#242 
 
In-Use Stability Studies and Associated 
Labeling Statements for Multiple-Dose 
Injectable Animal Drug Products 
 
Guidance for Industry 
 
Submit comments on this guidance at any time.  Submit electronic comments to 
https://www.regulations.gov.  Submit written comments to the Dockets Management Staff 
(HFA-305), Food and Drug Administration, 5630 Fishers Lane, Rm. 1061, Rockville, MD  
20852.  All comments should be identified with the docket number FDA-2016-D-4437. 
 
For further information regarding this document, contact AskCVM@fda.hhs.gov. 
 
Additional copies of this guidance document may be requested from the Policy and Regulations 
Staff (HFV-6), Center for Veterinary Medicine, Food and Drug Administration, 7500 Standish 
Place, Rockville, MD 20855, and may be viewed on the Internet at either 
https://www.fda.gov/animal-veterinary or https://www.regulations.gov 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Veterinary Medicine 
November 2020  Contains Nonbinding Recommendations 
 
 
Table of Contents 
 
I.  INTRODUCTION................................................................................................. 3 
II.  NEEDLE GAUGE AND NUMBER OF PUNCTURES .................................... 4 
A.  Needle Gauge ............................................................................................. 4 
B.  Theoretical Maximum Number of Punctures ........................................ 4 
III.  IN-USE STABILITY STUDY DESIGN ............................................................. 5 
A.  Testing for Chemically Preserved Products ........................................... 5 
B.  Testing for Self-Preserved Products ........................................................ 5 
C.  Use of Aged Product ................................................................................. 5 
IV.  FORMAT OF IN-USE LABELING STATEMENTS ....................................... 6 
A.  Time Limited ............................................................................................. 6 
B.  Puncture Limited ...................................................................................... 6 
C.  No In-Use Restrictions (Package Insert Statement Only) ..................... 7 
V.  OTHER DOSAGE FORMS/DELIVERY SYSTEMS ....................................... 7 
VI.  FILING STRATEGY ........................................................................................... 7 
VII.  REFERENCES ...................................................................................................... 8 
APPENDIX 1.  Body Weight/Needle Gauge Recommendations .................................. 9 
APPENDIX 2.  Example Calculation for Worst Case In-Use Stability Study     
Design ................................................................................................................... 10 
APPENDIX 3.  Second Example Calculation for Worst Case In-Use Stability    
Study Design ........................................................................................................ 11 
2  Contains Nonbinding Recommendations 
 
 
In-Use Stability Studies and Associated Labeling Statements 
for Multiple-Dose Injectable Animal Drug Products 
 
Guidance for Industry 
 
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 
 
The purpose of in-use stability testing is to establish a time period during which a multiple-dose 
drug product may be used while retaining acceptable quality specifications once the container is 
opened (e.g., after a container has been needle-punctured). For multiple-dose injectable drug 
products intended for use in humans, there is a volume limit of 30 mL in the multiple-dose 
container.  There is also a 28 day in-use period associated with multiple-dose injectable drug 
products intended for use in humans unless otherwise labeled, when supported by successful 
antimicrobial effectiveness testing (AET) per U.S. Pharmacopeial Convention (USP) <51> 
Antimicrobial Effectiveness Testing.  Multiple-dose injectable animal drug products have no 
volume limit and are often packaged in much larger containers.  In addition, some animal species 
weigh less than humans and, thus, individual doses are often smaller than those used in humans.  
As such, more punctures and a longer in-use period may be applicable to multiple-dose 
injectable animal drug products compared with their human counterparts.  All multiple-dose 
injectable animal drug products should have an in-use statement on the labeling.  This document 
serves to provide CVMs current thinking on how to formulate in-use statements for multiple-
dose injectable animal drug products as well as how to design and carry out in-use stability 
studies to support these in-use statements. 
 
