prEN ISO 11737-3

SLOVENSKI STANDARD
01-marec-2026
Sterilizacija izdelkov za zdravstveno nego - Mikrobiološke metode - 3. del:
Preskušanje bakterijskih endotoksinov (ISO 11737-3:2023)
Sterilization of health care products - Microbiological methods - Part 3: Bacterial
endotoxin testing (ISO 11737-3:2023)
Sterilisation von Produkten für die Gesundheitsfürsorge - Mikrobiologische Verfahren -
Teil 3: Prüfung bakterieller Endotoxine (ISO 11737-3:2023)
Stérilisation des produits de santé - Méthodes microbiologiques - Partie 3: Essai des
endotoxines bactériennes (ISO 11737-3:2023)
Ta slovenski standard je istoveten z: prEN ISO 11737-3
ICS:
07.100.10 Medicinska mikrobiologija Medical microbiology
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.

INTERNATIONAL ISO
STANDARD 11737-3
First edition
2023-06
Sterilization of health care products —
Microbiological methods —
Part 3:
Bacterial endotoxin testing
Stérilisation des produits de santé — Méthodes microbiologiques —
Partie 3: Essai des endotoxines bactériennes
Reference number
ISO 11737-3:2023(E)
ISO 11737-3:2023(E)
© ISO 2023
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Published in Switzerland
ii
ISO 11737-3:2023(E)
Contents Page
Foreword .v
Introduction . vi
1 Scope . 1
1.1 Inclusions. 1
1.2 Exclusions . 1
2 Normative references . 1
3 Terms and definitions . 1
4 General requirements . 7
5 Selection of products .8
5.1 General . 8
5.2 Selection of product units . 8
6 Methods for BET . 9
6.1 General . 9
6.2 Consideration of an applicable endotoxin limit . 10
6.2.1 Endotoxin limit . 10
6.2.2 Calculation of endotoxin limit for the extract solution . 10
6.2.3 Maximum valid dilution (MVD) . 10
6.3 Critical test parameters . 11
6.3.1 Temperature . 11
6.3.2 Time . 11
6.3.3 pH . 11
6.4 Equipment and materials . 11
6.5 Reagents .12
7 Method suitability for BET (BET validation) .12
7.1 General .12
7.2 Product and test method suitability .12
7.2.1 Gel-clot technique .12
7.2.2 Kinetic and end point methods (chromogenic and turbidimetric techniques) .13
7.3 Sample preparation . 14
7.3.1 General . 14
7.3.2 Solid health care products . 14
7.3.3 Aqueous health care products . 15
7.3.4 Sample interference .15
7.4 Reagent and analyst qualification . 15
7.4.1 Gel-clot technique reagent qualification . 15
7.4.2 Kinetic and end point method reagent qualification .15
7.4.3 Analyst qualification . 16
8 Routine testing, monitoring and interpretation of data .16
8.1 Routine testing . 16
8.1.1 Gel-clot limit test . 16
8.1.2 Gel-clot assay . 16
8.1.3 Kinetic and end point methods (chromogenic and turbidimetric) . 17
8.2 Monitoring (test frequency) . 17
8.3 Interpretation of results . 17
8.3.1 General . 17
8.3.2 Gel clot methods. 18
8.3.3 Kinetic and end point methods . 18
8.4 Data analysis . 18
8.5 Statistical methods . 18
9 Maintenance of the BET method .18
9.1 General . 18
iii
ISO 11737-3:2023(E)
9.2 Changes to either the product or manufacturing process, or both . 18
9.3 Changes to the BET method . 19
10 Alternatives to batch testing .19
10.1 General . 19
10.2 Criteria for establishing alternatives to batch testing . 19
10.3 Manufacturing process assessment . 20
10.3.1 Quality planning of manufacturing processes . 20
10.3.2 Process design .20
10.3.3 Process control . 20
10.4 Change control . 21
10.5 Maintenance of risk assessment . 21
Annex A (informative) Guidance on bacterial endotoxin testing (following the subclauses
in this document) .22
Annex B (informative) History and background on the bacterial endotoxins test (BET) .42
Annex C (informative) Guidance on out of specified limits (OSL) and failure investigation .46
Annex D (informative) Guidance on in-process monitoring of manufacturing processes or
component testing .50
Annex E (informative) Guidance on conducting a risk assessment to support alternatives
to batch testing .53
Annex F (informative) Typical assignment of responsibilities .58
Bibliography .60
iv
ISO 11737-3:2023(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 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.
