COMMITTEE USE ONLY :

IACUC ID#

INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE

APPLICATION FOR VERTEBRATE ANIMAL USE

NOTE: If this application proposes a study involving privately owned animals, please attach a copy of the owner consent form. If this application covers animal use for teaching, please attach a copy of the course syllabus.

Title of project:

Are you applying for or have you received Public Health Service (PHS), National Science Foundation (NSF), Department of Defense (DoD) or Veterans Administration (VA) funding for this animal use activity? Public Health Service funding includes all National Institutes of Health (NIH) agencies.

Approval is renewable annually for up to an additional two years. The IACUC office will notify you of the pending expiration date. We will ask you to complete the 2-page annual renewal form (https://research.ncsu.edu/sparcs/compliance/iacuc/iacuc-forms/).

Continuation of the approved animal usage beyond three years requires completion of a new application/protocol form and complete IACUC review.

Any changes to this approved protocol require the submission of a protocol amendment form (https://research.ncsu.edu/sparcs/compliance/iacuc/iacuc-forms/). Do not implement changes prior to IACUC review and approval.

Please retain a copy of your approved protocol. Submit a copy with your grant/proposal application to various University departmental or college research offices, or send a copy directly to the review group or project officer in the Funding Agency for your project.

IACUC OFFICE USE ONLY:

Approval: Expiration:

This institution has an Animal Welfare Assurance on file with OLAW (#D16-00214).

Revised 8/8/2018

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PLEASE – do not delete any sections of this form, even if they are not applicable to your project. CONTACT INFORMATION SECTION (We request this information for the purposes of IACUC review and after approval, for the purposes of contacting you and your staff in the event of a problem with your animal subjects.)

INSTRUCTIONS FOR CHECKBOXES : Re-formatted to be Mac-user-friendly. Right-click, select “checked.”

Principal Investigator(s):

Phone Number(s):

Department(s):

E-mail address:

Lab Contact (Technician) and phone number

Name of after-hours emergency contact:

After-hours emergency contact phone number:

***************************************************************************************************************************

Please complete the following questions only if you are renewing a previous protocol. If you are not applying for renewal, please skip down to question 1 below the table.

a)If the Committee has approved this use previously, please indicate the IACUC ID# of the previous protocol and its expiration date:

b)Procedures: Describe any unanticipated events (i.e. unexpected clinical signs, unexpected animal deaths directly related to the study) that may have occurred during the prior approval period, if not already reported to the IACUC or University Attending Veterinarian:

c) Animal Use: Please provide an accounting of your animal usage during the final year of prior IACUC approval in the chart below

Species/Strain (use common name if possible)

Category of Use (B, C, D or E)

Total Number Approved in each category and species for the entire 3-year period (check your originally approved protocol)

Total Number Used during the final year of protocol approval

OFFICE USE ONLY

IACUC ID#

*********************************************************************************************************************************

1. Is use of an NCSU animal facility or farm unit required?

Yes No

If no, explain why not (i.e. wildlife field studies, client owned). For wildlife field studies, give location. Be specific. Also, provide documentation of appropriate federal, state or local permits for conduct of the study at the site(s).

2. Identify the NCSU location and/or NCSU Animal Facility:

Room Number (if known):

3. Identify the NCSU location (building and room number) you plan to use for procedures, including surgeries, on live animals.

4. Will you transfer animals away from a University animal facility/unit?

Yes No Name of facility to which animals are transferring:

5. Will you transfer live animals at any time to a laboratory outside a University housing facility for a procedure (such as euthanasia for tissue collection)?

Yes No

If yes, how long will animals be in that location?

Building and Room Number:

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OFFICE USE ONLY

IACUC ID#

5. Do you plan to use or maintain animals at a non-University-owned site (i.e. outside of a campus animal housing facility)?

Yes No

Name of location/facility:

How long will animals be at that location?

If you plan to maintain animals in a laboratory or at a non-University-owned site for greater than 12 hours, provide justification. The IACUC may be required to review/inspect these arrangements. Contact the IACUC Office at 919.515.9532 to arrange for an inspection of the proposed animal housing area.

Justification:

6. For animals transported away from the animal facility to an investigator’s laboratory, to another NCSU site, or to any off-campus site, describe containment of animals and method of transport. The IACUC is required to inspect such vehicles prior to their use for transporting animals.

PERSONNEL INFORMATION SECTION

The IACUC Office will withhold final approval of your protocol until you and your listed personnel have completed the following two requirements:

1. REQUIREMENT FOR PERSONNEL WITH ANIMAL CONTACT:

Animal Contact Medical Questionnaire

Completion of a questionnaire is required for personnel who are involved in the direct care of vertebrate animals and their living quarters, and those individuals who have direct contact with animals (live or dead), their viable tissues, body fluids or wastes. For Animal Contact Program description, see: http://www.ncsu.edu/ehs/www99/right/handsMan/animal/animal.html. Form is located for download at http://www.ncsu.edu/ehs/www99/right/handsMan/animal/forms/AnimalContact.pdf

For employees who have submitted a previous questionnaire, a subsequent questionnaire may be required as described below:

a. Whenever a change in health status occurs that could increase their risk of health problems due to exposures to animals, such as developing a new onset asthma or contact allergy.

b. Whenever job descriptions change as they pertain to dealing with animal handling, e.g. from changing bedding to shaving animals.

c. Whenever personnel change the animal species or the biological materials handled.

