Pyrogen and Endotoxins Testing: Questions and Answers: Guidance for Industry

Administrative guidance, not enacted legislation. This captured publisher edition preserves the listed document and attachments; later developments are not independently consolidated.

Guidance for Industry
Pyrogen and Endotoxins Testing:
Questions and Answers
(Edition 2)
U.S. Department of Health and Human Services
Food and Drug Administration
Center for Drug Evaluation and Research (CDER)
Center for Biologics Evaluation and Research (CBER)
Center for Veterinary Medicine (CVM)
Center for Devices and Radiology Health (CDRH)
Office of Regulatory Affairs (ORA)
March 2026
Compliance

Contains Nonbinding Recommendations
Guidance for Industry
______________________________________________________________________________
Pyrogen and Endotoxins Testing:
Questions and Answers (Edition 2)
Additional copies are available from:
Office of Communication, Division of Drug Information
Center for Drug Evaluation and Research
Food and Drug Administration
Phone: 855-543-3784 or 301-796-3400; Fax: 301-431-6353; Email: druginfo@fda.hhs.gov
https://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/default.htm
or
Office of Communication, Outreach and Development,
Center for Biologics Evaluation and Research,
Food and Drug Administration
Phone: 800-835-4709 or 240-402-8010
Email: industry.biologics@fda.hhs.gov
http://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidanc
es/default.htm
or
Office of Policy
Center for Devices and Radiological Health
Food and Drug Administration
Email: CDRH-Guidance@fda.hhs.gov
https://www.fda.gov/medical-devices/device-advice-comprehensive-regulatory-
assistance/guidance-documents-medical-devices-and-radiation-emitting-products
or
Policy and Regulations Staff, Center for Veterinary Medicine;
Food and Drug Administration
Email: Ask CVM@fda.hhs.gov
http://www.fda.gov/AnimalVeterinary/GuidanceComplianceEnforcement/GuidanceforIndustry/def
ault.htm

Contains Nonbinding Recommendations
i
TABLE OF CONTENTS
I. 	INTRODUCTION..................................................................................................................... 1
II. 	BACKGROUND ....................................................................................................................... 2
III. 	QUESTIONS AND ANSWERS ............................................................................................... 2
1. 	How do I establish sampling for in-process testing and finished product release? .............. 2
2. 	When is retesting appropriate? ................................................................................................ 3
3. 	Is sample storage and handling important? ............................................................................ 3
4. 	Can finished product samples for analysis of bacterial endotoxins be pooled into a
composite sample prior to analysis? ........................................................................................ 3
5. 	May a firm use alternative assays to those in the USP for a compendial article?................ 4
6. 	What is the best process for transitioning from one alternate bacterial endotoxins test
(BET) method to another?........................................................................................................ 5
7. 	What happened to the endotoxins limit table in Appendix E of the 1987 Guidance? ......... 6
8. 	How can Quality by Design concepts support endotoxins limits? ........................................ 6
9. 	When is the USP Chapter <151> “Pyrogen Test” (the rabbit pyrogen test) appropriate? .. 6
10. 	How would an appropriate endotoxins limit be determined for a veterinary product that
targets multiple species? ........................................................................................................... 7
11. 	What are the endotoxins limits for medical devices? ............................................................. 7
12. 	What is the FDA’s expectation for regular screening of therapeutic drug products? ........ 8
13. 	Are control standard endotoxins still acceptable for use in running bacterial endotoxins
tests? ........................................................................................................................................... 8

