stainless steel, disk 2 cm in diameter, of which both sides have a grade 2B finish. The surfaces
shall be flat.
ShouldIf the coated surface without antimicrobial agent (the test surface) exist, it can be used as a
reference surface similar to inox and therefore can be used for log reduction calculation.
The reference surface used during the test mustshall be specified in the report.
4.3.2.2 Cleaning – disinfection— Disinfection/sterilization
The reference surfaces shall be cleaned and disinfected before use.
If a specific preparation protocol is applied to the test surface (see examples in Annex C),Annex C), the
same protocol should be applied to the reference surface (see 4.3.3.2).4.3.3.2). Otherwise, the protocol in
example 1 of Annex CAnnex C should be used.
No residual action or changes of the antimicrobial properties shall result from disinfection/cleaning
procedure on the reference surface.
The reference surface treatment used during the test mustshall be specified in the report.

4.3.3 Test surfaces
4.3.3.1 Identification and production
The characteristics (e.g. dimensions, thickness, etc.) and references of the finished product shall be
defined and specified in the final test report.
If the test surface is treated with a bactericide, the nature of the active substance(s) shall be specified to
the lab for safety and security reasons, and indicated in the final test report. For coated surfaces with
unknown porosity (e.g. paint), recovery of the microorganisms shall be checked by the laboratory,
comparing the coated surface with the antimicrobial agent and the reference surface (C0 vs T0) or the
coated surface with and without the antimicrobial agent, to ensure the accuracy of bacteria reduction
evaluation. Recovery of bacteria from the test surface shall not lose more than 1 log at T=0 compared to
the reference one.
The test surface shall be produced from the materials intended for the finished product claiming
bactericidal activity, and according to the same steps of the final production process. If it is impossible to
perform the steps on the same equipment, it is permitted to use a representative simulation of these
steps.
The test surface shall have flat surfaces and measure between 12 mm and 25 ± 2 mm on the sides or in
diameter. If it is impossible to cut the test surface into squares or disks of this size, other sizes and shapes
can be used. In this case, the actual dimensions used will be stated in the test report. Any changes made
to the protocol (recovery volume, etc.) due to the dimensions of the tested test surface shall be specified
in the test report.
© ISO 2022 – All rights reserved 5
...

FINAL
INTERNATIONAL ISO/FDIS
DRAFT
STANDARD 7581
ISO/TC 330
Evaluation of bactericidal activity of
Secretariat: AFNOR
a non-porous antimicrobial surface
Voting begins on:
2023-09-04 used in a dry environment
Voting terminates on:
2023-10-30
RECIPIENTS OF THIS DRAFT ARE INVITED TO
SUBMIT, WITH THEIR COMMENTS, NOTIFICATION
OF ANY RELEVANT PATENT RIGHTS OF WHICH
THEY ARE AWARE AND TO PROVIDE SUPPOR TING
DOCUMENTATION.
IN ADDITION TO THEIR EVALUATION AS
Reference number
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO-
ISO/FDIS 7581:2023(E)
LOGICAL, COMMERCIAL AND USER PURPOSES,
DRAFT INTERNATIONAL STANDARDS MAY ON
OCCASION HAVE TO BE CONSIDERED IN THE
LIGHT OF THEIR POTENTIAL TO BECOME STAN-
DARDS TO WHICH REFERENCE MAY BE MADE IN
NATIONAL REGULATIONS. © ISO 2023

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ISO/FDIS 7581:2023(E)
FINAL
INTERNATIONAL ISO/FDIS
DRAFT
STANDARD 7581
ISO/TC 330
Evaluation of bactericidal activity of
Secretariat: AFNOR
a non-porous antimicrobial surface
Voting begins on:
used in a dry environment
Voting terminates on:
COPYRIGHT PROTECTED DOCUMENT
© ISO 2023
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.
RECIPIENTS OF THIS DRAFT ARE INVITED TO
ISO copyright office
SUBMIT, WITH THEIR COMMENTS, NOTIFICATION
OF ANY RELEVANT PATENT RIGHTS OF WHICH
CP 401 • Ch. de Blandonnet 8
THEY ARE AWARE AND TO PROVIDE SUPPOR TING
CH-1214 Vernier, Geneva
DOCUMENTATION.