A list of all parts in the ISO 11737 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.
v
ISO 11737-3:2023(E)
Introduction
A pyrogen is any substance that can induce fever. Testing for pyrogens is required for release of many
health care products. Pyrogens can be classified into two groups: microbial (e.g. bacteria, fungi, viruses)
and non-microbial (e.g. drugs, device materials, steroids, plasma fractions; see the ISO 10993 series).
The predominant pyrogenic contaminants encountered in the manufacturing of health care products
are bacterial endotoxins, which are components of the cell walls of Gram-negative bacteria. Although
Gram-positive bacteria, fungi, and viruses can be pyrogenic, they do so through different mechanisms
(systemic effects) and to a lesser degree than Gram-negative bacteria. Only the Gram-negative bacterial
endotoxins test (BET) using amebocyte lysate reagents from Limulus polyphemus or Tachypleus
tridentatus is covered in this document. Other endotoxin detection methodologies, such as monocyte
activation and recombinant Factor C (rFc), are not included (see B.12) in this document.
Endotoxins are the molecular weight lipopolysaccharide (LPS) components of the outer cell wall of
Gram-negative bacteria, that can cause fever, meningitis, and a rapid fall in blood pressure if introduced
into the blood stream or certain other tissues of the body. The outer cell wall components, which are
composed primarily of proteins, phospholipids and LPS, are constantly released by the cell into the
surrounding environment. Endotoxins are ubiquitous in nature, stable, and small enough to pass
through conventional sterilizing filters. Sterilization processes will inactivate microorganisms on or
in products, but usually do not inactivate endotoxin on products. With controlled processes, endotoxin
contamination can be prevented.
The non-pyrogenicity of a health care product can be achieved through the following:
a) manufacturing techniques that prevent or control endotoxin contamination (e.g. contamination
with Gram-negative bacteria);
b) depyrogenation by endotoxin inactivation (e.g. dry heat) or physical removal (e.g. rinsing,
distillation, ultrafiltration).
The purpose of this document is to describe the requirements and guidance for testing for bacterial
endotoxins. This includes product required to be non-pyrogenic based on either intended use or
non-pyrogenic label claim, or both. Guidance is also provided on selection of product units, method
suitability, use of techniques for routine testing, interpretation of test results, and alternatives to batch
testing and risk assessment. Information on the following is provided in the annexes:
— guidance on bacterial endotoxin testing (Annex A);
— the history and background on the BET (Annex B);
— guidance on out of specified limits (OSL) and failure investigation (Annex C);
— guidance on in-process monitoring of manufacturing or component testing (Annex D);
— guidance on conducting a risk assessment to support alternatives to batch testing (Annex E);
— typical assignment of responsibilities (Annex F).
This document is based on ANSI/AAMI ST72. Several sections in this document have been restructured
and extended or changed from ANSI/AAMI ST72.
vi
INTERNATIONAL STANDARD ISO 11737-3:2023(E)
Sterilization of health care products — Microbiological
methods —
Part 3:
Bacterial endotoxin testing
1 Scope
1.1 Inclusions
This document specifies general criteria to be applied in the determination of bacterial endotoxins on or
in health care products, components or raw materials using bacterial endotoxins test (BET) methods,
using amebocyte lysate reagents.
1.2 Exclusions
1.2.1 This document is not applicable to the evaluation of pyrogens other than bacterial endotoxins.
Other endotoxin detection methodologies are not included (see B.12).
1.2.2 This document does not address setting specific endotoxin limit specifications.
2 Normative references
There are no normative references in this document.
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
bacterial endotoxins test
BET
assay for measuring bacterial endotoxins by combining an aqueous test sample or test sample extract
with Tachypleus amebocyte lysate (TAL) (3.41) or Limulus amebocyte lysate (LAL) (3.28) reagent and
measuring the resulting proportional reaction via visual, turbidimetric (3.42) or chromogenic techniques
(3.3)
3.2
batch
defined quantity of a product intended or purported to be uniform in character and quality produced
during a specified cycle of manufacture
[SOURCE: ISO 11139:2018, 3.21]
ISO 11737-3:2023(E)
3.3
chromogenic technique
bacterial endotoxins test (BET) (3.1) methodology that quantifies endotoxins on the basis of a measured
colour-producing reaction proportional to the interaction of Limulus amebocyte lysate (LAL) (3.28) and
endotoxin
3.4
control standard endotoxin
CSE
endotoxin standard preparation whose potency has been standardized against the Reference Standard
Endotoxin (RSE) (3.37) for a specific batch of Limulus amebocyte lysate (LAL) (3.28)
3.5
depyrogenation
process used to remove or deactivate pyrogenic substances to a specified level
Note 1 to entry: Pyrogenic substances include bacterial endotoxins.