Contains Nonbinding Recommendations
1
Guidance for Industry1 Pyrogen and Endotoxins Testing —
Questions and Answers (Edition 2)
This guidance represents the current thinking of the Food and Drug Administration (FDA or Agency)
on this topic. It does not establish any rights for any person and is not binding on FDA or the public.
You can use an alternative approach if it satisfies the requirements of the applicable statutes and
regulations. To discuss an alternative approach, contact the FDA staff responsible for this guidance
as listed on the title page.
I. 	INTRODUCTION
This guidance provides recommendations for biological product, drug, and device firms on FDA’s
current thinking concerning the testing recommendations and acceptance criteria in the United States
Pharmacopeia (USP) Chapter <85> “Bacterial Endotoxins Test”,2 USP Chapter <161> “Medical
Devices ‒ Bacterial Endotoxin and Pyrogen Tests”,3 and the Association for the Advancement of
Medical Instrumentation (AAMI) ST72:2002/R2010, Bacterial Endotoxins—Test Methodologies,
Routine Monitoring, and Alternatives to Batch Testing (AAMI ST72).4, 5 These three documents
describe the fundamental principles of the gel clot, photometric, and kinetic test methods, and
recommend that appropriate components and finished products be tested for the presence of pyrogens
and endotoxins.
This guidance does not cover the entire subject of pyrogen and endotoxins testing. 	Instead, it
addresses those issues that may be subject to misinterpretation and are not covered in compendial
procedures or in currently available guidance documents. 	You should already have a thorough
understanding of these documents when using this guidance.
FDA’s guidance documents, including this guidance, do not establish legally enforceable
responsibilities. 	Instead, guidances describe the Agency’s current thinking on a topic and should be
viewed only as recommendations, unless specific regulatory or statutory requirements are cited. 	The
use of the word should in Agency guidances means that something is suggested or recommended, but
not required.
1 This guidance has been prepared by the Division of Manufacturing and Product Quality, Office of Compliance, in the
Center for Drug Evaluation and Research (CDER) in cooperation with the Center for Biologics Evaluation and
Research (CBER), the Center for Veterinary Medicine (CVM), the Center for Devices and Radiological Health
(CDRH), and the Office of Regulatory Affairs (ORA) at the Food and Drug Administration.
2 United States Pharmacopeia (USP), 2018, Chapter <85> Bacterial Endotoxins Test.
3 USP, 2017, Chapter <161>, Medical Devices ‒ Bacterial Endotoxin and Pyrogen Tests.
4 Association for the Advancement of Medical Instrumentation (AAMI), 2002/R2010, Bacterial Endotoxins — Test
Methodologies, Routine Monitoring, and Alternative to Batch Testing.
5 Veterinary drug requirements parallel the human drug requirements in safety evaluation for pyrogenicity (21 CFR
514.1(b)(5)(v), and 514.1(b)(5)(vii)(b)).

Contains Nonbinding Recommendations
2
II. 	BACKGROUND
For more than 30 years, FDA has accepted the use of a Limulus Amoebocyte Lysate (LAL) test for
endotoxins in lieu of the rabbit pyrogens test. In a November 4, 1977, Federal Register notice (42 FR
57749), FDA described conditions for using LAL as a finished product test.6 	By 1983, FDA
indicated in guidance that an LAL test could be used as a finished product test for endotoxins. 	These
tests were described in a series of draft and final guidance documents. 	Guideline on Validation of the
Limulus Amebocyte Lysate Test as an End-Product Endotoxin Test for Human and Animal Parenteral
Drugs, Biological Products, and Medical Devices, was published in 1987 (the 1987 Guidance) and
Version 1 of this Guidance was published in 2012.
FDA has found that the published USP and AAMI documents describing methods and calculation of
pyrogen and endotoxins testing limits7 provide industry with appropriate information. The Agency
has withdrawn the 1987 Guidance because it no longer reflects the Agency’s current thinking on the
topic. 	However, because the compendial chapters and standards do not address certain regulatory
perspectives, FDA is providing supplemental information in this guidance to explain our current
thinking regarding the submission and maintenance of pyrogen and endotoxins testing for FDA-
regulated products.
III. 	QUESTIONS AND ANSWERS
1. 	How do I establish sampling for in-process testing and finished product release?
The current good manufacturing practice (CGMP) regulations for finished pharmaceuticals
require scientifically sound and appropriate sampling8 and the medical device quality
management system regulation requires validation using statistical techniques, as appropriate,
with rationale for sample sizes.9 Sampling information is addressed in AAMI ST72, but not
USP Chapter <85>. Firms should include sampling as part of their application documentation.
When sampling, firms should consider the potential for contamination in raw materials, in-
process materials, and the finished product. 	Specifically, firms should take into account
aspects of the manufacturing design, including consistency of a manufacturing process, impact
of in-process hold times, endotoxins removal steps, and finished product endotoxins
specifications. 	Sampling should be considered dynamic; firms should begin with maximum
coverage and adjust their procedures as they gain confidence in the prevention of endotoxins
in their manufacturing processes. 	Firms should update their regulatory filings when adjusting
6 “Licensing of Limulus Amebocyte Lysate, Use as an Alternative for Rabbit Pyrogen Test” (42 FR 57749, November 4,
1977).
7 USP, 2018, Chapter <85> Bacterial Endotoxins Test; USP, 2017, Chapter <161> Medical devices ‒ Bacterial Endotoxin
and Pyrogen Tests; Association for the Advancement of Medical Instrumentation (AAMI), 2002/R2010, Bacterial
Endotoxins—Test Methodologies, Routine Monitoring, and Alternatives to Batch Testing.
8 See 21 CFR 211.160.
9 See 21 CFR 820.7(b), which incorporates by reference ISO 13485:2016(E), Medical devices—Quality management
systems—Requirements for regulatory purposes, Third edition, March 1, 2016. ISO 13485:2016(E) Clause 7.5, and
Clause 8 and its subclauses require manufacturers to document verification plans, validation plans, and procedures for
validation of processes that include, as appropriate, statistical techniques with rationale for sample sizes.

