oSIST prEN ISO 11138-6:2026

• Abstract:

  This document specifies requirements for test organisms, inoculated carriers, biological indicators and test methods intended for use in assessing the performance of sterilizers and sterilization processes employing vaporized hydrogen peroxide as the sterilizing agent.; NOTE 1 Requirements for validation and control of vaporized hydrogen peroxide sterilization processes are provided by ISO 22441 and ISO 14937.; NOTE 2 National or regional regulations can provide requirements for workplace safety.

• Status: Not Published
• Public Enquiry End Date: 30-Jul-2026
• ICS: 11.080.01 - Sterilization and disinfection in general
• Technical Committee: VAZ - Healthcare
• Current Stage: 4020 - Public enquire (PE) (Adopted Project)
• Start Date: 21-May-2026
• Due Date: 08-Oct-2026
• Completion Date: 03-Aug-2026
• Ref Project: prEN ISO 11138-6 - Sterilization of health care products - Biological indicators - Part 6: Biological indicators for vaporized hydrogen peroxide sterilization processes (ISO/DIS 11138-6:2026)

• Referred By: SIST EN ISO 23936-4:2024 - Oil and gas industries including lower carbon energy - Non-metallic materials in contact with media related to oil and gas production - Part 4: Fiber-reinforced composite (ISO 23936-4:2024)
• Effective Date: 28-Jan-2026

oSIST prEN ISO 11138-6:2026 is the Slovenian adoption of ISO/DIS 11138-6:2026 from ISO, prepared by ISO/TC 198 on sterilization of health care products. It specifies requirements for test organisms, inoculated carriers, biological indicators and test methods used to assess sterilizers and sterilization processes that use vaporized hydrogen peroxide (VH2O2). Manufacturers, test laboratories and users use it to produce, verify and interpret biological indicators for VH2O2 processes.

What does oSIST prEN ISO 11138-6:2026 specify?

oSIST prEN ISO 11138-6:2026 specifies the requirements for biological indicators intended for VH2O2 sterilization processes. It sets the framework for manufacturing, resistance testing, culture conditions, carrier and packaging suitability, and the equipment used to generate reproducible test conditions.

The standard is organized into eight clauses and four annexes. Clause 1 gives the scope, Clauses 2 and 3 cover references and terms, Clauses 4 and 5 apply the general and specific manufacturing rules from ISO 11138-1, and Clauses 6 to 8 set the organism, resistance, culture, carrier and packaging requirements.

Annex	What it covers
Annex A	Normative method for determining resistance to VH2O2 sterilization
Annex B	Informative summary of method development and interlaboratory testing
Annex C	Informative calculation of the quantity of H2O2 solution to inject from chamber volume
Annex D	Normative VH2O2 resistometer performance requirements specific to the VH2O2 BI

The scope is limited to biological indicators used for sterilizers and sterilization processes with vaporized hydrogen peroxide as the sterilizing agent. It does not cover biological indicators used for hydrogen peroxide biodecontamination systems for rooms, enclosures or environmental spaces.

What are the key requirements of oSIST prEN ISO 11138-6:2026?

Manufacturing and general conditions

oSIST prEN ISO 11138-6:2026 applies the general manufacturing requirements of ISO 11138-1:2017, Clause 4, and the specific manufacturing requirements of ISO 11138-1:2017, Clause 5. It also applies ISO 11138-1:2017, Clause 7 for culture conditions and ISO 11138-1:2017, 5.2 and Annex B for carrier and primary packaging suitability.

In practice, this means the VH2O2 biological indicator must be made and cultured under the same general controls used across the ISO 11138 series, then shown to work with the chosen carrier and package in a VH2O2 process.

Test organism and population

Clause 6.1 gives the preferred test organism as spores of Geobacillus stearothermophilus. If another organism is used, its resistance suitability has to be determined. This matters because the indicator has to challenge the sterilization process in a controlled and defensible way.

Clause 6.2 sets the population requirements for inoculated carriers and biological indicators and requires the population to be determined using ISO 11138-1:2017, Annex A. The population also has to be stated with fine unit-by-unit increments, so the manufacturer can report lot characteristics consistently.

Resistance testing and lot reporting

Clause 6.3 says resistance shall be determined by the procedure in Annex A. Annex A is the core method for proving that the BI gives reproducible survival and kill responses under defined VH2O2 conditions.