Phone: [phone]
IN ADDITION TO THEIR EVALUATION AS
Reference number
Email: [e-mail]
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO­
ISO/FDIS 7581:2023(E)
Website: www.iso.org
LOGICAL, COMMERCIAL AND USER PURPOSES,
DRAFT INTERNATIONAL STANDARDS MAY ON
Published in Switzerland
OCCASION HAVE TO BE CONSIDERED IN THE
LIGHT OF THEIR POTENTIAL TO BECOME STAN­
DARDS TO WHICH REFERENCE MAY BE MADE IN
ii
  © ISO 2023 – All rights reserved
NATIONAL REGULATIONS. © ISO 2023

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ISO/FDIS 7581:2023(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative reference .1
3 Terms and definitions . 1
4 Apparatus, reagents and materials . 3
4.1 Test organisms . 3
4.2 Culture media and reagents. 3
4.3 Reference and test surfaces . 4
4.3.1 General . 4
4.3.2 Reference surfaces . 4
4.3.3 Test surfaces . . 5
4.4 Apparatus and materials . 6
5 Preparation of the test organism suspensions . 7
5.1 Stock cultures of the test bacteria . 7
5.2 Working culture of the test bacteria . 7
5.3 Test suspensions . 8
5.4 Quantifying of bacterial test suspensions . 8
5.5 Calculation of the weighted mean bacteria concentration in the test suspensions. 8
6 Evaluation method of the bactericidal activity of a non-porous surface and its
efficacy .10
6.1 Obligatory experimental conditions . 10
6.2 Test procedure . 10
6.2.1 Sampling . 10
6.2.2 Test (T and T ) . 10
0 xh
6.2.3 Reference surfaces (C and C ) . 11
0 xh
6.2.4 Validation (V ). 11
n
7 Results . .12
7.1 Determination of the number of viable bacteria .12
7.2 Verification of the methodology. 13
7.3 Expression of results . .13
7.3.1 Reduction . 13
7.3.2 Conclusion: Bactericidal activity of non-porous surfaces . 14
8 Test report .14
Annex A (normative) Corresponding obligatory reference strains .16
Annex B (informative) Standard test report of the measurement of the bactericidal activity
of non-porous surfaces .17
Annex C (informative) Examples of surface preparation protocols .20
Annex D (informative) Neutralizers .21
Annex E (informative) Technical guide to depositing the inoculum .23
Bibliography .25
iii
© ISO 2023 – All rights reserved

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ISO/FDIS 7581:2023(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 non­governmental, 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 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 330, Surfaces with biocidal and
antimicrobial properties.
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/FDIS 7581:2023(E)
Introduction
The surfaces in our environment can constitute significant reservoirs of bacteria and a risk to health
that is essential to control.
In healthcare establishments, healthcare-associated infections (HAI) are a major cause of mortality
and disability the world over. HAI affects 5 % to 15 % of hospital patients in developed countries and
can affect 9 % to 37 % of patients admitted to intensive care units. Each year, about 1 in 25 U.S. hospital
patients is diagnosed with at least one infection related to hospital care alone. At least five million
HAI are estimated to occur in acute care hospitals in Europe, causing 135 000 deaths per year and
approximately 25 million additional days spent in hospital, representing a financial cost of €13 billion
to €24 billion.
In public transport, the risks of contamination between travellers are extremely high due to the
frequency and number of contacts points. In establishments receiving the public, all common areas and
collective spaces constitute a risk of the spread of pathogens microorganisms.
In the agri­food sector, microbial hazards are the highest risk amongst those that can impact
consumer health, above malnutrition and chemical contaminants. In addition to the impact on health,
the consequences of a case of contamination can be disastrous for the image and sustainability of a
company. For instance:
— an estimated 600 million – almost 1 in 10 people in the world – fall ill after eating contaminated
food and 420 000 die every year, resulting in the loss of 33 million healthy life years [disability-
adjusted life years];
— US$110 billion is lost each year in productivity and medical expenses resulting from unsafe food in
low- and middle-income countries;
— children under 5 years of age carry 40 % of the foodborne disease burden, with 125 000 deaths
every year;
— diarrhoeal diseases are the most common illnesses resulting from the consumption of contaminated
food, causing 550 million people to fall ill and 230 000 deaths every year.