Contains Nonbinding Recommendations
3
procedures. 	For drugs and biological products, these in-process changes should be submitted
in a supplement or annual report, as appropriate.10 For devices, a 30-day notice11 may be
appropriate.12
2. 	When is retesting appropriate?
When conflicting results occur within a test run, firms should consult USP Chapter <85>, Gel-
Clot Limit Test, Interpretation, for guidance on repeat testing. 	As specified in Chapter <85>,
if the test failure occurred at less than the maximum valid dilution (MVD), the test should be
repeated using a greater dilution not exceeding the MVD. 	A record of this failure should be
included in the laboratory results. 	If a test is performed at the MVD and an out-of-
specification (OOS) test result occurs that cannot be attributed to testing error, the lot should
be rejected.13 	All testing procedures, including those for retesting within the above limits,
should be specified in advance in written standard operating procedures approved by the
firm’s quality control unit.
3. 	Is sample storage and handling important?
Yes. 	The ability to detect endotoxins can be affected by storage and handling. 	Firms should
establish procedures for storing and handling (which includes product mixing) samples for
bacterial endotoxins analysis using laboratory data that demonstrate the stability of assayable
endotoxins content. 	Protocols should consider the source of endotoxins used in the study,
bearing in mind that purified bacterial endotoxins might react differently from native sources
of endotoxins.
4. 	Can finished product samples for analysis of bacterial endotoxins be pooled into a
composite sample prior to analysis?
Yes. 	With some exceptions (see below), finished drug product units may be pooled into a
composite sample and assayed for bacterial endotoxins. 	The composite sample may be
represented by the entire unit or partial aliquots (equal volumes) of finished product containers
from one manufactured lot of aqueous-based pharmaceuticals. 	Pooling would generally be
accepted for small-volume parenterals (those with volumes of 100 mL or less) as long as the
MVD is adjusted to a proportional, lower value because of the potential for diluting a unit
containing harmful levels of endotoxins with other units containing lower, less harmful, levels
of endotoxins. 	This “adjusted MVD” is obtained by dividing the MVD computed for an
individual sample by the total number of samples to be pooled. 	FDA suggests pooling no
more than three units per composite in keeping with the concept of testing representative
10 See 21 CFR 314.81(b)(2)(iv), 314.98, and 601.12.
11 See 21 CFR 814.39(f).
12 For guidance regarding when and how to submit a 30-day notice, please refer to guidance for industry and FDA staff
30-Day Notices, 135-Day Premarket Approval (PMA) Supplements and 75-Day Humanitarian Device Exemption
(HDE) Supplements for Manufacturing Method or Process Changes (December 2019).
13 For guidance regarding how to examine results that are above acceptable limits, please refer to guidance for industry
Investigating Out-of-Specification (OOS) Test Results for Pharmaceutical Production (May 2022). Although this
guidance is not intended to address biological assays, many of the concepts in the guidance are applicable to bacterial
endotoxins testing..

