ACURO Animal Use Appendix (ver.
14 July 2020)
Appendix for Animal Use in
Research, Development, Testing, Evaluation, and Training
Animal Care and Use Review Office (ACURO)
US Army Medical Research and Development Command
*DoD-funded animal work must NOT be initiated until the awardee receives ACURO approval.*
*Animal work initiated without ACURO approval is noncompliant and may not be funded.*
*ACURO will ONLY review protocols that have been approved by your Institutional Animal
Care and Use Committee (IACUC). *
Institutions using United States Department of Defense (DoD) funds to support the use of animals in
research, development, testing, and evaluation (RDT&E) or training must adhere to DoD requirements
for administrative review and approval by ACURO.
Provide electronic copies (searchable PDF format preferred) of the following documents to ACURO for
review and approval:
A. Appendix. A completed copy of this ACURO Animal Use Appendix (note: if submitting multiple
protocols, each IACUC-approved protocol must have a separate ACURO Appendix).
B. Protocol. A copy of the fully approved institutional animal use protocol that describes all DoD-
funded animal use. Note: If the protocol expires within the next 90 days, please wait to submit until
your protocol is renewed.
C. Approval. Documentation of initial protocol approval.
D. Amendments. A copy of existing protocol amendments applicable to this DoD award and
documentation of approval for each.
Questions regarding ACURO protocol review processes should be directed to ACURO at
Phone: 301-619-6694
Email: Usarmy.detrick.medcom-usamrmc.other.acuro@health.mil
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ACURO Animal Use Appendix (ver. 14 July 2020)
Administrative Data
1. DoD Funding of Protocol
Check one to indicate if all activities described in the submitted protocol are funded by DoD.
Fully funded by DoD: All of the experiments described in the accompanying animal use protocol are
☐ funded by this DoD award.
Partially funded by DoD: This animal use protocol contains some experiments that are NOT funded by
☐ this DoD award.
• Note: ACURO review covers only those aspects of the protocol related to work funded by DoD.
• The copy of the protocol submitted MUST include highlighting of all information relevant to the
DoD-funded portion of the study.
2. Key Personnel & Administrative Data
Complete all sections of the table with the requested information. Consult with your IACUC office as needed.
DoD Award PI Name
DoD Award PI Email & Telephone
DoD Funding Source
DoD Grant/Award/Proposal Number
Institution where animal studies are
conducted
Protocol PI Name
Protocol PI Email & Telephone
IACUC Protocol Title
IACUC Protocol Number
☐ If this is a triennial de novo rewrite submission and the protocol number has changed from the original
protocol, check this box and enter the original protocol number here: Click here to enter text.
Attending Veterinarian (AV) Name
AV Email & Telephone
IACUC Point of Contact (PoC) Name
IACUC PoC Email & Telephone
Institutional Grants Manager Name
Grants Manager Email & Telephone
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ACURO Animal Use Appendix (ver. 14 July 2020)
Animal Usage
3. Total Number of Animals to be used by Species and USDA Pain/Distress Category
Required information:
a) Fill in the table for each species used, listing all species of animals used in DoD-funded research and the
highest expected USDA Pain/Distress Category for all animals.
b) Note: please list species (by common or scientific name) only. It is NOT necessary to provide a breakdown
by strain, substrain, breed, etc.
c) Note: DoD requires this USDA pain category information for all animal species, including mice, rats, and
other species not regulated by USDA.
d) See brief definitions below. Full definitions can be found on APHIS form 7023.
a. Column/Category B: Animals being bred or held for use in research but not yet used for such
purposes
b. Column/Category C: Animals that will experience no more than slight or momentary pain or distress
c. Column/Category D: Animals that will potentially experience pain or distress for which appropriate
anesthetic, analgesic, or tranquilizing drugs WILL be used
d. Column/Category E: Animals that will potentially experience pain or distress for which appropriate
anesthetic, analgesic, or tranquilizing drugs WILL NOT be used
HIGHEST USDA
SPECIES PAIN/DISTRESS CATEGORY TOTAL NUMBER
(B, C, D, or E)
4. Justification for Unalleviated Pain or Distress
Required information (only for USDA Category E studies):
a) Provide in the box below a detailed justification for any unalleviated pain or distress.
