Guidance for Industry 
 
Container and Closure System Integrity 
Testing in Lieu of Sterility Testing as a 
Component of the Stability Protocol for 
Sterile Products 
 
 
 
 
 
 
 
 
 
For questions on the content of the guidance, contact CBERs Office of Compliance and 
Biologics Quality at 301-827-3031; CDERs Office of Pharmaceutical Science at 301-796-1228; 
CDRHs Office of Device Evaluation at 240-276-3747; or CVMs Office of New Animal Drug 
Evaluation at 301-827-6963.   
 
 
 
 
 
 
 
 
 
 
 
U. S. Department of Health and Human Services 
Food and Drug Administration 
Center for Biologics Evaluation and Research  
Center for Drug Evaluation and Research  
Center for Devices and Radiological Health  
Center for Veterinary Medicine  
February 2008
       
 
Guidance for Industry 
 
Container and Closure System Integrity 
Testing in Lieu of Sterility Testing as a 
Component of the Stability Protocol for 
Sterile Products 
 
Additional copies of this guidance are available from: 
 
Office of Communication, Training and Manufacturers Assistance, HFM-40 
Center for Biologics Evaluation and Research 
Food and Drug Administration 
1401 Rockville Pike, Suite 200N, Rockville, MD 20852-1448 
Internet: http://www.fda.gov/cber/guidelines.htm 
Phone:  800-835-4709 or 301-827-1800 
or 
Office of Training and Communication 
Division of Drug Information, HFD-240 
Center for Drug Evaluation and Research 
Food and Drug Administration 
5600 Fishers Lane, Rockville, MD 20857  
Phone:  301-827-4573 
Internet:  http://www.fda.gov/cder/guidance/index.htm 
or 
Division of Small Manufacturers, International, and Consumer Assistance (DSMICA), HFZ-220 
Center for Devices and Radiological Health 
Food and Drug Administration 
1350 Piccard Drive, Rockville, MD 20850 
Phone:  800-638-2041 
Internet:  http://www.fda.gov/cdrh/guidance.html 
Email:  dsmica@cdrh.fda.gov 
Fax: 240-276-3151 
or 
Communications Staff, HFV-12 
Center for Veterinary Medicine (CVM) 
Food and Drug Administration 
7519 Standish Place 
Rockville, MD 20855 
Internet at http://www.fda.gov/cvm/guidance/published.htm
 
    Contains Nonbinding Recommendations   
 
Table of Contents 
 
I.  PURPOSE AND SCOPE................................................................................................1 
II.  INTRODUCTION...........................................................................................................2 
III.  DEFINITIONS................................................................................................................3 
IV.  BACKGROUND.............................................................................................................3 
V.  ALTERNATIVES...........................................................................................................4 
VI.  IMPLEMENTATION....................................................................................................5 
VII.  APPLICATION SUBMISSION....................................................................................5 
 
 
 
 
i    Contains Nonbinding Recommendations   
 
GUIDANCE FOR INDUSTRY1
 
Container and Closure System Integrity Testing in Lieu of Sterility 
Testing as a Component of the Stability Protocol for Sterile 
Products 
 
This guidance represents the Food and Drug Administrations (FDAs) 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 appropriate FDA staff.  If you cannot identify the appropriate FDA staff, 
 
call the appropriate number listed on the title page of this guidance.
 
I.  PURPOSE AND SCOPE 
 
This guidance document provides recommendations to you, manufacturers, for using methods 
other than sterility testing to confirm container and closure system integrity as a part of the 
stability protocol for sterile biological products, human and animal drugs, and medical devices.  
This guidance document finalizes the draft guidance of the same title dated January 1998 
(January 28, 1998, 63 Federal Register (FR) 4272). 
 
Manufacturers of drugs and biologics purporting to be sterile must test each batch or lot, as the 
case may be, to ensure that the product in question conforms to sterility requirements.  21 CFR 
211.167(a); 21 CFR 610.12.  Such drugs and biologics are also subject to stability testing 
requirements.  21 CFR 211.166.  The stability testing requirements include maintaining a written 
testing program designed to assess stability characteristics.  Manufacturers of medical devices 
must validate processes, including sterilization for a device purporting to be sterile.  21 CFR 
820.75.  Stability testing should be part of the design validation of such devices.  In vitro 
diagnostic products for human use are required to be labeled with stability information. 21 CFR 
809.10.  For products labeled as sterile, we consider sterility to be a stability characteristic.   
 