[SOURCE: ISO 11139:2018, 3.77]
3.6
direct contact
medical device or medical device component that comes into physical contact with body tissue
[SOURCE: ISO 10993-1:2018, 3.6]
3.7
end product
product samples that have completed the entire manufacturing process
Note 1 to entry: For the purposes of this document, end-product testing can be performed prior to sterilization
(pre-sterilization samples) or after sterilization (post-sterilization samples). For limitations see 5.2.6.
3.8
endotoxin
bacterial endotoxin
lipopolysaccharide (LPS)(3.29) component of the cell wall of Gram-negative bacteria that is heat stable
and elicits a variety of inflammatory responses in animals and humans
[SOURCE: ISO 11139:2018, 3.101]
3.9
endotoxin limit
maximum allowable amount of endotoxin present on the product or in a product extraction solution
3.10
endotoxin unit
EU
international unit
IU
standard unit of measure for endotoxin activity initially established relative to the activity contained
in 0,2 ng of the Reference Standard Endotoxin (RSE) (3.37) Lot EC-2 [US Pharmacopeia (USP) standard
reference material]
Note 1 to entry: Currently, the US RSE EC-6, USP Lot G, and the World Health Organization’s primary international
[45]
endotoxin standard (IS) are sub-lots of the same endotoxin preparation, making the EU and IU equal .
ISO 11737-3:2023(E)
3.11
end point
most dilute concentration of a test or control solution for which a positive reaction for bacterial
endotoxin is observed
Note 1 to entry: This definition is used for concentration dependent bacterial endotoxin testing, in contrast to
dilution dependent end point methods described in A.6.1.1.
3.12
enhancement
bacterial endotoxins test (BET) (3.1) anomaly in which a non-endotoxin related factor, usually
attributable to a characteristic of the test sample, elicits a test reaction greater than the amount of
endotoxin present
3.13
gel-clot technique
bacterial endotoxins test (BET) (3.1) methodology that quantifies or detects endotoxin on the basis of
a clot-producing reaction proportional to the interaction of Limulus amebocyte lysate (LAL) (3.28) and
endotoxin
3.14
geometric mean end point
antilog of the average of the logarithmic values with respect to the end points (3.11) from replicate
dilution series converted back to a base 10 number used to establish the central tendency or typical
value from a test solution
3.15
health care product
medical device, including in vitro diagnostic medical device, or medicinal product, including
biopharmaceutical
[SOURCE: ISO 11139:2018, 3.132]
3.16
indirect contact
medical device or medical device component through which a fluid or gas passes, prior to the fluid or
gas coming into physical contact with body tissue (in this case the medical device or medical device
component itself does not physically contact body tissue)
[SOURCE: ISO 10993-1:2018, 3.11]
3.17
inhibition
bacterial endotoxins test (BET) (3.1) anomaly in which a non-endotoxin related factor, usually attributable
to a characteristic of the test sample, elicits a test reaction less than the amount of endotoxin present
3.18
method suitability
inhibition/enhancement test
test used to determine whether a particular sample contains interfering factors that diminish its
accuracy by introducing enhancement (3.12) or inhibition (3.17) into the test system
3.19
interference
interfering factor observed in the performance of the test that exceeds the acceptable threshold for
a given bacterial endotoxins test (BET) (3.1) technique (e.g. positive product control that indicates a
detected endotoxin level less than 50 % or greater than 200 % or ±2 lambda)
3.20
intraocular, adj.
located or occurring within or administered through the eye
ISO 11737-3:2023(E)
3.21
interfering factors
non-endotoxin related factor, usually attributable to a characteristic of the test sample, that causes
inhibition (3.17) or enhancement (3.12)
3.22
intravascular, adj.
located or occurring within or administered through the heart or blood vessels
3.23
intralymphatic, adj.
located or occurring within or administered through a lymph vessel
3.24
intrathecal, adj.