It should be noted that this current thinking pertains to both generic drug products and pioneer 
drug products regardless of whether the pioneer reference listed new animal drug (RLNAD) 
currently has an in-use statement on the labeling. CVM considers all in-use statements to be 
data-driven. Thus, the in-use statements on pioneer and generic drug products may differ as each 
is based on the chemistry and manufacturing controls data generated for each individual 
application (see sections 512(d)(1)(C) and 512(c)(2)(A)(i) of the Federal Food, Drug, and 
Cosmetic Act, respectively). 
 
It should also be noted that this document outlines general recommendations from CVM for the 
design of in-use stability studies and associated labeling statements for multiple-dose injectable 
animal drug products. Some drug products and their intended use pose unique challenges and 
will be handled on a case-by-case basis. CVM recommends that the appropriate Target Animal 
Division (TAD)/Division of Generic Animal Drugs (DGAD) and Division of Manufacturing 
3  Contains Nonbinding Recommendations 
 
Technologies (DMT) be contacted early in the in-use stability study planning process, to ensure 
that all concerns unique to each drug product are addressed before the study begins and labeled 
in-use statements are finalized. 
 
In general, FDAs guidance documents 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.  NEEDLE GAUGE AND NUMBER OF PUNCTURES  
 
All target species and labeled dosage(s) should be considered when determining the number of 
punctures and needle size to be used for an in-use stability study.  If multiple container/closure 
sizes are available for a single drug product, a single in-use stability study may be used to 
represent more than one container size if identical elastomeric stopper formulations are used for 
all container/closure systems.  In these cases, CVM generally considers the smallest stopper size 
to be the worst case.  Care should be taken to ensure that the needle gauge and number of 
punctures represents the worst case for the entire group of containers represented. 
 
A.  Needle Gauge 
 
In general, the in-use stability study should consider all intended species and routes of 
administration and use the overall largest needle gauge normally used in practice.  If 
special circumstances exist or questions remain regarding the needle gauge to be used for 
the in-use stability study, you should discuss this with the appropriate DMT team.  
Appendix 1 contains recommended needle gauges for major animal species, and 
Appendices 2 and 3 contain example calculations of the needle gauge and number of 
punctures to be used for a worst-case in-use stability study of a multiple-dose injectable 
animal drug product intended for use in multiple species. 
 
B.  Theoretical Maximum Number of Punctures 
 
The theoretical maximum number of punctures should be used for in-use stability studies 
whenever possible and should be based on the intended species receiving the maximum 
number of doses per container.  If the stopper cannot withstand the theoretical maximum 
number of punctures, CVM may recommend an in-use statement restricting the 
maximum number of punctures or the use of multi-dosing equipment,1 or both.  
 
You may propose, with proper justification (e.g., anticipated or historical clinical/field 
usage data), to perform the in-use stability study using less than the theoretical maximum 
number of punctures.  If CVM agrees with this proposal, then a labeling statement 
restricting the maximum number of punctures may not be needed. 
 
 
                                           
1 See section IV.B. Puncture Limited for additional recommendations if the use of multiple-dosing equipment is proposed. 
4  Contains Nonbinding Recommendations 
 
 
III.  IN-USE STABILITY STUDY DESIGN 
 
Containers (market container/closure system) of drug product should be punctured and stored at 
the approved or proposed labeled storage conditions.  The length of time that the punctured 
product containers are stored will directly relate to the in-use statement on the labeling.  CVM 
views performing all of the punctures at the beginning of the storage period as representing the 
worst case; however, CVM will also accept properly justified product puncture intervals that 
simulate those which occur in practice.  Performing the maximum number of punctures and 
withdrawing product each time the container is punctured would leave little or no drug product 
in the container for testing.  Thus, punctures of the container that do not include withdrawal of 
drug product are acceptable, but there should be contact between the needle and drug product for 
each puncture.  The drug product should then be tested at time points of your choosing, and 
specific tests should be carried out as outlined below. 
 
A.  Testing for Chemically Preserved Products 
 
If the drug product contains a chemical preservative system, full stability testing should 
be monitored at each test interval, with the following considerations: 
  Assay of the preservative should be part of the testing parameters, and the 
minimum preservative assay acceptance criterion should be supported by AET 
data at the lowest acceptable preservative concentration. 
  Sterility Testing and Bacterial Endotoxins Testing are not necessary. 
 