Contains Nonbinding Recommendations
4
beginning, middle, and end finished product containers. 	If this reduction in MVD results in
an inability to overcome product-related assay interference because of an insufficient dilution,
then the samples should be tested individually.
Finished medical devices may also be pooled into a composite sample and assayed for
bacterial endotoxins. 	Testing for medical devices should be conducted using rinsing/eluting
and sampling techniques as described in ISO 10993-114 and ISO 10993- 12,15 as also used for
inhibition/enhancement. 	Sampling can be adjusted for special situations. 	After a suitable
eluate/extract pool is obtained from a finished production lot, this pooled extract should be
kept under conditions appropriate for stability until it is tested in duplicate.
FDA recommends that pooled samples be a composite of aseptically removed aliquots (after at
least 30 seconds of vigorous mixing) from each of the product containers.16 	In this way, the
original, individual containers will be available for possible retesting in the event the pooled
sample displays an OOS result.
Some product types should not be pooled. 	Two examples are drug products that have an
initial low MVD (see discussion above of “adjusted MVD”) and products that are
manufactured as a suspension, because sample aliquot homogeneity may present significant
interference issues. 	FDA also does not recommend pooling in-process samples from different
in-process stages of the manufacturing process because it may be difficult to ensure the
homogeneity of these materials.
5. 	May a firm use alternative assays to those in the USP for a compendial article?
Yes, firms may use alternative methods and/or procedures if they provide advantages in terms
of accuracy, sensitivity, precision, selectivity, or adaptability to automation or computerized
data reduction, and in other special circumstances. 	Such alternative procedures and methods
should be validated as described in the USP General Chapter <1225>, “Validation of
Compendial Procedures”,17, 18 and should be shown to achieve equivalent or better results.19
When a difference appears or in the event of a dispute, the final decision is made based upon
the USP compendial gel-clot method unless otherwise indicated in the monograph for the
product being tested.20
Below is an example of an alternative assay.
Monocyte Activation Type Pyrogen Test
Product-specific validation is necessary to establish whether a particular test substance or
material is appropriate for evaluation of the monocyte activation method. 	The validation
should include, but is not limited to, interference testing, accurate detection of pyrogen in
14 ISO 10993-1:2009 Biological evaluation of medical devices Part 1: Evaluation and testing in the risk management
process.
15 ISO 10993-12:2007 Biological evaluation of medical devices Part 12: Sample preparation and reference materials.
16 See 21 CFR 211.84(c)(3).
17 USP General Notices and Requirements 6.30 Alternative and Harmonized Methods and Procedures.
18 USP, 2017, Chapter <1225> Validation of Compendial Procedures.
19 USP General Notices and Requirements 6.30 Alternative and Harmonized Methods and Procedures.
20 USP, 2018, Chapter <85> Bacterial Endotoxins Test.

Contains Nonbinding Recommendations
5
individual test samples, and, for devices, ability of test system to provide direct contact to the
monocytes.
6. 	What is the best process for transitioning from one alternate bacterial endotoxins
test (BET) method to another?
The transition between tests that measure the same entity can be made by comparing the two
tests to verify the equivalence of the new method.21 	The comparison of the limit of detection
and inhibition/enhancement is fundamental. The sensitivity of the new method can be
evaluated on spiked product samples.22 	In addition to using spiked samples, a battery of field
samples of product found to be positive may be a good source for comparing results from the
methods. 	The method validation should also attempt to address the variability found in the
normal use of the method and the manufacturing environment (e.g., source materials or
seasonal changes).23
For drug, animal drug, and biological products, the transition to a new method should be
submitted in a prior approval supplement (PAS). 	Alternatively, once a firm has established a
general method for making the transition between tests, it may submit the method for review
in a PAS—comparability protocol (CP). 	The CP should describe, in detail, the methods used
to transition between assays and the acceptance criteria used to establish the equivalence of the
new method. 	After approval of the CP, results of implementation of the CP may be reported
in a reduced reporting category (21 CFR 314.81, 314.98, and 601.12). 	The firm should
maintain the study protocol, final report, and all data at the facility for FDA review. 	The firm
should confirm the filing process with the appropriate review division before submitting a CP.
For Class III devices, the transition to a new assay may be appropriate for a 30-day notice filed
under 21 CFR 814.39(f).24 	Manufacturing changes for Class I and II devices must be in
accordance with the quality management system regulation, 21 CFR part 820, which
incorporates ISO 13485:2016 by reference. 	Design control, production and process control
requirements can be found at ISO 13485:2016 Clause 7, including subclauses 7.3 and 7.5.
For devices, a 30-day notice may be appropriate for changes to quality control testing used on
incoming components, raw materials, the in-process device, or the finished device, including
performing end-product pyrogen testing on nonsterile samples prior to sterilization.25
Manufactures of medical devices should demonstrate a sensitivity that is consistent with the
route of administration for the device and the type of body contact. Manufacturers may be
able to use another endotoxin test after demonstrating a reproducible correlation between
methods and the USP reference standard.
21 USP, 2017, Chapter <1225> Validation of Compendial Procedures.
22 International Conference on Harmonisation (ICH) Q2(R1) Validation of Analytical Procedures: Text and Methodology
(1994); USP, 2017, Chapter <1225> Validation of Compendial Procedures.
23 Thompson, M. et al., 2002, “Harmonized Guidelines for Single-Laboratory Validation of Methods of Analysis: IUPAC
Technical Report,” Pure Appl. Chem., Vol. 74, No. 5, pp. 835-855.
24 See guidance for industry and FDA staff, Modifications to Devices Subject to Premarket Approval (PMA) - The PMA
Supplement Decision-Making Process (December 2008).
25 See 21 CFR 814.39(f).