The drive to control microbial risks extends to many other sectors (transport, pharmaceuticals,
aeronautics, cosmetics, the phytosanitary sector, services, etc.), and even to the entire industrial
manufacturing sector.
Pathogenic agents can be transmitted in a variety of ways, such as via food and water, via air (aerosols),
via body contact with infected person and via surfaces contaminated by body secretions and fomites.
Healthcare-associated infections, or infections acquired in healthcare settings are the most frequent
adverse event in healthcare delivery worldwide. Hundreds of millions of patients are affected by
healthcare-associated infections worldwide each year, leading to significant mortality and financial
losses for health systems. Of every 100 hospitalized patients at any given time, 7 in developed and 10 in
developing countries will acquire at least one healthcare-associated infection. Such infections annually
account for 37 000 attributable deaths in Europe and potentially many more that can be related, and
they account for 99 000 deaths in the USA. Amongst the available tools for reducing microbial risks,
surfaces with biocidal properties can help to control cross­contaminations when used in combination
with standard cleaning and disinfecting practice.
This document was written to address the need to demonstrate the biocidal efficacy of a surface under
ambient conditions close to the conditions found in the field. The method described in this document
simulates the contamination of a surface by a microdroplet. For example, this pathway of contamination
is representative of a surface contamination following a sneeze (microdroplet post sneeze might have
a mobility of several meters). The temperatures and humidity required for the test were defined in
relation to conditions that are representative of the atmosphere in the healthcare facilities.
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ISO/FDIS 7581:2023(E)
Numerous non­porous surfaces claim to perform a bactericidal function:
— surfaces of materials that have been treated with or include a biocidal product in order to give the
material bactericidal properties, either temporarily or permanently.
— surfaces of materials, such as certain metals, that claim to have intrinsic bactericidal properties.
The method prescribes the representative basic strains to be tested. Additional strains may be tested,
depending on the intended use. For a given use, further experiments including in use condition (soiling
substances, ageing, adaption of environmental condition) are needed for demonstrating bactericidal
activity of the tested surface as claimed.
This method will be revised in the near future to include interfering substances and to adapt strains/
efficacy/contact time specifications to the needs of different sectors, in addition to the medical area.
Ageing simulation testing will be produced soon by ISO/TC 330.
Surfaces with bactericidal properties supplement, but do not replace, the regular use of surface­
treatment products, such as detergents, detergent­disinfectants and surface disinfectants which must
be demonstrated to be compatible with maintaining the surface bactericidal activity.
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FINAL DRAFT INTERNATIONAL STANDARD ISO/FDIS 7581:2023(E)
Evaluation of bactericidal activity of a non-porous
antimicrobial surface used in a dry environment
WARNING — Persons using this document shall have technical microbiological knowledge.
1 Scope
This document specifies the test conditions and the levels of activity to determine the bactericidal
activity of non-porous surfaces used in a dry environment. It defines a protocol to validate the
bactericidal character of a surface and to measure its performance. It is not intended to be used to
substantiate cleaning or disinfecting properties.
This document is applicable to surfaces claiming to have an activity against vegetative bacteria. The
obligatory test conditions are defined in this document. It does not apply to porous surfaces.
It does not refer to methods for testing the toxicological and ecotoxicological properties of the surfaces.
This document is used to measure bactericidal action, not bacteriostatic activity of a surface.
2 Normative reference
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.
EN 12353, Chemical disinfectants and antiseptics — Preservation of test organisms used for the
determination of bactericidal (including Legionella), mycobactericidal, sporicidal, fungicidal and virucidal
(including bacteriophages) activity
3 Terms and definitions
For the purposes of this document, the following terms and definitions 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
bactericidal activity
capability of a surface to produce a reduction in the number of viable bacterial cells of representative
test organisms (3.6) under defined conditions without soil load, nor ageing test nor test conditions
adaptation to a specific sector.