B.  Testing for Self-Preserved Products 
 
If the drug product contains no chemical preservative(s) and is designated self-
preserving, full stability testing should be monitored at each test interval, with the 
following considerations: 
  AET should be performed at the end of the in-use period. 
  Sterility Testing may be substituted for AET.  If sterility testing is substituted for 
AET, at a minimum there should be one-time AET data (or similar microbial 
challenge study data), to support the exclusion of AET during the in-use stability 
study. 
  Bacterial Endotoxins Testing is not necessary. 
 
C.  Use of Aged Product 
 
While CVM does not currently have an expectation that an aged drug product, at or near 
expiry, be used during in-use stability studies, careful consideration should be given to 
terminally sterilized products or any other products that have shown or may be suspected 
to show any adverse trending (i.e., impurities that increase to near the upper specification 
limit or API assay that trends downward near the lower specification limit) under normal 
stability storage conditions.  If any of these events are anticipated, early discussion with 
5  Contains Nonbinding Recommendations 
 
DMT is recommended, as the use of an aged drug product for the in-use stability studies 
may be recommended. 
 
IV.  FORMAT OF IN-USE LABELING STATEMENTS 
 
The in-use statement on the labeling should be supported by acceptable data generated during the 
in-use stability study, as outlined above. 
 
A.  Time Limited 
 
If the in-use stability study is carried out with the theoretical maximum number of 
punctures (or a fewer number of punctures has been justified as outlined in section II.B. 
Theoretical Maximum Number of Punctures above), and the longest timeframe 
investigated is less than the product expiry, the following in-use statement is 
recommended for the labeling: Use within XX timeframe 2 of first puncture. 
 
B.  Puncture Limited 
 
If the in-use stability study is carried out with less than the theoretical maximum number 
of punctures (and a justification, as outlined in section II.B. Theoretical Maximum 
Number of Punctures above, has not been accepted by CVM), and the longest timeframe 
investigated is less than the product expiry, the following in-use statement is 
recommended for the labeling: Use within XX timeframe 2 of first puncture and puncture 
a maximum of YY 2 times. 
 
If the in-use stability study is carried out with less than the theoretical maximum number 
of punctures, and the longest timeframe investigated is the product expiry, the following 
in-use statement is recommended for the labeling: Puncture a maximum of YY 2 times. 
 
If the in-use study cannot be carried out using the maximum theoretical number of 
punctures, and a puncture limiting statement is required, the in-use statement should, in 
most cases, also include language pertaining to the use of multi-dosing equipment 
(typically for food animal drugs).  CVM recognizes the breadth of multi-dosing 
equipment available to the end-user of animal drug products (e.g., automatic dosing 
device, multiple-dose syringe, draw-off spike/needle), and as such, suggests the following 
statement be added to the end of the in-use statement: 
 
If more than YY 2 punctures are anticipated, the use of multi-dosing equipment is 
recommended.  When using a draw-off spike or needle with bore diameter larger than 
ZZ, 2 discard any product remaining in the vial immediately after use. 
 
                                           
2 XX timeframe represents the in-use timeframe supported by the in-use stability study, YY represents the maximum number 
of punctures supported by the in-use stability study, and ZZ represents the needle gauge used for the in-use stability study.  For 
example, Use within 30 days of first puncture, Use within 30 days of first puncture and puncture a maximum of 10 times, 
Puncture a maximum of 10 times, or If more than 10 punctures are anticipated, the use of multi-dosing equipment is 
recommended.  When using a draw-off spike or needle with bore diameter larger than 16-gauge, discard any product remaining 
in the vial immediately after use. 
6  Contains Nonbinding Recommendations 
 
If the use of multi-dosing equipment is proposed for a new sterile injectable animal 
product packaged in a multiple-dose container, CVM may recommend using this 
equipment during target animal safety and field effectiveness studies.  CVM recommends 
that you discuss the use of multi-dosing equipment with the appropriate TAD early in 
your development process. 
 