The standard requires both an All Survive test and an All Kill test. Each uses not less than 50 replicates, and the manufacturer must report the results for each lot. For All Survive, every replicate has to be positive, although one unexpected negative can be followed by additional testing on at least 100 more samples; if no further unexpected results appear, the All Survive requirement is confirmed. For All Kill, the test must be completed and reported, and if there are more than 10 positives, the resistometer performance must be checked against its specification.

Clause 6.3.4 also allows additional concentration levels between those defined in Annex A to be tested and reported. Clause 6.3.5 allows D-value testing using the manufacturer’s recommended method. In practice, this gives manufacturers some flexibility, but the core resistance claims still have to rest on the prescribed resistance method.

Annex A test cycle

Annex A defines the resistance test conditions used to evaluate VH2O2 biological indicators. The chamber and sample holder are conditioned to 50 °C ± 1 °C, the biological indicators are preconditioned to 23 °C ± 2 °C, and the test cycle then uses a controlled evacuation, VH2O2 admission and 120 s exposure period.

Annex A also sets separate exposure pressure conditions for the All Survive and All Kill stages, and it requires concentration, pressure and chamber temperature to be measured and recorded throughout the exposure period. In practical terms, the method is designed to make the resistance result traceable to the actual process conditions, not just to a nominal cycle setting.

Annex D resistometer performance

Annex D sets the performance checks for the VH2O2 resistometer used with this BI. It requires a method for measuring the quantity of sterilant injected, defines accuracy and resolution targets for mass or volume measurement, and requires chamber volume to include all tubing opened to the chamber.

Annex D also requires temperature mapping of the chamber wall at 50 °C ± 1 °C, with a minimum recorded temperature of at least 45 °C. The vaporization test must show that the sterilant vaporizes without condensing in the empty chamber or test system, and the last three of four runs must stay within the stated pressure and concentration tolerances. This is the control that shows the test equipment can actually produce the conditions needed for valid BI resistance testing.

What terms does oSIST prEN ISO 11138-6:2026 define?

• Biological indicator (BI) - a prepared test article used to challenge a sterilization process and check whether the process inactivates the test organism.
• Inoculated carrier - a carrier that has been loaded with microorganisms and is used as the test article in the resistance method.
• Vaporized hydrogen peroxide (VH2O2) - hydrogen peroxide used in vapor form as the sterilizing agent.
• Resistometer - test equipment or a test sterilizer used to create and control reproducible sterilization conditions for BI resistance testing.
• All Survive test - a test in which every article in the exposed group still shows surviving microorganisms after the cycle.
• All Kill test - a test in which the microorganisms in each exposed article are completely inactivated after the cycle.
• D-value - a resistance value that may be tested using the manufacturer’s recommended method.
• Self-contained biological indicator (SCBI) - a biological indicator design discussed in the method-development material, where the BI is self-contained.

Who uses oSIST prEN ISO 11138-6:2026?

The standard is used by BI manufacturers, sterilizer manufacturers, sterilization laboratories, and healthcare product suppliers that validate or monitor VH2O2 sterilization processes. Quality, regulatory and procurement teams use it to judge whether a BI lot, a test method or a resistometer setup is appropriate for VH2O2 work.

It is also relevant to users who need evidence that a VH2O2 process can be challenged and measured in a consistent way. That includes work on lot release, process validation, routine monitoring and equipment qualification.

Which standards are used with oSIST prEN ISO 11138-6:2026?

• ISO 11138-1:2017 - provides the general requirements, terms, culture conditions and the core population determination method used throughout this part.
• ISO 11138-7 - gives guidance on the selection, use and interpretation of biological indicators.
• ISO 22441:2022 - provides requirements for the development, validation and routine control of low temperature VH2O2 sterilization processes.
• ISO 14937:2009 - covers general requirements for characterizing a sterilizing agent and developing, validating and routinely controlling a sterilization process for medical devices.
• ISO 18472:2018 - sets performance requirements for the test equipment used to establish the response of biological and chemical indicators to critical process variables.

What does the oSIST prEN ISO 11138-6:2026 document contain?

