Guidance for Industry 

Pyrogen and Endotoxins Testing: 

Questions and Answers 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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 Radiological Health (CDRH) 

Office of Regulatory Affairs (ORA) 

 
June 2012 

Compliance 
 
 
   
Guidance for Industry 

Pyrogen and Endotoxins Testing: 

Questions and Answers 

 
Additional copies are available from: 

Office of Communications, Division of  Drug Information,  WO51, Room  2201 
 
10903 New Hampshire Ave., Silver Spring, MD   20993 
  
Phone: 301-796-3400;  Fax: 301-847-8714 
 
druginfo@fda.hhs.gov 
  
http://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/default.htm  
or 

 Office of Communication, Outreach  and Development, HFM-40  

Center for Biologics Evaluation and Research, Food  and Drug  Administration 

1401 Rockville Pike, Rockville, MD   20852-1448  

ocod@fda.hhs.gov 

http://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/default.htm    
(Tel)  800-835-4709 or 301-827-1800 
 
or 

Office of Communication, Education and Radiation Programs 

Division of  Small Manufacturers, International  and Consumer Assistance 
 
Center for Devices and Radiological  Health; Food  and Drug Administration 

10903 New Hampshire Ave., WO66-4613, Silver Spring, MD   20993 
  
http://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/GuidanceDocuments/default.htm  
Email: dsmica@cdrh.fda.gov, Fax:  301.827.8149 
 
(Tel)  Manufacturers Assistance: 800.638.2041 or 301.796.7100,  International Staff Phone: 301.796.5708 
 
or 

Communications Staff, HFV-12 

Center for Veterinary Medicine; Food  and Drug  Administration 

7519  Standish  Place, Rockville, MD  20855 
 
(Tel) 240-276-9300 
 
http://www.fda.gov/AnimalVeterinary/GuidanceComplianceEnforcement/GuidanceforIndustry/default.htm  
 
 
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 Radiological Health (CDRH) 

Office of Regulatory Affairs (ORA) 

 
June 2012 

Compliance 
 
 
   
TABLE OF CONTENTS 

 
 
 
I.   INTRODUCTION............................................................................................................. 1 
 
II.   BACKGROUND ............................................................................................................... 2 
 
III.   QUESTIONS AND ANSWERS....................................................................................... 2 
 
1. 	  How do I establish a sampling plan 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?.............................................................................................4
  
5. 	  May a firm use alternative assays to those in the USP for a compendial article?....................5
  
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?.............................................7
  
9. 	  When is the USP Chapter <151> Pyrogenicity Test (the rabbit pyrogen test) appropriate? .7
  
10. 	  How would an appropriate endotoxins limit be determined for a veterinary product that 

targets multiple species?................................................................................................................8
  
11. 	  What are the endotoxins limits for medical devices?..................................................................8
  
12. 	  What is the FDAs expectation for regular screening of therapeutic drug products? ............9 
 
13. 	  Are control standard endotoxins still acceptable for use in running BETs?..........................10
  
   
Contains Nonbinding Recommendations 
 
1
 
Guidance for Industry  
Pyrogen and Endotoxins Testing  

Questions and Answers 

 
 
 
This guidance represents the Food and Drug Administration's (FDA's) current thinking on this topic.  It 
does not create or confer any rights for or on any  person and does not operate to bind FDA or the public.  
You can use an alternative approach if the approach satisfies the requirements of the applicable statutes 
and regulations.  If you want to discuss an alternative approach, contact the FDA staff responsible for 
implementing this guidance.  If you cannot identify the appropriate FDA staff, call the appropriate 
number listed on the title page of this guidance.  
 
 
 
I.  INTRODUCTION  
 
This guidance provides recommendations for biological product, drug, and device firms on 
FDAs current thinking concerning the testing recommendations and acceptance criteria in the 
United States Pharmacopeia (USP) Chapter <85> Bacterial Endotoxins Test,2 USP Chapter 
<161> Transfusion and Infusion Assemblies and Similar Medical Devices,3 and the Association 
for the Advancement of Medical Instrumentation (AAMI) ST72:2002/R2010, Bacterial 
EndotoxinsTest 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. 
 
                                                 
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), 2011, Chapter <85>, Bacterial Endotoxins Test.    

3 USP, 2011, Chapter <161>, Transfusion and  Infusion Assemblies and Similar Medical Devices.   

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 
 
FDAs guidance documents, including this guidance, do not establish legally enforceable 
responsibilities. Instead, guidances describe the Agencys 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.  
 
 
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.  The 
last guidance document, 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).   
 
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.  We also note the continued development of USP Chapters <85> and <161> and 
FDA guidance documents.  The Agency has withdrawn the 1987 Guidance because it no longer 
reflects the Agencys 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 a sampling plan for in-process testing and finished product 
release? 
 
The current good manufacturing practice (CGMP) regulations for finished 
pharmaceuticals and the medical device quality system regulations require development 
of controls that include scientifically sound and appropriate sampling plans.8,9    
 
                                                 
6 Licensing of Limulus Amebocyte Lysate, Use as an Alternative for Rabbit Pyrogen Test (42  FR 57749, 

November 4, 1977). 

7 USP, 2011, Chapter <85>, Bacterial Endotoxins Test; USP, 2011, Chapter <161>, Transfusion and  Infusion  

Assemblies and Similar Medical Devices, Association for the Advancement of Medical Instrumentation (AAMI), 
 
2002/R2010, Bacterial EndotoxinsTest Methodologies, Routine Monitoring, and  Alternatives to Batch Testing.   