C.  No In-Use Restrictions (Package Insert Statement Only) 
 
If the in-use stability study is carried out with the theoretical maximum number of 
punctures (or a labeling statement restricting the maximum number of punctures is not 
warranted per a justification as outlined in section II.B. Theoretical Maximum Number of 
Punctures above), and the longest timeframe investigated is the product expiry, then no 
time-limited or puncture-limited in-use statement is needed for the drug product.  
However, the following statement should be added to the package insert portion of the 
labeling: When used as labeled, there is no limit on the number of punctures throughout 
the full expiry period. 
 
V.  OTHER DOSAGE FORMS/DELIVERY SYSTEMS 
 
Other dosage forms and/or delivery systems will be handled on a case-by-case basis. In these 
cases, justification should be provided to CVM for the number of punctures and needle gauge 
proposed for the in-use stability study.  While CVMs basic recommended in-use statement 
language has been outlined in this document, other dosage forms and delivery systems present 
the possibility for other language to be proposed and or recommended.  In any of these cases, 
early communication with CVM (DMT and the appropriate TAD) is recommended so that 
concurrence on acceptable in-use stability study design and/or labeling language can be obtained. 
 
VI.  FILING STRATEGY 
 
For any new multiple-dose injectable animal drug products, the supporting in-use stability study 
data should be included as part of the original chemistry, manufacturing, and controls (CMC) 
Technical Section submitted to the investigational or generic investigational new animal drug 
application (J/INAD) or the original submission to a traditional new animal drug application 
(NADA) or abbreviated new animal drug application (ANADA), and an appropriate in-use 
statement should be included on the proposed labeling.  CVM also encourages the submission of 
an in-use stability study protocol to DMT early in the drug development process, so that 
concurrence can be obtained before study commencement. 
 
There are some previously approved products that, under CVMs current thinking, should 
include an in-use statement but currently do not.  As major changes (e.g., manufacturing site 
transfers, new stopper type) or other changes that require labeling changes are submitted to these 
applications, you should also generate in-use stability study data and submit the data and updated 
labeling, including an in-use statement, to that supplement. 
 
7  Contains Nonbinding Recommendations 
 
When in-use stability study information is submitted in a supplement to an approved NADA or 
ANADA, the submission classification in most cases will be determined as follows (decisions 
will be made on a case-by-case basis): 
  For in-use stability study data and updated labeling submitted to CVM in addition to a 
major or moderate change, the filing category is determined by the major or moderate 
change. 
  For any submission containing only in-use stability study data and updated labeling to 
include a supported in-use statement, the filing category will be Supplement  
Changes Being Effected in 30 Days (CBE-30).  
  If the sponsor has already received approval of in-use stability study data and updated 
labeling and is extending the in-use period with new data and updated labeling, or the 
sponsor has previously submitted a proposed in-use stability study to DMT and 
received protocol concurrence, the filing category will be Supplement  Changes 
Being Effected (Immediate CBE). 
 
For approved animal drug products that already have an approved in-use statement, if changes 
are made to the storage temperature or expiry period that would impact the current in-use 
statement, you should reassess the in-use statement and submit revised labeling to CVM for 
review as necessary. 
 
VII. REFERENCES 
 
1.  USP <1>, Injections 
https://www.usp.org/ 
 
2.  USP <51>, Antimicrobial Effectiveness Testing 
https://www.usp.org/ 
 
3.  CVM GFI #5, Drug Stability Guidelines 
https://www.fda.gov/regulatory-information/search-fda-guidance-documents/cvm-gfi-5-drug-
stability-guidelines 
 
4.  CVM GFI #83, Chemistry, Manufacturing, and Controls Changes to an Approved 
NADA/ANADA 
https://www.fda.gov/regulatory-information/search-fda-guidance-documents/cvm-gfi-83-
chemistry-manufacturing-and-controls-changes-approved-nadaanada 
 