Contains Nonbinding Recommendations
6
7. 	What happened to the endotoxins limit table in Appendix E of the 1987
Guidance?
The endotoxins limit table is out of date due to the increase in numbers of dosage (regimes)
and drug strengths since the publication of the 1987 Guidance. 	The appropriate way to
establish the endotoxins limit is to use the calculation methods provided in the USP or AAMI
standards. 	Monograph limits may also not account for current product strengths or dosage
regimes; these should also be checked using the calculations recommended in the standards.
If there are several components in a finished product, then the overall endotoxins limit for
parenterally-administered products should not exceed the overall threshold limit specified in
the USP <85> “Bacterial Endotoxins Test”, regardless of an individual component endotoxins
limit. 	Intrathecally-administered products, ophthalmics, or devices (see question 11 for
devices) may have endotoxins limits that are not based on the calculation for parenterally
administered products. 	FDA encourages firms to check with the appropriate office or review
division about these products.
8. 	How can Quality by Design concepts support endotoxins limits?26
When implementing Quality by Design concepts, the strategy for endotoxins testing should be
based upon product and process understanding in combination with risk management to ensure
consistent final product quality. 	The appropriate in-process testing should be used to evaluate
the production process areas at risk of endotoxins formation or incursion. 	Many firms already
have programs for monitoring incoming ingredients and components, including the processing
water, for endotoxins contamination. 	The finished product release specification should be
considered when determining in-process limits for each phase of manufacturing tested. 	For
purposes of evaluating the relative risk of product contamination, quantitative testing may be
preferable to limit testing to facilitate product quality trending and to identify and correct
excursions before they exceed the specification and cause product failure. 	An endotoxins limit
should be justified on a case-by-case basis, and will be evaluated as a part of each relevant
marketing application or supplement.
9. 	When is the USP Chapter <151> “
Pyrogen Test” (the rabbit pyrogen test)
appropriate?
For certain biological products, 21 CFR 610.13(b) requires a rabbit pyrogen test. 	The
requirement in 21 CFR 610.13(b) may be waived if a method equivalent to the rabbit pyrogen
test is demonstrated in accordance with 21 CFR 610.9.
For human and animal drugs, some USP monographs still require a rabbit pyrogen test. Even
with such monographs, a firm may substitute an endotoxins test or alternative cell- based test
if the firm can demonstrate equivalent pyrogen detection. 	The appropriate FDA review
division will consider alternative methods, such as monocyte activation, on a case-by-case
basis.
26 ICH Harmonised Tripartite Guideline, Q8(R2) Pharmaceutical Development, Step 4 Version (Nov 2009).

Contains Nonbinding Recommendations
7
For devices and drug materials, firms should assess the risk of the presence of non- endotoxin
pyrogens. 	If the risk assessment indicates that non-endotoxin pyrogens may be present, it may
be more appropriate to use the rabbit pyrogen test.
Bacterial endotoxins assays are subject to a variety of interferences related to the physical and
chemical properties of the test article. 	Where such interferences cannot be mitigated through
sample dilution (up to the MVD) or other validated means of sample preparation, firms should
use the rabbit pyrogen test.
10. 	How would an appropriate endotoxins limit be determined for a veterinary
product that targets multiple species?
For a veterinary product labeled for use in multiple species, the limit should be based on the
maximum product dose used on the smallest species. 	If the label indicates that the product
may be used on juvenile and adult animals, the juvenile is considered the worst case. 	If the
weight of the animal is required to calculate the dose, firms should use an average weight for
that species. 	For a listing of some average animal weights, see the FDA guidance for industry
Demonstrating Bioequivalence for Soluble Powder Oral Dosage Form Products and Type A
Medicated Articles Containing Active Pharmaceutical Ingredients Considered to Be Soluble in
Aqueous Media (June 2023). 	For animal weights not listed in that guidance, please contact
FDA’s Center for Veterinary Medicine.
11. 	What are the endotoxins limits for medical devices?
The Center for Devices and Radiological Health (CDRH) has adopted the USP Endotoxin
Reference Standard and endotoxin limits for medical device extracts expressed in EU/mL as
stated in USP Chapter <161> “Medical devices ‒ Bacterial Endotoxin and Pyrogen Tests”.
This USP chapter provides the endotoxin limits for medical devices within its scope. 	These
endotoxin limits for a medical device are dependent on the intended use of the device and
what the device contacts (e.g., blood, the cardiovascular system, cerebrospinal fluid,
intrathecal routes of administration, permanently implanted devices, and devices implanted
subcutaneously).27
For medical devices, using the extraction volume recommendations described below, the
endotoxin limit is 0.5 EU/mL or 20 EU/device for products that directly or indirectly contact
the cardiovascular system and lymphatic system. 	For devices in contact with cerebrospinal
fluid, the endotoxin limit is 0.06 EU/mL or 2.15 EU/device. 	For devices that are in direct or
indirect contact with the intraocular environment, a lower endotoxins limit may apply. Please
contact the appropriate review division for specific recommendations.
The process of preparing an eluate/extract for testing may vary from device to device. Some
medical devices can be flushed, some may have to be immersed, while others may need
disassembly. 	Unless otherwise directed by another compendial standard, our recommended
rinse volumes include the following: (1) each of the 10 test units should be rinsed with 40 mL
of non-pyrogenic water; (2) for unusually small or large devices, the surface area of the device
that contacts the patient may be used as an adjustment factor in selecting the rinse or extract
volume. 	The endotoxins limit can be adjusted accordingly. 	In any case, the rinse/extract
27 USP, 2017, Chapter <161> Medical devices ‒ Bacterial Endotoxin and Pyrogen Tests.