The purpose of stability testing is to provide evidence on how the quality of a substance or 
product varies with time under the influence of a variety of environmental factors such as 
temperature, humidity, and light, which enables you to establish or modify recommended storage 
conditions, retest periods, and shelf life or dating period, as the case may be.2  This guidance 
document applies only to the replacement of the sterility test with an appropriate container and 
closure system integrity test in the stability written testing program (referred to in this guidance 
                                                 
1 This guidance document was prepared by an intercenter working group with representatives from the Center for 
Biologics Evaluation and Research, Center for Devices and Radiological Health, Center for Drug Evaluation and 
Research, and the Center for Veterinary Medicine. 
2  "Dating period" being the term used for biologics, as defined at 21 CFR 600.3(l), and "shelf life" being the term 
used for other drugs. 
1    Contains Nonbinding Recommendations   
as the "stability protocol"), recommending an alternative to sterility testing for supporting the 
continued capability of containers to maintain sterility.  The guidance document does not apply 
to sterility testing methods for product sterility testing prior to release, as container and closure 
system integrity tests cannot demonstrate a product's initial sterility. 
 
This guidance document provides information that we recommend you consider when you 
propose using alternative methods to sterility testing to confirm the integrity of a container and 
closure system throughout the product's shelf life or dating period.  The recommendations in this 
guidance document apply to both pre- and post-approval stability protocols for sterile biological 
products, human and animal drugs, including investigational and bulk drugs.  For medical 
devices, the recommendations in this guidance document apply to stability protocols for those 
devices labeled as sterile. 
 
If you currently perform sterility testing as a stability-indicating test as part of a stability 
protocol, you may continue to do so.   If your product is approved for an alternative to sterility 
testing as a component of your stability protocol, this document is not intended to recommend 
additional testing requirements. 
 
FDAs guidance documents, including this guidance, do not establish legally enforceable 
responsibilities.  Instead, guidances describe the FDAs 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 FDAs guidances means that something is suggested or 
recommended, but not required. 
 
II.  INTRODUCTION 
 
Products labeled as sterile are expected to be free from viable microbial contamination 
throughout the product's entire shelf life or dating period.  For products labeled as sterile, we 
consider sterility to be a stability characteristic.  As a result, the stability protocol should include 
confirmation of continuing sterility throughout the product's shelf life or dating period.    The 
minimum sterility testing generally performed as a component of the stability protocol for sterile 
products is at the initial time point (release) and final testing interval (i.e., expiration).  
Additional testing is often performed at appropriate intervals within this time period (e.g., 
annually).  However, as discussed below sterility tests for the purpose of demonstrating 
continuing sterility have limitations, with respect to the method's reliability, accuracy, and the 
conclusions that may be derived from the results.  Because of the limitations of sterility tests 
described below, sterility tests are not recommended as a component of a stability program for 
confirming the continued sterility throughout a product's shelf life or dating period.  Alternative 
methods may be more reliable in confirming the integrity of the container and closure system as 
a component of the stability protocol for sterile products. 
 
This guidance document does not suggest specific test methods and acceptance criteria (except 
for references to USP methods), nor does it provide comprehensive lists of tests.  You should 
determine these details based on good scientific principles for each specific container and closure 
system taking into consideration particular product formulations and, where applicable, routes of 
administration. 
2    Contains Nonbinding Recommendations   
 
III.  DEFINITIONS 
 
The definitions presented here are for the purposes of this guidance only. 
 
A container and closure system refers to the entirety of packaging components that together 
contain and protect the product. 
 
A packaging component means any single part of a container and closure system.  Typical 
components are containers (e.g., ampules, vials, bottles), container liners, closures (e.g., screw 
caps, stoppers), closure liners, stopper overseals, container inner seals, administration ports (e.g., 
on large-volume parenterals), overwraps, administration accessories, and container labels. 
 
IV.  BACKGROUND 
 
Sterility tests have long been used to verify that products maintain their sterility throughout the 
product's entire shelf life or dating period.  However, sterility testing has scientific and practical 
limitations, which are well known.  Some of these are: 
 
1.  Sterility tests will only detect viable microorganisms present at the time of the test; 
2.  Viable organisms present at the time of the test can only be detected if they are 
capable of growth in the specified culture media; 
3.  Sterility tests may be subject to potential interference due to adventitious microbial 
contamination introduced at the time of testing, resulting in false positive readings; 
and 
4.  Sterility tests are always destructive of the samples tested and do not offer the 
opportunity to reexamine the same samples in the event of either positive or negative 
findings. 
 