8 See 21 CFR 211.160. 

9 See 21 CFR 820.70,  and  21 CFR 820.250.  
 
  2
  Contains Nonbinding Recommendations 
 
Sampling plan information is addressed in AAMI ST72, but not USP Chapter <85>.  
Firms should include a sampling plan as part of their application documentation.  In the 
sampling plan, 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. The sampling plan should be considered dynamic; firms 
should begin with maximum coverage and adjust their sampling plans as they gain 
confidence in the prevention of endotoxins in their manufacturing processes.  Firms 
should update their regulatory filings when adjusting sampling plans.  For drugs and 
biological products, in-process changes to sampling plans are annual reportable 
changes.10  For devices, a 30-day notice11 may be appropriate for in-process changes to 
the sampling plan.12  
 
2.   When is retesting appropriate? 
 
When conflicting results occur within a test run, firms should consult USP Chapter <85>, 
Gel Clot Limits 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 firms 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 
                                                 
10 See 21 CFR 314.81(b)(2)(iv). 

11 21 CFR 814.39(f). 

12 For guidance regarding when and how to submit a 30-day  notice, please refer to the FDA 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 (issued April 13, 2011). 

http://www.fda.gov/downloads/MedicalDevices/DeviceRegulationandGuidance/GuidanceDocuments/UCM080194.p 

df.
  
13  For guidance regarding how to examine results that are above acceptable limits, please refer to the FDA guidance  
for industry  on  Investigating Out-of-Specification (OOS) Test Results for Pharmaceutical Production. Although this 
guidance is not intended to address biological assays, many of the concepts in the guidance are applicable to  
bacterial endotoxins testing.  We update guidance  documents periodically.  To make sure you have the most recent 
version of a guidance, check the FDA Drugs guidance  Web  page at  
http://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/default.htm. 
  3
  Contains Nonbinding Recommendations 
 
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 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 
                                                 
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). 
 
  4
  Contains Nonbinding Recommendations 
 
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 are two examples of alternative assays.  
  
    (1) Recombinant Horseshoe Crab Factor C Assay   
 
If a manufacturer chooses to use a recombinant factor C-based assay, then method 
validation should be in accordance with the requirements of USP Chapter <85>, 
Bacterial Endotoxins Test, as described in the section for Photometric 
Quantitative Techniques, and USP Chapter <1225>, Validation of Compendial 
Procedures. 21  
 
(2) 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 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 (e.g., LAL cascade) can be 
made by comparing the two tests to verify the equivalence of the new method. 22  The 
comparison of the limit of detection and inhibition/enhancement is fundamental.  The 
                                                 
17  USP, General Notices 6.30. Alternative and  Harmonized Methods and Procedures. 

18 USP, 2011, Chapter <1225>, Validation  of  Compendial Procedures. 

19  USP, General Notices 6.30. Alternative  and Harmonized Methods and  Procedures. 

20 USP, 2011, Chapter <85>, Bacterial Endotoxins Test. 

21 USP, 2011, Chapter <1225>, Validation  of  Compendial Procedures. 

22 USP, 2011, Chapter <1225>, Validation  of  Compendial Procedures. 

  5
  Contains Nonbinding Recommendations 
 
sensitivity of the new method can be evaluated on spiked product samples. 23  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). 24    
 
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 PAScomparability 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 directed to be reported in a reduced reporting category 
(SupplementChanges Being Effected or Annual Report or Special Report (21 CFR 
314.80)). 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 requires a 30-day notice filed under 21 CFR 814.39(e).  See FDAs guidance, 
Modifications to Devices Subject to Premarket Approval (PMA) - The PMA Supplement 
Decision-Making Process, 
http://www.fda.gov/downloads/MedicalDevices/DeviceRegulationandGuidance/Guidanc 
eDocuments/UCM089360.pdf. Manufacturing changes for Class I and II devices should 
be in accordance with the quality system  regulation, 21 CFR part 820.  Design control, 
production and process control requirements can be found at 21 CFR 820.30, 21 CFR 
820.70, 21 CFR 820.72, and 21 CFR 820.75. 
 
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 use another endotoxin test after demonstrating a reproducible 
correlation between methods and the USP reference standard.  
 
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 
                                                 
23  International Conference on Harmonisation (ICH) Q2(R1) Validation of  Analytical Procedures: Text  and 

Methodology (1994); USP, 2008, Chapter  <1225>, Validation of  Compendial Procedures. 

24 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. 

25 21 CFR 814.39(f). 

  6
  Contains Nonbinding Recommendations 
 
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 limit requirements 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> Pyrogenicity 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  (2009). 
  7
  Contains Nonbinding Recommendations 
 
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 draft guidance for industry on Waivers of In Vivo Demonstration of Bioequivalence 
of Animal Drugs in Soluble Powder Oral Dosage Form Products and Type A Medicated 
Articles. For animal weights not listed in that guidance, please contact FDAs 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 limits for medical device extracts expressed in EU/mL.  USP 
Chapter <161> Transfusion and Infusion Assemblies and Similar Medical Devices 
provides the limits for medical devices within its scope.  The endotoxins limit for a 
medical device is 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 
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 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 
                                                 
27 USP, 2011, Chapter <161>, Transfusion and  Infusion Assemblies and Similar Medical Devices. 
  8
  Contains Nonbinding Recommendations 
 
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 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 LAL testing that deviates 
significantly from this guidance or recognized standard, 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 should 
be submitted.  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 FDAs 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 LAL test.  However, in some product 
formulations, the ingredients interfere with the LAL 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 
  9
  Contains Nonbinding Recommendations 
 
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 an LAL reagent manufacturer for use with 
a specific lot of reagent 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 LAL 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. 
 
 
 
  10