located, or occurring within or administered through the space under the arachnoid membrane of the
brain or spinal cord
3.25
kinetic method
photometric quantitative techniques (turbidimetric or chromogenic) for bacterial endotoxins test (BET)
(3.1)
3.26
LAL reactive material
LAL-RM
Limulus amebocyte lysate reactive material
any non-endotoxin compound that will activate the Limulus amebocyte lysate (LAL) (3.28) clotting
cascade and cause enhancement (3.12)
3.27
lambda
λ
labelled sensitivity of a Limulus amebocyte lysate (LAL) (3.28) gel-clot reagent, expressed in EU/ml or,
for chromogenic or turbidimetric tests, the lowest point (endotoxin concentration) on the referenced
standard curve
3.28
Limulus amebocyte lysate
LAL
reagent extracted from amebocytes taken from hemolymph of the horseshoe crab, Limulus polyphemus,
that reacts with endotoxin, to form a gelatinous clot and is used to estimate endotoxin levels in bacterial
endotoxins test (BET) (3.1) methods
Note 1 to entry: The term LAL is sometimes used to describe Tachypleus amebocyte lysate (TAL) (3.41), as both
are similar lysates that are used in the BET. They also are often generically referred to as “lysate”.
3.29
lipopolysaccharide
LPS
Gram-negative bacterial cell wall component composed of lipid A, a core polysaccharide, and an O-side
chain
3.30
maximum valid dilution
MVD
maximum amount a sample can be diluted, or the total extraction volume used relative to the sensitivity
of a bacterial endotoxins test (BET) (3.1) in which the specified endotoxin limit (3.9) can be detected
ISO 11737-3:2023(E)
3.31
medical device
instrument, apparatus, implement, machine, appliance, implant, reagent for in vitro use, or software
material or other similar or related article, intended by the manufacturer to be used, alone or in
combination, for human beings, for one or more of the specific medical purpose(s) of:
— diagnosis, prevention, monitoring, treatment or alleviation of disease;
— diagnosis, monitoring, treatment, alleviation of or compensation for an injury;
— investigation, replacement, modification or support of the anatomy or of a physiological process;
— supporting or sustaining life;
— control of conception;
— disinfection of medical devices;
— providing information by means of in vitro examination of specimens derived from the human
body;
and does not achieve its primary intended action by pharmacological, immunological or metabolic
means, but which may be assisted in its intended function by such means
Note 1 to entry: Products which can be considered to be medical devices in some jurisdictions, but not in others
include:
— items specifically intended for cleaning or sterilization of medical devices;
— pouches, reel goods, sterilization wrap, and reusable containers for packaging of medical devices for
sterilization;
— disinfection substances;
— aids for persons with disabilities;
— devices incorporating either animal or human tissues, or both;
— devices for in vitro fertilization or assisted reproduction technologies.
[SOURCE: ISO 11139:2018, 3.166]
3.32
non-pyrogenic, adj.
not inducing a fever
Note 1 to entry: Describes an item or product that contains endotoxin levels that conform to specified limits.
3.33
out of specified limits
OSL
sample with a valid bacterial endotoxins test (BET) (3.1) result that exceeds a product endotoxin limit
(3.9) specification
Note 1 to entry: The term OSL applies only within the context of this document and does not imply compliance
with any other regulatory guidance dealing with out of specification (OOS) results.
3.34
product positive control
PPC
sample spiked with a known amount of endotoxin used for confirmation that the product being tested
is not subject to interfering factors (3.21)
ISO 11737-3:2023(E)
3.35
pyrogen
substance that induces a fever
3.36
pyrogenic, adj.
inducing a fever
Note 1 to entry: Describes an item or product that contains endotoxin levels above specified limits.
3.37
Reference Standard Endotoxin
RSE
US Pharmacopeia (USP) endotoxin reference standard that has a defined potency of 10 000 USP EUs per
vial
3.38
repeat test
analysis of additional product samples from a previously tested batch or another batch
3.39
retest
reanalysis of previously tested product samples or product sample preparation
3.40
standard control series
serial dilution series of Reference Standard Endotoxin (RSE) (3.37) or control standard endotoxin (CSE)
(3.4) used to verify Limulus amebocyte lysate (LAL) (3.28) sensitivity
3.41
Tachypleus amebocyte lysate
TAL
reagent extracted from amebocytes taken from hemolymph of the horseshoe crab, Tachypleus
tridentatus, which reacts with endotoxin, to form a gelatinous clot and is used to estimate endotoxin
levels in bacterial endotoxins test (BET) (3.1) methods
Note 1 to entry: The term TAL is sometimes used to describe Limulus amebocyte lysate (LAL) (3.28), as both are
similar lysates that are used in the BET. They also are often generically referred to as “lysate”.