b) The justification should provide the rationale for withholding pain relieving medication, including, as
applicable:
a. Procedures or conditions that will result in unalleviated pain or distress
b. Outcomes or parameters to be measured that can be altered by use of pain relieving medication
c. Expected effect of pain relieving medications on those outcomes
i. Note: When applicable, justification should address commonly used pain-relieving
medications (e.g., NSAIDs, opioids, local anesthetics, etc).
ii. When possible, provide references from the scientific literature or data from previous
studies that demonstrate the adverse effect on specific study variables to be measured.
d. Why the effects on these outcomes will compromise the validity of study results
e. Why you believe the confounding effects of pain-relieving medications will be more severe than the
confounding effects of unrelieved pain
c) If this is NOT a category E study, enter “N/A.”
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ACURO Animal Use Appendix (ver. 14 July 2020)
5. Animal Procurement
Check the boxes to answer the questions and provide documentation requested, if applicable.
A. Does the protocol involve USDA regulated animal species? (Note: most mice &
☐ Yes ☐ No rats bred for use in research are NOT USDA regulated.). If “No” then check “N/A”
for question B.
B. Do you obtain animals from a supplier that holds a current USDA license?
☐ Yes ☐ No
Note: If supplier claims exemption from USDA licensing, ACURO requires
confirmation from your IACUC that the supplier has met the exemption criteria
☐ N/A
outlined in the USDA Animal Welfare Regulations.
C. Will wildlife species be used?
☐ Yes ☐ No
If “yes” provide ACURO copies of all capture & use licenses and permits.
D. Will privately-owned animals be used for RDT&E or training?
☐ Yes ☐ No If “yes” provide a copy of the owner consent form that will be used. The form must
inform the owner that the animal will be used in DoD-funded research.
Animal Care and Use Program Information
6. Institutional Accreditations
a) Check the appropriate boxes for the institution where the animal research will be conducted.
b) Unaccredited, Unassured Institutions in the United States: If you check “no” for both 6.i and 6.ii below,
submit a statement signed by the Institutional Official stating that the care and use of animals will be
conducted in accordance with the standards of the current edition of the National Research Council’s Guide
for the Care and Use of Laboratory Animals and applicable Federal and DoD regulations.
c) Your institution’s IACUC office or attending veterinarian can assist with this information, as needed.
i. AAALAC International Accreditation
☐ Yes ☐ No Animal work is being performed at an AAALAC International accredited institution.
ii. Public Health Service Animal Welfare Assurance Statement
Animal work is being performed at an institution that holds a current assurance with
☐ Yes ☐ No
the U.S. Public Health Service Office of Laboratory Animal Welfare (OLAW).
7. Institution’s Veterinary Care Program
For institutions that are NOT AAALAC accredited, provide in the box below a description of the institution’s
program of veterinary care. The description should address all of the following, as applicable:
a) Description of routine care of animals by vivarium staff, including daily observation of animals
b) Description of animal care on weekends & holidays and provision of emergency care to animals
c) If the facility uses USDA covered species and your attending veterinarian is part time, confirm that there is a
written program of veterinary care
d) For animals on your studies, describe how health and welfare issues are communicated to a veterinarian.
e) If AAALAC accredited, enter “N/A.”
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ACURO Animal Use Appendix (ver. 14 July 2020)
**IMPORTANT: read these instructions before filling out sections 8-27**
Details are provided in the grey instructions box for each section below on the required information that
must be provided for ACURO review.
Each section below MUST indicate the correct page number in the IACUC-approved protocol that
provides the required information. Ensure page numbers are clearly and correctly marked on the
protocol document. If your protocol document does not have page numbers, save the document as a PDF
and use the page numbering indicated in the PDF reader.
If the protocol provides all the required information for a given item, leave the corresponding “Required
information not provided in the protocol” field blank.
If any of the required information is NOT provided in the protocol, add the required information to the
“Required information not provided in the protocol” field for each item. Do NOT copy information from
the protocol into sections 8-27 of this Appendix form.
If any section is not applicable to your protocol, enter “N/A” for “Page number in protocol.”