The document contains the scope, normative reference, terms, manufacturing requirements, organism and resistance rules, culture conditions, and carrier and packaging requirements for VH2O2 biological indicators. It also contains a normative resistance test method in Annex A, supported by a calculation for sterilant quantity in Annex C and resistometer performance requirements in Annex D.

Annex A gives the test cycle conditions, including the sequence of preconditioning, evacuation, exposure and recovery. Annex C provides an ideal-gas-based way to relate chamber volume, pressure and injected solution quantity, with a table that links chamber pressure to the corresponding sterilant dose and concentration.

Annex D describes the checks used to confirm that the resistometer can create the required VH2O2 conditions reproducibly. Annex B summarizes the method-development work and the interlaboratory testing that led to the method in Annex A.

SLOVENSKI STANDARD
01-julij-2026
Sterilizacija zdravstvenih izdelkov - Biološki indikatorji - 6. del: Biološki indikatorji
za sterilizacijske postopke z uparjenim vodikovim peroksidom (ISO/DIS 11138-
6:2026)
Sterilization of health care products - Biological indicators - Part 6: Biological indicators
for vaporized hydrogen peroxide sterilization processes (ISO/DIS 11138-6:2026)
Sterilisation von Produkten für die Gesundheitsfürsorge - Biologische Indikatoren - Teil 6:
Biologische Indikatoren für Sterilisationsverfahren mit Wasserstoffperoxid (ISO/DIS
11138-6:2026)
Stérilisation des produits de santé - Indicateurs biologiques - Partie 6: Indicateurs
biologiques pour la stérilisation au peroxyde d'hydrogène (ISO/DIS 11138-6:2026)
Ta slovenski standard je istoveten z: prEN ISO 11138-6
ICS:
11.080.01 Sterilizacija in dezinfekcija na Sterilization and disinfection
splošno in general
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

DRAFT
International
Standard
ISO/DIS 11138-6
ISO/TC 198
Sterilization of health care
Secretariat: ANSI
products — Biological indicators —
Voting begins on:
Part 6: 2026-05-11
Biological indicators for vaporized
Voting terminates on:
2026-08-03
hydrogen peroxide sterilization
processes
ICS: 11.080.01
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENTS AND APPROVAL. IT
IS THEREFORE SUBJECT TO CHANGE
AND MAY NOT BE REFERRED TO AS AN
INTERNATIONAL STANDARD UNTIL
PUBLISHED AS SUCH.
This document is circulated as received from the committee secretariat.
IN ADDITION TO THEIR EVALUATION AS
BEING ACCEPTABLE FOR INDUSTRIAL,
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USER PURPOSES, DRAFT INTERNATIONAL
STANDARDS MAY ON OCCASION HAVE TO
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BE CONSIDERED IN THE LIGHT OF THEIR
POTENTIAL TO BECOME STANDARDS TO
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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 SUPPORTING DOCUMENTATION.
Reference number
ISO/DIS 11138-6:2026(en)
DRAFT
ISO/DIS 11138-6:2026(en)
International
Standard
ISO/DIS 11138-6
ISO/TC 198
Sterilization of health care
Secretariat: ANSI
products — Biological indicators —
Voting begins on:
Part 6:
Biological indicators for vaporized
Voting terminates on:
hydrogen peroxide sterilization
processes
ICS: 11.080.01
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENTS AND APPROVAL. IT
IS THEREFORE SUBJECT TO CHANGE
AND MAY NOT BE REFERRED TO AS AN
INTERNATIONAL STANDARD UNTIL
PUBLISHED AS SUCH.
This document is circulated as received from the committee secretariat.
IN ADDITION TO THEIR EVALUATION AS
BEING ACCEPTABLE FOR INDUSTRIAL,
© ISO 2026
TECHNOLOGICAL, COMMERCIAL AND
USER PURPOSES, DRAFT INTERNATIONAL
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
STANDARDS MAY ON OCCASION HAVE TO
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Published in Switzerland Reference number
ISO/DIS 11138-6:2026(en)
ii
ISO/DIS 11138-6:2026(en)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 General manufacturing requirements . 1
5 Specific manufacturing requirements . 1
6 Test organism, population and resistance . 2
6.1 Test organism .2
6.2 Population .2
6.3 Resistance .2
6.3.1 Determination of resistance .2
6.3.2 All Survive test .2
6.3.3 All Kill test . .2
6.3.4 Additional concentration levels .3
6.3.5 Testing for D-value.3
7 Culture conditions . 3
8 Carrier and packaging . 3
Annex A (normative) Method for determination of resistance to VH2O2 sterilization . 4
Annex B (informative) Summary of method development for the determination of biological
indicator resistance to vaporized hydrogen peroxide sterilization . 6
Annex C (informative) Quantity of H O solution to inject based on chamber volume .13
2 2
Annex D (normative) VH2O2 resistometer performance requirement specific to the VH2O2 BI .15
Bibliography . 17

