prEN ISO 11138-6

• 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
• Publication Date: 01-Nov-2027
• ICS: 11.080.01 - Sterilization and disinfection in general
• Technical Committee: CEN/TC 102 - Sterilizers for medical purposes
• Drafting Committee: CEN/TC 102/WG 7 - Biological and chemical indicators
• Current Stage: 4060 - Closure of enquiry - Enquiry
• Start Date: 03-Aug-2026
• Due Date: 23-Dec-2025
• Completion Date: 03-Aug-2026
• Ref Project: oSIST prEN ISO 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)

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

prEN ISO 11138-6 is the CEN draft for biological indicators used with vaporized hydrogen peroxide (VH2O2) sterilization processes for health care products. It specifies the test organisms, inoculated carriers, biological indicators and test methods used to assess sterilizer and sterilization process performance. Manufacturers, validation laboratories and users can use it to prepare, test and review VH2O2 biological indicators in a consistent way.

What does prEN ISO 11138-6 specify?

prEN ISO 11138-6 specifies requirements for biological indicators intended for VH2O2 sterilization processes, and it excludes biological indicators used for hydrogen peroxide biodecontamination systems for rooms, enclosures or environmental spaces. The document is organized into Clauses 1 to 8, with four annexes that separate the core requirements from the detailed test method, background information and equipment checks.

Annex	What it covers
Annex A	Normative method for determining resistance to VH2O2 sterilization
Annex B	Informative summary of how the resistance method was developed
Annex C	Informative calculation of sterilant quantity from chamber volume
Annex D	Normative VH2O2 resistometer performance requirements specific to the BI

Clauses 4 to 8 cover manufacturing requirements, test organism and resistance requirements, culture conditions, and carrier and packaging suitability. That structure matters because it shows where the document relies on ISO 11138-1 and where it adds VH2O2-specific requirements.

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

• Clauses 4 and 5 apply the general and specific manufacturing requirements of ISO 11138-1. In practice, a supplier cannot treat this part as a stand-alone document - it builds on the general biological indicator manufacturing rules in Part 1.

• Clause 6.1 identifies spores of Geobacillus stearothermophilus as the preferred test organism, and any other organism has to have its resistance suitability demonstrated. For users and buyers, this means product claims should be tied to a known organism with evidence that it behaves appropriately in VH2O2 exposure.

• Clause 6.2 requires a defined population for inoculated carriers and biological indicators, with the population stated in a controlled increment. This matters in practice because the declared population is part of lot-to-lot consistency and of how resistance data are interpreted.

• Clause 6.3.1 and Annex A require resistance to be determined by the defined VH2O2 method using a resistometer or another test sterilizer with precise measurement and process control. The practical point is that resistance data must come from a reproducible vapor process, not from a simplified exposure that does not represent the sterilization mechanism.

• Clauses 6.3.2 and 6.3.3 require both All Survive and All Kill testing, each with at least 50 replicates, and the manufacturer has to report the results for each lot. All Survive means every replicate still shows surviving microorganisms after exposure; All Kill means the microorganisms in each replicate are completely inactivated. In practice, this gives a lot-specific check that the BI behaves as expected at both ends of the resistance range.

• Clause 6.3.2.4 and 6.3.3.4 define follow-up actions for unusual results. A single negative result in an All Survive set can be investigated with additional samples, and more than 10 positives in All Kill triggers verification that the resistometer is working according to specification. This helps distinguish a real product issue from an equipment or process problem.

• Clause 6.3.4 and 6.3.5 allow additional concentration levels between the levels defined in Annex A, and they allow D-value testing by the manufacturer’s recommended method. For manufacturers, this means extra characterization data can be added, but the core resistance method and reporting expectations still apply.

• Clause 7 and Clause 8 rely on ISO 11138-1 for culture conditions and require carrier and primary packaging suitability to be shown for VH2O2 use. That is important because the carrier and package have to survive the process and still let the BI perform as intended.

What Annex A adds

Annex A defines the test cycle for resistance determination. It requires chamber pre-conditioning, pre-conditioning of the BIs, controlled injection of VH2O2 solution, a fixed exposure sequence, and recording of concentration, pressure and chamber temperature throughout exposure. It also distinguishes the exposure conditions used for All Survive and All Kill, so the test can demonstrate both persistence and inactivation under defined vapor conditions.

What Annex D adds

Annex D adds VH2O2-specific performance checks for the resistometer. It requires defined measurement of the sterilant dose, mapping of chamber temperature, verification that the chamber can meet the cycle specification, and checks that the vaporization step produces the expected pressure and concentration behavior. In practice, Annex D is what supports confidence that the test equipment is not creating misleading resistance results.

What terms does prEN ISO 11138-6 define?

• Vaporized hydrogen peroxide (VH2O2) - hydrogen peroxide used in vapor form as the sterilizing agent covered by this document.
• Biological indicator (BI) - a prepared inoculated carrier or related indicator system used to assess the performance of a sterilization process.
• Inoculated carrier - a carrier loaded with microorganisms so that resistance to the sterilization process can be measured.
• Resistometer - test equipment or a test sterilizer that can measure and control process conditions precisely enough for BI resistance testing.
• All Survive - a test outcome in which every replicate still contains surviving microorganisms after exposure to the test cycle.
• All Kill - a test outcome in which every replicate is completely inactivated after exposure to the test cycle.
• D-value - the resistance value that can be determined by the manufacturer’s recommended method for this BI.

Who uses prEN ISO 11138-6?

prEN ISO 11138-6 is used by BI manufacturers, sterilizer manufacturers, test laboratories, validation specialists, quality managers and buyers of sterilization-related products. It supports the design and qualification of biological indicators, the development and verification of VH2O2 sterilization processes, and lot-by-lot review of BI performance. It is also relevant to organizations that need to compare sterilizer performance or document that a VH2O2 process has been characterized using a recognized method.

Which standards are used with prEN ISO 11138-6?

• ISO 11138-1:2017 - provides the general requirements, terms, manufacturing rules, culture conditions, and the population method that this part applies.
• ISO 22441:2022 - provides requirements for the development, validation and routine control of low temperature VH2O2 sterilization processes, which is the process family referenced by this part.
• ISO 14937:2009 - gives general requirements for characterization of a sterilizing agent and development, validation and routine control of a sterilization process.
• ISO 11138-7 - gives guidance on selection, use and interpretation of results when biological indicators are used.
• ISO 18472:2018 - sets performance requirements for biological and chemical indicator test equipment, which Annex D builds on for VH2O2 BI testing.

What does the prEN ISO 11138-6 document contain?

The document contains the general manufacturing clauses, the VH2O2-specific requirements for test organisms, population and resistance, and the culture and packaging provisions taken with ISO 11138-1. It also contains a normative resistance test method in Annex A, with controlled chamber conditioning, vapor exposure and result reporting. Annex B explains how the method was developed through interlaboratory testing, which helps readers understand why the final cycle conditions were chosen. Annex C provides the calculation approach for sterilant quantity versus chamber volume, including a correspondence table between pressure and concentration, and Annex D sets the equipment checks needed to verify resistometer performance for this BI.

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,
TECHNOLOGICAL, COMMERCIAL AND
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
ISO/CEN PARALLEL PROCESSING
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on
BE CONSIDERED IN THE LIGHT OF THEIR
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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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