**Do NOT copy text from the IACUC protocol into sections 8-27.**
Study Design
8. Summary of Study Objectives
Required information: Brief description in language understandable to a nonscientist of how animals will be
used to achieve the overall and specific objectives of the protocol
● Page number in protocol: enter page number(s)
● Required information not provided in the protocol:
9. Rationale for Using Animals
Required information: Explanation of why it is necessary to use animals to achieve study objectives
● Page number in protocol: enter page number(s)
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ACURO Animal Use Appendix (ver. 14 July 2020)
● Required information not provided in the protocol:
10. Animal Model Rationale
Required information:
a) Explanation of why this particular animal model is the most appropriate model to answer the scientific
question (e.g., what unique physiologic, morphologic, or genetic characteristics does this specific animal
species, strain, or breed possess that make it the optimal model?)
b) If multiple species are used, include justification for each species used.
● Page number in protocol: enter page number(s)
● Required information not provided in the protocol:
11. Rationale for Number of Animals to be Used (Statistical Justification).
Required information:
a) Description of the rationale for the number of animals used, including, as applicable:
a. Methodology used to determine group size and total number of animals
b. Description of power-based sample size determination, or
c. Legal mandates that require specific group sizes or total number of animals for certain types of
testing
● Page number in protocol: enter page number(s)
● Required information not provided in the protocol:
12. Consideration of Alternatives to Procedures that Cause Pain or Distress (Required for all
species of animals used in USDA Category D or E)
Required information:
a) If any animals are listed in USDA Category D or E, the description must include a narrative description of
methods and sources used to determine that alternatives were not available for the procedures that may
cause pain or distress.
b) Note: DoD regulations require this information for all animal species, including those not subject to USDA
regulation.
c) If there are NO animals listed under USDA Category D or E, mark this section “N/A.”
● Page number in protocol: enter page number(s) or n/a (category B or C)
● Required information not provided in the protocol:
13. Experimental Design
Required information:
a) Explanation of experimental design that will allow reviewers to understand and follow the progress of
animals through the proposed procedures
b) Clearly understandable account of numbers of animals and their distribution into experimental groups
a. Inclusion of flow charts illustrating experimental design and summary tables of the experimental
groups is encouraged.
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ACURO Animal Use Appendix (ver. 14 July 2020)
c) Scientific justification for the use of only one sex of animal, when applicable
d) Description of the projected experimental endpoints, including maximum time on study for animals
e) Note: Animal numbers described in this section must be consistent with animal numbers described
elsewhere in the protocol and in this Appendix.
● Page number in protocol: enter page number(s)
● Required information not provided in the protocol:
Surgery and Other Potentially Painful Procedures
14. Description of Surgical and Other Potentially Painful Procedures
Required information:
a) Description of each type of surgical or other potentially painful procedure planned in sufficient detail that
someone with appropriate training in surgery and animal use could repeat it with minimal variation
b) Statement regarding whether or not aseptic technique will be used for surgical procedures. If aseptic
technique is not practiced, provide detailed justification.
c) For the purposes of this question, a potentially painful procedure is one that may produce a level or duration
of pain that would normally require the use of anesthetics or analgesics.
d) If surgery or other potentially painful procedures will NOT be conducted, enter “N/A.”
● Page number in protocol: enter page number(s) or n/a
● Required information not provided in the protocol:
15. Immediate and Long-Term Post-procedural Monitoring/Observations/Treatment
Required information:
a) For any surgical or other potentially-painful procedures, provide a description of immediate (during
anesthetic recovery), intermediate (1-3 days post-procedural), and long-term post-procedural monitoring,
including frequency of monitoring and how you will monitor for and address any potential pain/distress.
b) Typical signs of pain or distress specific to the animal species and procedures being performed and plans to
mitigate the pain or distress, including timing of administration of pain relieving medications
c) If used, provide a copy of any pain scoring sheets.
d) If surgery or other potentially painful procedures will NOT be conducted, enter “N/A.”
● Page number in protocol: enter page number(s) or n/a
● Required information not provided in the protocol:
16. Multiple Major Survival Surgeries
Required information:
a) Scientific justification for multiple major survival surgeries performed on the same animal
b) If multiple major survival surgeries will NOT be performed on the same animal, write “N/A.”