iii
ISO/DIS 11138-6:2026(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 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 198, Sterilization of health care products, in
collaboration with the European Committee for Standardization (CEN) Technical Committee CEN/TC 102,
Sterilizers and associated equipment for processing of medical devices, in accordance with the Agreement on
technical cooperation between ISO and CEN (Vienna Agreement).
A list of all parts in the ISO 11138 series can be found on the ISO website.
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
ISO/DIS 11138-6:2026(en)
Introduction
ISO 11138-1 specifies production, labelling, test methods and performance requirements for the
manufacture of biological indicators including inoculated carriers and suspensions intended for use in
validation and monitoring of sterilization processes. This document gives specific requirements for those
biological indicators intended for use in vaporized hydrogen peroxide (VH2O2) sterilization processes.
The ISO 11138 series represents the current “state-of-the-art” according to the experts representing
manufacturers, users and regulatory authorities involved in developing these documents. The intent is not
to promote the use of biological indicators where such use is not advised, but rather to provide common
requirements for the production of biological indicators that are known to be in use today.
Requirements for the validation and control of VH2O2 sterilization are provided in ISO 22441.
NOTE There can be other national or regional standards covering requirements for sterilization or biological
indicators.
Guidance on selection, use and interpretation of results when using biological indicators can be found in
ISO 11138-7.
v
DRAFT International Standard ISO/DIS 11138-6:2026(en)
Sterilization of health care products — Biological
indicators —
Part 6:
Biological indicators for vaporized hydrogen peroxide
sterilization processes
1 Scope
This document specifies requirements for test organisms, inoculated carriers, biological indicators and test
methods intended for use in assessing the performance of sterilizers and sterilization processes employing
vaporized hydrogen peroxide (VH2O2) as the sterilizing agent.
NOTE Requirements for validation and control of VH2O2 sterilization processes are provided in ISO 22441.
This document does not cover biological indicators used for hydrogen peroxide (H O ) biodecontamination
2 2
systems such as those used for rooms, enclosures or environmental spaces.
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 11138-1:2017, Sterilization of health care products — Biological indicators — Part 1: General requirements
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 11138-1 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/
4 General manufacturing requirements
The general manufacturing requirements of ISO 11138-1:2017, Clause 4 shall apply.
5 Specific manufacturing requirements
The specific manufacturing requirements of ISO 11138-1:2017, Clause 5 shall apply.

ISO/DIS 11138-6:2026(en)
6 Test organism, population and resistance
6.1 Test organism
6.1.1 The preferred test organisms shall be spores of Geobacillus stearothermophilus.
6.1.2 If a test organism other than Geobacillus stearothermophilus is used, the suitability of the resistance
of that test organism shall be determined.
NOTE Annex A of ISO 22441:2022 provides factors for consideration in selecting microorganisms.
6.2 Population
6.2.1 For inoculated carriers and biological indicators, the population shall be greater than or equal to 1,0
× 10 .
n
6.2.2 The population shall be stated with increments less than or equal to 0,1×10 per unit (e.g. per ml of
suspension, per inoculated carrier or per biological indicator).
6.2.3 The population shall be determined in accordance with ISO 11138-1:2017, Annex A.
6.3 Resistance
6.3.1 Determination of resistance
Resistance shall be determined per the procedure defined in Annex A.
NOTE See ISO 11138-7 for information on use and applications of BIs.
6.3.2 All Survive test
6.3.2.1 All Survive means a result from the culture of a group of inoculated carriers or biological indicators
where every test article is found to contain surviving microorganisms after exposure to the test cycle.
6.3.2.2 Meeting the All Survive test result criteria is a requirement of this document. All Survive test
results must be reported by the biological indicator manufacturer for each lot.
6.3.2.3 Not less than 50 replicates shall be used for the All Survive test. All replicates must be positive to
meet the requirement (except as noted in 6.3.2.4).
NOTE The number of units run per exposure depends on both the capacity and the operating characteristics of
the resistometer being used. It can be necessary to run several exposures at the survival time in order to test the total
number of units required.
6.3.2.4 For lots with a single negative biological indicator in a Survival Test, an additional 100 samples
(minimum) may be tested. If no additional unexpected results are obtained, the All Survive requirement is
confirmed.
6.3.3 All Kill test
6.3.3.1 All Kill means a result from the culture of a group of inoculated carriers or biological indicators
where the population of microorganisms from each test article is found to have been completely inactivated
after exposure to the test cycle.