 
8  Contains Nonbinding Recommendations 
 
 
APPENDIX 1.  Body Weight/Needle Gauge Recommendations 
 
Average  Average 
Largest needle  Largest needle 
Animal 1  body  body 
gauge-solution  gauge-suspension 
weight (lbs)  weight (kg) 
Dogs  45  20.5  20 2  20 2 
Cats  10  4.5  20 2  20 2 
Horses  1000  450  18 3  18 3 
Cattle  1000  450  16 4  16 4 
Calves  130  60  18  18 
Growing beef 
steers and heifers 
300-900  135-400  16 4  16 4 
on pasture or in 
drylot/grow yard 
Replacement dairy 
175-1300  80-600  16 4  16 4 
heifers 
Growing beef 
steers and heifers 
800-1300  360-600  16 4  16 4 
fed in confinement 
for slaughter 
Lactating dairy 
800-1600  360-730  16 4  16 4 
cows 
Swine  260  118  16 4  16 4 
Nursing piglets   10  4.5  18  18 
Nursery pigs  40  18  18  18 
Growing pigs  110  50  16  16 
Finishing pigs  220  100  16 4  16 4 
Sows  440  200  16 4  16 4 
Chickens  6  2.7  20  20 
Laying hens  3.7  1.7  20  20 
Broiler chickens  6.2  2.8  20  20 
Turkey  22  10  20  20 
Sheep  100  45  18  18 
 
1 Specific classes of animals, notated with italics in the table, should be used if specified on the labeling or a robust 
justification is provided to CVM.  Otherwise the general category information for each species should be applied. 
2 An 18-gauge needle may be needed to draw up viscous materials and should be considered worst case for higher 
viscosity solutions or suspensions for use in dogs and cats. 
3 A 16-gauge needle may be needed to draw up viscous materials, especially those that need to be administered 
quickly, and should be considered worst case in these situations for use in horses. 
4 For highly viscous formulations, a 14-gauge needle should be considered worst case for use in large cattle or 
swine. 
 
9  Contains Nonbinding Recommendations 
 
 
APPENDIX 2.  Example Calculation for Worst Case In-Use Stability Study Design 
 
Labeling indicates multiple species:  cattle and swine 
 
Product = 80 mL vial (50 mg/mL drug substance concentration), true solution 
 
Dosage information from labeling:  0.5 mg/kg for both cattle and swine 
 
The average weight of cattle is 450 kg: 
 
 
 
The number of doses contained in a vial for cattle is calculated as follows: 
 
 
 
The 4.5 mL dose for cattle would result in 18 punctures.  The recommended needle gauge for 
cattle is 16 gauge. 
 
Average weight of swine for food production is 118 kg: 
 
 
 
The number of doses contained in a vial for swine is calculated as follows: 
 
 
 
The 1.18 mL dose for swine represents the smallest dose for all labeled species and would result 
in 68 punctures.  The recommended needle gauge for swine is 16 gauge. 
 
Justified needle size:  16 gauge, as this is the largest needle size recommended for all intended 
species. 
 
Worst Case Study Design: 
 
68 punctures carried out using a 16-gauge needle 
10  Contains Nonbinding Recommendations 
 
 
APPENDIX 3.  Second Example Calculation for Worst Case In-Use Stability Study Design 
 
Labeling indicates multiple species: cattle and horses 
 
Product = 80 mL vial (50 mg/mL drug substance concentration), true solution 
 
Dosage information from labeling: 0.5 mg/kg for cattle and 0.25 mg/kg for horses 
 
The average weight of cattle is 450 kg: 
 
 
 
The number of doses contained in a vial for cattle is calculated as follows: 
 
 
 
The 4.5 mL dose for cattle would result in 18 punctures. The recommended needle gauge for 
cattle is 16 gauge. 
 
The average weight of horses is 450 kg:  
 
 
 
The number of doses contained in a vial for horses is calculated as follows: 
 
 
 
The 2.25 mL dose for horses represents the smallest dose for all labeled species and would result 
in 36 punctures. The recommended needle gauge for horses is 18 gauge. 
 
Justified needle size: 16 gauge, as this is the largest needle size recommended taking into 
consideration all intended species. 
 
Worst Case Study Design: 
 
36 punctures carried out using a 16-gauge needle 
 
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