● Page number in protocol: enter page number(s) or n/a
● Required information not provided in the protocol:
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ACURO Animal Use Appendix (ver. 14 July 2020)
Other Procedures / Technical Methods
17. Paralytic Agents
Required information:
a) Description of how paralytic agents will be used
b) Justification for use
c) Monitoring methods used to ensure animals are adequately anesthetized while under the influence of
paralytics
d) Note: the use of paralytic agents without anesthesia is prohibited.
e) Note: paralytic agents include neuromuscular blocking agents (such as pancuronium, rocuronium, gallamine,
succinylcholine, and others) that produce generalized muscle paralysis without loss of consciousness.
General anesthetics (e.g., isoflurane, ketamine, etc.) are NOT considered paralytic agents.
f) If paralytics will NOT be used, enter “N/A.”
● Page number in protocol: enter page number(s) or n/a
● Required information not provided in the protocol:
18. Genotyping
Required information:
a) Description of any methods used for sample collection for genotyping or other DNA analysis, including:
a. Age of animals at sampling
b. Amount and type of tissue obtained for sampling
c. Anesthetic and/or analgesic use and criteria for use
b) If NO tissue samples are collected for genotyping, enter “N/A.”
● Page number in protocol: enter page number(s) or n/a
● Required information not provided in the protocol:
19. Adjuvants
Required information:
a) Adjuvants to be used and the plan for their use
b) Expected adverse effects
c) Frequency and method of injection site monitoring
d) Response plan (e.g., endpoints, treatment, etc.) in the event of an adverse reaction
e) Any use of adjuvants that have significant potential to induce pain (e.g., Complete Freund’s Adjuvant) must
be addressed in Section 12 of this appendix (Consideration of Alternatives to Procedures that Cause Pain or
Distress).
f) If adjuvants will NOT be used, enter “N/A.”
● Page number in protocol: enter page number(s) or n/a
● Required information not provided in the protocol:
20. Use of Non-pharmaceutical Grade (NPG) Agents in Animals
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ACURO Animal Use Appendix (ver. 14 July 2020)
Required information:
a) NPG agents to be used in animals are identified as such in the protocol
b) If NPGs are to be used but are not identified as NPG in the protocol, provide communication from IACUC
that NPG usage is approved.
c) If NPG agents will NOT be used, enter “N/A”
● Page number in protocol: enter page number(s) or n/a
● Required information not provided in the protocol:
21. Prolonged Restraint
Required information:
a) Note: this section is for prolonged or long-term physical restraint only; it is NOT intended for short-term
actions such as brief restraint for routine blood collection, etc.
b) Justification and detailed description of any prolonged restraint to be used during the study (e.g., primate
chairs, restraint boards, etc.)
c) Duration of restraint and frequency of animal observations
d) Procedures for habituation or training of animals to the device prior to the restraint, including the plan for
management of animals that fail to adapt to restraint
e) If prolonged restraint will NOT be used, enter “N/A.”
● Page number in protocol: enter page number(s) or n/a
● Required information not provided in the protocol:
22. Behavioral Testing or Behavioral Modification Techniques
Required information:
a) Description of any behavioral tests or behavioral modification methods, including use of aversive stimuli
(such as foot shocks), food or water regulation, etc.
b) Description of how animal welfare will be ensured, including acclimation if applicable
c) For food or water regulation, provide the following information:
a. Describe any plans for temporary removal of animals from the food/water regulation regimen and
criteria for reinstating food/water regulation.
b. Include endpoint criteria related to food/fluid regulation (e.g., % weight loss, hydration status, etc.)
d) If behavioral tests or behavioral modification will NOT be used, enter “N/A.”
● Page number in protocol: enter page number(s) or n/a
● Required information not provided in the protocol:
23. Exceptions/exemptions/departures from the Guide or Animal Welfare Regulations
Required information: Description of and appropriate justification for any proposed exceptions to the Animal
Welfare Regulations or to “must” standards of the Guide (e.g., enrichment restrictions in primates, cage space
restrictions, etc.)
● Page number in protocol: enter page number(s) or n/a
● Required information not provided in the protocol:
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ACURO Animal Use Appendix (ver. 14 July 2020)
24. Other Procedures (e.g., antemortem sample collection, electrocardiograms, imaging
procedures, stress induction, etc.)
Required information:
a) Description of all other procedures that will be performed while conducting this research but have not been
addressed elsewhere in this Appendix
b) If the protocol involves induction of stress for experimental purposes, describe duration, severity, and
endpoint criteria for the procedures.
c) If no other procedures will be conducted, enter “N/A.”