ISO/DIS 11138-6:2026(en)
6.3.3.2 Completing the All Kill test is a requirement of this standard. Meeting the All Kill criteria (all units
negative) is not required. All Kill test results must be reported by the biological indicator manufacturer for
each lot.
6.3.3.3 Not less than 50 replicates shall be used for the All Kill test.
NOTE The number of units run per exposure depends on both the capacity and the operating characteristics of
the resistometer being used. It can be necessary to run several exposures at the kill time in order to test the total
number of units required.
6.3.3.4 If there are >10 positives, verification that the resistometer is working according to specification
shall be performed.
6.3.4 Additional concentration levels
Additional concentration levels that fall between the levels defined in Annex A and particularly A.3.13 and
A.3.14 can be tested and reported by the BI manufacturer.
6.3.5 Testing for D-value
In addition to the resistance determination requirements in 6.3.1, testing for D-value can be performed
according to the BI manufacturer’s recommended method. ISO 11138-1 provides further information on
methods.
7 Culture conditions
The culture condition requirements of ISO 11138-1:2017, Clause 7 apply.
8 Carrier and packaging
The suitability of the carrier and primary packaging materials for biological indicators for use in VH2O2
sterilization processes shall be demonstrated in accordance with the requirements of ISO 11138-1:2017, 5.2
and Annex B.
ISO/DIS 11138-6:2026(en)
Annex A
(normative)
Method for determination of resistance to VH2O2 sterilization
A.1 Principle
This method is intended to define reproducible test conditions for the evaluation of the resistance of
biological indicators (inoculated carriers) for assessing the performance of VH2O2 processes. This method
requires the use of a resistometer or other test sterilizer that is capable of precise measurement and process
control.
A.2 Materials
Hydrogen peroxide (H O ) solution: nominal 59 % weight/weight +/- 1 % by weight.
2 2
A.3 Test procedure
A.3.1 Chamber pre-conditioning: before initiating a test cycle, the sample holder and the sterilizer chamber
shall be equilibrated to the required operating temperature (50 °C ± 1 °C).
A.3.2 Pre-condition the biological indicators to 23 °C ± 2 °C.
A.3.3 Calculate the amount of nominal 59 % H O by weight solution to be injected into the exposure
2 2
chamber based on the volume of the sterilizer chamber.
NOTE See Annex C for information on the quantity of H O sterilant solution to be injected
2 2
A.3.4 Conditioning cycle: if applicable run one or more warm up cycles to bring the test system to steady
state including the sample holder before the first test cycle. No BI test samples or data collection required.
A.3.5 Load the BI test samples into the chamber in a manner which minimizes temperature loss.
A.3.6 Immediately after loading the BI test samples initiate the test cycle according to Table A.1.
Table A.1 — Test cycle conditions
Variable Value
Starting liquid H O concentration Target 59 % w/w ± 1 % w/w
2 2
Chamber operating temperature 50 °C ± 1 °C
Pre-exposure evacuation time + Hold Time <8 min
Evacuation pressure 0,07 kPa (±0,01 kPa)
0,5 Torr (±0,10 Torr)
Time to achieve set point pressure or concentration of ≤10 s
VH2O2 (come-up period)
VH2O2 exposure pressure – All Survive (see NOTE) Minimum 0,27 kPa ± 0,05 kPa
(Minimum 2 Torr ± 0,4 Torr)
VH2O2 exposure pressure – All Kill (See NOTE) Minimum 1,47 kPa ± 0,08 kPa
(Minimum 11 Torr ± 0,6 Torr)
ISO
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