Some Centers of FDA have sought to address these shortcomings and communicated the 
information to you in the guidance document:  "Guidance for Industry for the Submission 
Documentation for Sterilization Process Validation in Applications for Human and Veterinary 
Drug Products" (December 3, 1993, 58 FR 63996).  Section V. A., page 16 of the guidance 
states: 
 
The  ability  of  the  container-closure  system  to  maintain  the  integrity  of  its 
microbial barrier, and, hence, the sterility of a drug product throughout its shelf 
life, should be demonstrated. [...] As previously stated, sterility testing at the 
initial  time  point  is  not  considered  sufficient  to  demonstrate  the  microbial 
integrity of a container-closure system. [...]. 
 
In the Federal Register of July 10, 1996 (61 FR 36466), we published a notice regarding the final 
guideline entitled "Quality of Biotechnological Products: Stability Testing of 
Biotechnological/Biological Products", prepared under the auspices of the International 
Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals 
3    Contains Nonbinding Recommendations   
for Human Use (ICH Final Guideline).3  The ICH Final Guideline is intended to provide 
guidance to applicants regarding the type of stability studies that should be provided in support 
of marketing applications for biotechnological/biological products. 4   
 
V.  ALTERNATIVES 
 
Alternatives to sterility testing as part of the stability protocol, such as replacing the sterility test 
with container and closure system integrity testing, might include any properly validated 
physical or chemical container and closure system integrity test (e.g., bubble tests, 
pressure/vacuum decay, trace gas permeation/leak tests, dye penetration tests, seal force or 
electrical conductivity and capacitance tests, etc.), or microbiological container and closure 
system integrity tests (e.g., microbial challenge or immersion tests).  Such tests may be more 
useful than sterility testing in demonstrating the potential for product contamination over the 
products shelf life or dating period.  The advantages of using such container and closure system 
integrity tests in lieu of sterility tests in the stability protocol for sterile products include: 
 
1.  Such alternate methods may detect a breach of the container and/or closure system 
prior to product contamination; 
2.  Some of the alternate methods used to evaluate container and closure integrity can 
conserve samples that may be used for other stability tests; 
3.  Alternative test methods may require less time than sterility test methods which 
require at least seven days incubation; and 
4.  The potential for false positive results may be reduced with some alternative test 
methods when compared to sterility tests. 
                                                 
3 For veterinary products, generally, see: VICH GL17, "Stability Testing of New Biotechnological/Biological 
Veterinary Medicinal Products," 66 FR 19177, April 13, 2001, and VICH GL3(R), "Stability Testing of New 
Veterinary Drug Substances and Medicinal Products (Revision)," 71 FR 19525, April 14, 2006. 
 
4 The ICH Final Guideline is intended to supplement the tripartite ICH guideline entitled, Stability Testing of New 
Drug Substances and Products, the notice for which is published in the Federal Register of September 22, 1994 (59 
FR 48754). This guideline reflects formal scientific principles for stability testing of human drugs, and provides a 
general indication of the information to be generated on product stability, but leaves sufficient flexibility to 
encompass the variety of different practical approaches required for specific scientific situations and characteristics 
of the materials being evaluated.   
 
A revision of the September 22, 1994, ICH guideline with the same title (Q1A (R)) (66 FR 56332, November 7, 
2001) was issued to add information to certain sections and to provide clarification to other sections. The most 
important changes were as follows:  (1) stress testing of the drug substance was moved from the glossary to the main 
text; (2) the text on test procedures was made consistent with ICH guidance  Q6A Specifications:  Test Procedures 
and Acceptance Criteria for New Drug Substances and New Drug Products:  Chemical Substances (65 FR 83041, 
December 29, 2000)), and relevant cross-references to other ICH guidelines were introduced; (3) the text on testing 
frequency was amended for accelerated testing conditions; (4) storage conditions are described in more detail, and 
testing at low temperature and testing of aqueous liquids in semipermeable containers are specifically addressed; (5) 
the postapproval commitment is clearly described; and (6) the guidance was made editorially consistent.  A second 
revision of the guideline, Guidance for Industry Q1A (R2) Stability Testing of New Drug Substances and 
Products, (68 FR 65717, November 21, 2003) was issued to harmonize intermediate storage conditions for zones I 
and II with long-term storage conditions for zones III and IV.    
 