Note 1 to entry: Note to entry: At the moment this document does not include soiling or ageing condition.
3.2
bacteriostatic activity
capability of a surface to inhibit the growth of viable bacterial cells of representative test organisms
(3.6) under defined conditions
Note 1 to entry: The term "bacteriostatic activity" cannot be used for claims according to this document.
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ISO/FDIS 7581:2023(E)
3.3
additional condition
test conditions that are optional and not obligatory, that can be used for additional claims of bactericidal
activity (3.1) regarding a surface
3.4
cleaning
all operations that achieve a level of cleanliness, appearance, comfort and hygiene
Note 1 to entry: Such operations use, to varying degrees, the following combined factors: chemical action,
mechanical action, temperature, duration of action
3.5
neutralizer
chemical agent or formulation that suppresses the residual microbicidal activity of a product or active
substance from the surface to be tested for a specific test but does not inactivate or inhibit the test
organism (3.6)
3.6
test organism
strain of a microorganism selected to evaluate the antimicrobial activity of a surface for a standardized
test
3.7
bactericidal surface
surface that irreversibly kill vegetative bacteria under defined conditions
Note 1 to entry: The adjective “bactericidal” corresponds to the noun “bactericide”.
3.8
reference surface
surface without any bactericidal activity (3.1) or properties, that is used to evaluate the quantity of
culturable bacteria present at the moment when the bactericidal activity of the test surface (3.9) is
evaluated
3.9
test surface
surface claiming bactericidal activity (3.1)
Note 1 to entry: This method is suitable for testing any type of non-porous surfaces (such as metals, plastic, glass,
coated surfaces, etc.) as long as recovery of bacteria from the test surface shall not lose more than 1 log at T=0.
Note 2 to entry: Very hydrophobic surfaces, for which a drying time greater than 10 min (see 6.2.2) is necessary,
cannot be tested according this method.
3.10
ambient light
light corresponding to a maximum value of 2 000 lux
Note 1 to entry: Any specific light or light spectrum (e.g. strong light, UV, etc.) is not considered as ambient.
3.11
porous surface
surface permeable to water, air, or other fluids
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ISO/FDIS 7581:2023(E)
4 Apparatus, reagents and materials
4.1 Test organisms
Bactericidal activity shall be evaluated using the following four strains:
— Pseudomonas aeruginosa ATCC 15442 = CIP 103-467;
— Staphylococcus aureus ATCC 6538 = CIP 4.83;
— Enterococcus hirae ATCC 10541 = CIP 5855;
— Escherichia coli ATCC 10536 = CIP 54127.
The reference strain numbers in other culture collections shall be in accordance with the strains
specified in Annex A.
The activity data can be completed with other strains using the experimental design described in
this document, varying the conditions to meet the needs of the intended -practice application(s). If
additional strains are used, they shall be incubated under optimum growth conditions (temperature,
time, atmosphere) to be recorded in the test report.
Their suitability for supplying the inoculum and controls with a sufficient concentration shall
be verified. If these additional test strains are not classified at a culture collection centre, their
identification characteristics shall be stated. In addition, they shall be held by the testing laboratory or
national culture collection under a reference for a five-year period.
NOTE ISO/TC 330 is currently adapting the method in terms of strains/efficacy levels/contact times/
interfering substances according to different sectors’ needs and to include not only the medical area.
4.2 Culture media and reagents
The reagents shall be of analytical grade and/or appropriate for microbiological purposes. They shall
be free from substances that are toxic or inhibitory to the test organisms.
4.2.1 Water, which shall be free from any substances that are toxic or that inhibit the bacteria. It shall
be freshly glass-distilled water or water for injection or possibly deionized or demineralized water.
Sterilize in the autoclave (4.4.1).
NOTE If the water is sterilized during the sterilization of the reagents, this is not necessary.
4.2.2 Microbial suspension diluents
Tryptone-salt solution:
Tryptone, pancreatic digest of casein .  1,0 g
Sodium chloride                        8,5 g
Water (see 4.2.1)                    1,000 ml
Sterilize in the autoclave (see 4.4.1). After sterilization, the pH of the medium shall be equivalent to
7,0 ± 0,2 when measured at 20 °C.