3.42
turbidimetric technique
bacterial endotoxins test (BET) (3.1) methodology that quantifies or detects endotoxin on the basis of a
measured turbidity reaction proportional to the interaction of Limulus amebocyte lysate (LAL) (3.28)
and endotoxin
3.43
validation
confirmation, through the provision of objective evidence, that the requirements for a specific intended
use or application have been fulfilled
Note 1 to entry: The objective evidence needed for a validation is the result of a test or other form of determination
such as performing alternative calculations or reviewing documents.
Note 2 to entry: The word “validated” is used to designate the corresponding status.
Note 3 to entry: The use conditions for validation can be real or simulated.
[SOURCE: ISO 11139:2018, 3.313]
ISO 11737-3:2023(E)
3.44
verification
confirmation, through the provision of objective evidence, that specified requirements have been
fulfilled
Note 1 to entry: The objective evidence needed for a verification can be the result of an inspection or of other
forms of determination such as performing alternative calculations or reviewing documents.
Note 2 to entry: The word “verified” is used to designate the corresponding status.
[SOURCE: ISO 11139:2018, 3.314]
3.45
water for bacterial endotoxins test
WBET
purified water employable as a solvent, diluent, and/or extractant that is non-reactive with the lysate
employed at the detection limit of the reagent, and does not elicit interference (3.19) with methodology
in use (typically Limulus amebocyte lysate (LAL) (3.28) reagent water, water for injection, or other
appropriate solution meeting these requirements)
4 General requirements
4.1 The development, validation and routine control of products with acceptable endotoxin levels
are critical elements in the realization of some types of health care products. To ensure the consistent
implementation of the requirements specified in this document, the necessary processes shall be
established, implemented and maintained. Processes of particular importance in relation to the
development, validation and routine endotoxin control of a process include but are not limited to:
— control of documentation, including records,
— assignment of management responsibility,
— provision of adequate resources, including competent human resources and infrastructure,
— control of product provided by external parties,
— identification and traceability of product throughout the process, and
— control of non-conforming product.
NOTE ISO 13485 covers all stages of the life cycle of medical devices in the context of quality management
systems for regulatory purposes. National and/or regional regulatory requirements for the provision of health
care product can require the implementation of a full quality management system and the assessment of that
system by a recognized conformity assessment body.
4.2 A process shall be specified for the calibration of all equipment, including instrumentation for
test purposes, used in meeting the requirements of this document.
ISO 11737-3:2023(E)
5 Selection of products
5.1 General
5.1.1 The types of products required or labelled to be non-pyrogenic and the associated bacterial
endotoxin limits shall be determined and be consistent with the intended clinical application.
Products should not be labelled as ‘pyrogen free’ because complete freedom from bacterial endotoxins
cannot be demonstrated by testing due to the detection limits inherent in current test methods. The
term ‘non-pyrogenic’ should be used.
NOTE 1 See A.5.1.1 and Annex B for risks associated with endotoxins and for commonly used limits.
NOTE 2 National regulatory requirements can apply regarding non-pyrogenic labelling.
5.1.2 For some products, higher endotoxin limits can be justifiable, with additional supporting data
depending on the risk/benefit of the device. Likewise, for other products, more stringent limits can be
required (e.g. devices with intrathecal contact).
5.1.3 Product required or labelled to be non-pyrogenic shall require explicit substantiation employing
a suitable BET method. Such substantiation shall include at least one of the following:
— end-product testing for each batch;
— alternative-to-batch testing (see Clause 10 and Annex E).
5.1.4 All parts of products required or labelled to be non-pyrogenic shall be included in the testing
process. The exclusion of any part of the product shall be justified and documented (e.g. a handle or a
power cord).
5.1.5 There are health care products that have portions of the product that are sealed and as such do
not come into contact with the patient. Such portions of the product that do not have patient contact are
not required or intended to be non-pyrogenic, and may be excluded from endotoxin testing.
5.1.6 For products for which a claim of non-pyrogenicity applies only to a portion of the product (e.g.
the fluid path in an administration set for intravenous infusion), endotoxin testing does not apply to
the portions of product not intended to be non-pyrogenic. A statement about the portion of the product
to which the claim applies (such as ‘non-pyrogenic fluid path’) shall be supported by appropriate
evaluation of components and surfaces relevant to that portion of the product.
5.1.7 For multi-component kit products for which a claim of either non-pyrogenicity or label claim, or
both, applies to only a portion of the kit, endotoxin testing does not apply to the portions of the kit not
intended to be non-pyrogenic. The non-pyrogenic portions of the kit shall be supported by appropriate
documented rationale.