Contains Nonbinding Recommendations
8
procedure should not result in a greater dilution of endotoxin than recommended in USP <85>.
For inhibition/enhancement testing, both the rinse/extract solution and the device
eluate/extract should be tested.
Examples of medical devices with testing or interference challenges include devices that are
coated with anticoagulant, contain heavy metals, or that have particulates. 	In these situations,
treatments for interferences can include digestion, dilution, and addition of buffers,
centrifugation, or filtration.
During the same surgical procedure or placement in the same surgical site, multiple units of
the same device from one manufacturer should generally meet the same endotoxins limit as a
single device administered during the procedure. 	In instances where multiple units of the
same device are known or intended for use in a single procedure, manufacturers should justify
any deviation from the overall endotoxins limit identified in this guidance.
When a manufacturer of medical devices plans to use testing that deviates significantly from
this guidance or recognized standards, a premarket notification (510(k)) under section 510(k)
of the Federal Food, Drug, and Cosmetic Act (the Act) or a premarket approval application
(PMA) supplement under section 515 of the Act may be required. 	Significant deviations
include, but are not necessarily limited to: 	higher endotoxin concentration release criteria,
sampling from fewer than three (3) lots for inhibition/enhancement testing, lesser sensitivity to
endotoxins, and a device rinsing protocol resulting in greater dilution of endotoxins than that
recommended in this guidance.
12. 	What is the FDA’s expectation for regular screening of therapeutic drug
products?
Ideally, the undiluted product should be screened as long as there is no interfering/enhancing
property within the test. 	However, in some product formulations, the ingredients interfere
with the test. 	For such formulations, the USP recommends that the product be diluted to
overcome interference or enhancement properties. 	The calculated MVD is the dilution of a
sample at which the endotoxins limit would be detected, but it should not be the regular testing
dilution. 	When product interference is encountered during development, FDA recommends
that the firm determine the lowest product dilution that would neutralize the interfering
condition.
FDA recommends that firms begin subsequent product screening at a product dilution just
above the level that neutralized the interference. 	For example, if the product has an MVD of
1:100, and the product displays inhibition at the 1:10, but not at the 1:20, it may be best to
screen product at 1:30. 	If bacterial endotoxins are detected at this level, then the firm should
conduct full enumeration with the product to titrate the true amount of endotoxins.
13. 	Are control standard endotoxins still acceptable for use in running bacterial
endotoxins tests?
Control standard endotoxins (CSEs) are endotoxin preparations other than the international or
national reference standards that are traceable in their calibration to the international reference
endotoxins standard. 	CSEs may be secondary or tertiary standards and are usually
manufactured and certified by a reagent manufacturer for use with a specific lot of reagent

Contains Nonbinding Recommendations
9
under defined assay conditions. 	CSEs have become an accepted source for preparation of
standard curve calibrators and as assay controls, and have provided a cost saving to end users
and helped to preserve the inventory of primary standards. 	FDA encourages the continued
use of CSEs that are suitably calibrated to the international reference endotoxins standard.