● Page number in protocol: enter page number(s) or n/a
● Required information not provided in the protocol:
Endpoints & Euthanasia
25. Humane Early Endpoints
Required information:
a) Description of potential adverse outcomes, including adverse effects due to experimental manipulations,
agents administered, or any other potential adverse effects not already addressed elsewhere in this Appendix
b) Description of specific, objective criteria that will be used to determine early removal from the study for
humane reasons prior to the planned study endpoint (e.g., percentage of weight loss, tumor size that inhibits
movement, number of abdominal taps, abdominal distension, loss of locomotion, hypothermia, decreased
food or water consumption, decreased activity, pain nonresponsive to analgesics, etc.)
● Page number in protocol: enter page number(s) or n/a
● Required information not provided in the protocol:
26. Death as an Endpoint
Required information:
a) Scientific justification for any proposal in which animals are allowed to die as a result of the experimental
procedures without the benefits of early euthanasia or treatment to alleviate pain
b) Explanation of why endpoints prior to death are not adequate to achieve the study objectives
● Page number in protocol:enter page number(s) or n/a
● Required information not provided in the protocol:
27. Euthanasia Methods
Required information:
a) Description of all methods of euthanasia to be used in this study, including methods used to confirm death
b) Scientific justification for use of any euthanasia method not consistent with the most recent edition of the
AVMA Guidelines for the Euthanasia of Animals
● Page number in protocol: enter page number(s) or n/a
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ACURO Animal Use Appendix (ver. 14 July 2020)
● Required information not provided in the protocol:
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ACURO Animal Use Appendix (ver. 14 July 2020)
Animal Use Assurances
28. Protocol PI Assurances:
U.S. DoD and Federal regulations specifically require several written assurances from the PI. Carefully read each
item and sign below.
As the Principal Investigator on this protocol, my signature below confirms the following:
A. Painful Procedures: Pain and distress in animals will be limited to what is unavoidable in the conduct of
scientifically valuable research; analgesic, anesthetic, or tranquilizing drugs will be used when appropriate to
minimize pain or distress; and a veterinarian was consulted regarding any painful procedures in this protocol.
B. Animal Use: The animals authorized for use in this protocol will be used only in the activities and manner
described herein unless a modification is approved by the IACUC, or equivalent animal welfare body, and
ACURO prior to implementation.
C. Duplication of Effort: I have made a reasonable, good faith effort to ensure that this protocol is not an
unnecessary duplication of previous experiments.
D. Statistical Assurance: I have consulted with a qualified individual who evaluated the experimental design
with respect to the statistical analysis and determined that the most appropriate number of animals needed for
scientific validity will be used.
E. Training: I verify that the personnel performing the animal procedures, manipulations, or observations
described in this protocol are technically competent and qualified and have been properly trained to ensure that
animals will experience no unnecessary pain or distress as a result of the procedures/manipulations.
F. Responsibility: I assure that all individuals associated with this project will demonstrate the appropriate
regard for the health, comfort, welfare, and well-being of the research animals. I pledge to implement animal use
alternatives where feasible, and conduct humane and lawful research.
G. Scientific Review: This proposed animal use protocol has received appropriate peer scientific review, and
is consistent with good scientific research practice.
H. Wildlife studies (if applicable): All research involving wildlife will be performed in compliance with all
applicable laws and regulations and with all required permits or licenses.
I. Animal procurement: All animals will be procured in compliance with all applicable laws and regulations.
Furthermore, USDA regulated animals will be obtained only from USDA licensed suppliers unless exempted
under the provisions of the Animal Welfare Regulations.
J. Disposition. Animals used in RDT&E or training will have a nonterminal disposition (e.g., adoption,
retirement, inter-institutional transfer) whenever possible.
K. Privately-owned animals (if applicable). If privately-owned animals are used for research, owner consent
will be obtained, and owners will be informed that the study is supported by the Department of Defense.
L. Annual review of protocols: Our IACUC conducts annual continuing review of animal use protocols
when required by the Animal Welfare Regulations or other applicable regulations.
*SUBMISSION IS NOT COMPLETE WITHOUT PROTOCOL PI’s SIGNATURE ON THIS PAGE*
Protocol PI Printed Name Protocol PI Signature Date
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