5.2 Selection of product units
5.2.1 The sampling criteria for selection of product units for endotoxin testing are based on the
premise that the manufacturing process, as well as the processes identified in 4.1, are controlled (refer
to A.2).
NOTE See Annex D for guidance on in-process monitoring of manufacturing processes or component testing.
ISO 11737-3:2023(E)
5.2.2 The selection of product units for testing shall be based on criteria defined in a sampling plan
that includes an assessment of components and processing. This rationale should consider the following:
a) applicable regulatory requirements;
b) assessment of risk;
c) historical performance;
d) manufacturing process validation;
e) statistical considerations.
5.2.3 There are two types of sampling plans: batch testing and alternatives to batch testing.
5.2.3.1 For batch testing, non-pyrogenicity is confirmed through the use of end-product testing.
The batch may be defined as each production lot or a product intended or purported to be uniform in
character and quality produced during a specified cycle of manufacture. This should be supported with
documented rationale or risk assessment (refer to A.5.2 for guidance on the number of samples).
5.2.3.2 Alternatives to batch testing may be used if it has been demonstrated that the manufacturing
process and materials are suitably controlled. If alternatives to batch testing are performed, a risk
assessment to evaluate the criteria used to establish the sampling plan shall be performed (see
Clause 10 and Annex E).
5.2.4 Samples selected for testing shall include all factors that can affect or contribute to the levels of
endotoxin.
5.2.5 Samples used for endotoxin testing can be selected from routine production, products that have
been rejected for other production quality issues that have no effect on endotoxin content, or surrogate
samples that are representative of the full manufacturing process and representative of product
endotoxin levels.
5.2.6 Samples may be obtained prior to sterilization (pre-sterilization) or after sterilization (post-
sterilization). Post-sterilization samples encompass all the factors that can affect the product or
the endotoxin test. When pre-sterilization samples are selected for testing, the acceptability of the
samples in representing the endotoxin level on sterilized product shall be justified and documented.
The program for ongoing testing should consistently reflect either pre- or post-sterilization samples.
Guidance is provided in A.5.2.6 for assessing the acceptability of pre-sterilization testing.
NOTE For products that support microbial growth, see A.5.2.6.
5.2.7 In the testing of multi-component kits (procedure packs) or sets of individual products within
the same sterile barrier system, depending upon how the product is used, there are instances where
each component may be evaluated individually and other instances where the entire contents may
be considered as a single entity. Consideration of a set or a kit as a single unit shall address sample
preparation in adherence to method requirements and the applicable endotoxin limit. The total volume
of extraction fluid used for the subcomponents should not exceed the maximum extraction volume
determined by the MVD.
6 Methods for BET
6.1 General
6.1.1 There are currently three commonly accepted BET techniques. The choice of technique
should be based upon an assessment of the laboratory’s capability, experience, sample throughput
ISO 11737-3:2023(E)
requirements, data handling requirements, and the nature of the test sample. The current techniques
and associated methods are:
a) gel-clot techniques: limit test and assay methods;
b) chromogenic photometric technique: end point method;
NOTE A turbidimetric end point is available but is not commonly used.
c) chromogenic and turbidimetric photometric techniques: kinetic methods.
Information on each of these methods is presented in A.6.
6.1.2 The selected method shall be determined to be suitable as specified in Clause 7. Continued
suitability shall be confirmed as specified in Clause 9.
6.2 Consideration of an applicable endotoxin limit
6.2.1 Endotoxin limit
The endotoxin limit defines the maximum allowable amount of endotoxin present on the product or in
a product extract solution.
6.2.2 Calculation of endotoxin limit for the extract solution
The endotoxin limit for the extract solution in endotoxin units per ml (EU/ml) shall be calculated as
shown in Formula (1) as:
()KN()
(1)
V
where
K is the product endotoxin limit;
N is the number of devices tested;
V is the total volume of the extract or rinse (ml) that can be adjusted for the size and configu-
ration of the device(s).
Product endotoxin limits can be reported in terms of EU/ml, provided an appropriate specified volume
for rinsing/immersing has been determined to not exceed the MVD. Reporting the results in EU/device
takes into account the initial extraction volume.
6.2.3 Maximum valid dilution (MVD)
6.2.3.1 Products can sometimes interfere with a BET, resulting in inhibition or enhancement due to
the presence of interfering factors. The interfering factors shall be assessed. A common technique us
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