4    Contains Nonbinding Recommendations   
 
Occasionally, applicants have proposed the use of a preservative effectiveness test in lieu of the 
appropriate sterility test for products containing antimicrobial preservatives.  However, these 
tests only measure the effectiveness of preservatives against a panel of five different test 
organisms.  This method cannot confirm product sterility since it does not confirm the presence 
or absence of contamination, but rather only demonstrates the microbiological effectiveness of 
the preservative system against the five test organisms in question.  For these reasons, 
preservative effectiveness tests are not acceptable alternative tests for monitoring container and 
closure system integrity or for demonstrating maintenance of sterility.  However, such tests are 
appropriate to perform as part of the stability protocol on multi-dose containers at the end of the 
product's shelf life or dating period, to verify antimicrobial preservative effectiveness and 
preservative content. 
 
VI.  IMPLEMENTATION  
 
When seeking to implement container and closure system integrity testing as an alternative to 
sterility testing as a component of the stability protocol for sterile products, we recommend that 
you consider the following: 
 
1.  A container and closure system integrity test may replace sterility testing in a stability 
program at time points other than the product sterility test prior to release; 
2.  Container and closure system integrity tests do not replace sterility testing methods 
for product sterility testing prior to release; 
3.  Any validated container and closure system integrity test method should be 
acceptable provided the method uses analytical detection techniques appropriate to 
the method and is compatible with the specific product being tested.  A test method is 
adequately validated if it has been proven through scientifically accepted studies to be 
capable of detecting a breach in container and closure system integrity; and  
4.  An appropriate container and closure system integrity test should be conducted 
annually and at expiration, or as otherwise required by applicable regulations. 
 
VII.  APPLICATION SUBMISSION 
 
For new marketing applications for sterile products, we recommend that you include container 
and closure system integrity tests in your stability protocol.  Pending new marketing applications 
may be amended prior to approval. 
 
If you wish to incorporate a validated container and closure system integrity test to demonstrate 
the continued capability of containers to maintain sterility for an approved product,5 you must 
submit the appropriate application supplement, as follows: 
 
                                                 
5  As previously mentioned, if you currently perform sterility testing as a stability-indicating test as part of a stability 
protocol, you may continue to do so.  Also, if your product is approved for an alternative to sterility testing as a 
component of your stability protocol, this document is not intended to impose additional testing requirements. 
5    Contains Nonbinding Recommendations   
Human drugs:  Submit methods and data labeled Special Supplement - Changes Being 
Effected for new and abbreviated new drug applications under  314.70. 
 
Animal drugs:  Submit methods and data as Supplement - Changes Being Effected under  
514.8. 
 
Biological products:  Submit a supplement with proper validation data in support of the 
proposed change as a Supplement - Changes Being Effected under  601.12. 
 
Medical devices:  Submit a PMA supplement with proper validation data in support of the 
proposed change under  814.39. 
 
You should include in your application supplement a discussion of what the test method 
evaluates and how it is applicable to microbial integrity.  You should select methods that are 
appropriate to the product in question, and validate all test methods.  Validation of particular 
methods should be specific to the product container and closure system or product type.  Several 
alternative container and closure system integrity test methods exist, as described above.  We 
encourage you to develop innovative methods of testing the integrity of specific container and 
closure systems. 
 
The use of media-filled containers is generally acceptable for initial validation studies.  It may be 
acceptable to use media-filled containers instead of product-filled containers for testing during 
the product's shelf life or dating period, if the product contains a material, such as a preservative, 
which would bias the results of the container and closure system integrity test.  If you propose to 
use media-filled containers for some or all of the testing, you should include data in your 
application supplement to support this request. 
 
If you manufacture a number of products that use the same type of container and closure system, 
you may validate your integrity test method using a bracketing matrix.6  It is not necessary to 
perform validation studies on each product. 
 
The number of samples to be tested should be statistically appropriate.  Samples which pass 
container and closure system integrity testing may be further utilized in the stability testing for 
that specific test period or interval; however, the test should be non-destructive and the sample 
unaltered by the container and closure testing method itself.  Samples should not, however, be 
tested for container and closure system integrity at one time interval (e.g., 12 months), and then 
stored for further stability testing at later time periods (e.g., 24 months). 
                                                 
6 See ICH guidance Q1D Bracketing and Matrixing Designs for Stability Testing of New Drug Substances and 
Products (68 FR 2339, January 16, 2003).  
6