4.2.3 Liquid for bacteria recovery and for membrane rinsing
4.2.3.1 Composition
Tryptone, pancreatic digest of casein                   1,0 g
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ISO/FDIS 7581:2023(E)
Sodium chloride                                       8,5 g
Polysorbate 80                                        5,0 g
Water (see 4.2.1)                                          1 000 ml
4.2.3.2 Preparation
a) Dissolve the sodium chloride and tryptone in the water (see 4.2.2). Add the Polysorbate 80, mix and
complete up to 1 000 ml with water
b) distribute into smaller adapted flasks. Sterilize in the autoclave.
c) If necessary, add a neutralizer to the recovery liquid
The use of a validated neutralizer of the respective antimicrobial is compulsory to warrant a correct
test time for bacteria recovery and for membrane rinsing (see 4.2.3): The functionality of the respective
neutralizer shall be validated beforehand. The neutralizer:
— shall be validated for the tested surface, as per 6.2.4 and it shall be sterile;
— shall be mentioned in the test report.
Examples of neutralizers are given in Annex D.
4.2.4 Agar for bacteria maintenance and counting [tryptone soya agar (TSA)]
Use agar to preserve the bacterial strains and count the viable bacteria.
Tryptone, pancreatic digest of casein         .  15,0 g
Soya peptone, papaic digest of soybean meal .  5,0 g
Sodium chloride               5,0 g
Agar                15,0 g
Water (see 4.2.1)              1 000 ml
Sterilize in the autoclave. After sterilization, the pH of the medium shall be equivalent to 7,2 ± 0,2
when measured at 20 °C. Pre pored plate TSA are acceptable if they meet the composition and pH value
described in 4.2.4.
4.3 Reference and test surfaces
4.3.1 General
The surfaces shall be used only once.
4.3.2 Reference surfaces
4.3.2.1 Description
Inox 304 stainless steel, disk 2 cm in diameter, of which both sides have a grade 2B finish. The surfaces
shall be flat.
If the coated surface without antimicrobial agent (the test surface) exist, it can be used as a reference
surface similar to inox and therefore can be used for log reduction calculation.
The reference surface used during the test shall be specified in the report.
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ISO/FDIS 7581:2023(E)
4.3.2.2 Cleaning — Disinfection/sterilization
The reference surfaces shall be cleaned and disinfected before use.
If a specific preparation protocol is applied to the test surface (see examples in Annex C), the same
protocol should be applied to the reference surface (see 4.3.3.2). Otherwise, the protocol in example 1
of Annex C should be used.
No residual action or changes of the antimicrobial properties shall result from disinfection/cleaning
procedure on the reference surface.
The reference surface treatment used during the test shall be specified in the report.
4.3.3 Test surfaces
4.3.3.1 Identification and production
The characteristics (e.g. dimensions, thickness, etc.) and references of the finished product shall be
defined and specified in the final test report.
If the test surface is treated with a bactericide, the nature of the active substance(s) shall be specified
to the lab for safety and security reasons, and indicated in the final test report. For coated surfaces
with unknown porosity (e.g. paint), recovery of the microorganisms shall be checked by the laboratory,
comparing the coated surface with the antimicrobial agent and the reference surface (C0 vs T0) or the
coated surface with and without the antimicrobial agent, to ensure the accuracy of bacteria reduction
evaluation. Recovery of bacteria from the test surface shall not lose more than 1 log at T=0 compared to
the reference one.
The test surface shall be produced from the materials intended for the finished product claiming
bactericidal activity, and according to the same steps of the final production process. If it is impossible
to perform the steps on the same equipment, it is permitted to use a representative simulation of these
steps.
The test surface shall have flat surfaces and measure between 12 mm and 25 ± 2 mm on the sides
or in diameter. If it is impossible to cut the test surface into squares or disks of this size, other sizes
and shapes can be used. In this case, the actual dimensions used will be stated in the test report. Any
changes made to the protocol (recovery volume, etc.) due to the dimensions of the tested test surface
shall
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