PRINCIPAL INVESTIGATOR: Certify that you and your staff and working students have completed this requirement check this box:

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2. REQUIREMENT FOR INSTITUTIONAL COMPLIANCE WITH THE FEDERAL ANIMAL WELFARE ACT (REGULATORY COMPLIANCE TRAINING): All faculty, staff and students working with animals in research and teaching at NCSU must complete this training.

Principal Investigators, Postdoctoral Researchers, Veterinary Medical Residents, and Graduate Students must complete the NCSU Tutorial within the AALAS Learning Library; contact Paula DeLong (paula_delong@ncsu.edu) for login information.

NCSU Animal care staff, research staff and student workers are required to complete the NCSU Animal Subjects Tutorial. Go to this site for instructions or assistance: NCSU Animal Welfare Regulatory Compliance Training.

PRINCIPAL INVESTIGATOR: Certify that you and your staff and working students have completed this requirement check this box:

INSTRUCTIONS FOR IDENTIFYING PERSONNEL:

IN THE TABLE BELOW

List all personnel in your research group who will care for and work with the animals independently (without direct, in-lab supervision). Please list the Principal Investigator, even if he/she will not work directly with the animals. Follow this guidance:

1) Name and each individual’s unity ID – this is the ID they use to log in to their NCSU email account; for example, John Smith’s unity ID might be “jsmith142.” For

individuals external to NCSU, provide an email address for contact. See “NOTE” below.

2) Describe animal-related experience and training, for proposed procedures, in sufficient detail to document for the IACUC and outside auditing agencies (i.e. USDA, OLAW, AAALAC, etc.) that individuals are qualified; listing of degrees is not sufficient.

3) Indicate their roles in the project, i.e. the function of individuals. For example, indicate their role as “Principal Investigator,” or “Researcher” (list technicians, undergraduates, graduate students and post-docs as “researchers”).

4) Provide specific information about procedures to be performed by these individuals, especially euthanasia, and if applicable, anesthesia and/or surgery.

NOTE: If including personnel not employed by or not enrolled as students at NCSU, please provide an email address for each external individual so that the IACUC Office may contact them about completion of the required animal welfare regulatory compliance training and/or enrollment in the NCSU Occupational Health and Safety Program for Personnel with Animal Contact.

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Name and unity ID (see guidance in item 1 above)

NCSU Position Title

Animal Care and Use Related Experience (see guidance in item 2 above)

Roles in this project (see guidance in item 3 above)

Procedures to be performed (see guidance in item 4 above)

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BY SUBMISSION OF THIS PROTOCOL, THE PRINCIPAL INVESTIGATOR AGREES:

That she/he will abide by North Carolina State University policies for the care and use of animals; the provisions of the ILAR Guide for the Care and Use of Laboratory Animals (http://grants.nih.gov/grants/olaw/Guide-for-the-Care-and-Use-of-Laboratory-Animals.pdf) and/or the Federation of Animal Science Societies Guide for the Use of Agricultural Animals in Research and Teaching (https://www.aaalac.org/about/Ag_Guide_3rd_ed.pdf), (as applicable, and all federal, state and local laws and regulations governing the use of animals in research; and that he/she understands that emergency veterinary care will be administered to animals showing evidence of pain or illness, in addition to routine veterinary care as prescribed for individual species in the Standard Operating Procedures;

That all manipulations involving live animals will be performed under her/his supervision or that of another qualified individual listed on this protocol;

That all personnel having direct animal contact, including the investigator, have been trained in humane and scientifically acceptable procedures in animal handling, administration of anesthetics, analgesics, and euthanasia to be used in this project, and have completed the required regulatory compliance training (http://research.ncsu.edu/sparcs/compliance/iacuc/iacuc-training/), or are under the direct (in-lab) supervision of an individual who is aware of University policies and federal regulations via completion of the required regulatory compliance training; and that employees will be allowed adequate time to attend training sessions;

That personnel with animal or animal tissue contact participate in the Occupational Health and Safety Program;

That this proposed animal use does not unnecessarily duplicate previous activities;

That she/he will obtain approval from the IACUC before initiating any changes in this study, including changes in personnel or location of animal use;

That she/he will notify the IACUC and the attending veterinarian regarding any unexpected study results that adversely affect the animals, including any unanticipated pain or distress, morbidity or mortality.

Animal care provided by personnel other than staff under the direct supervision of the investigator, requires that the investigator personally discuss the project in detail with the supervisory staff for animal care personnel. This is required to ensure that personnel understand the potential exposures and mutually agree upon the required protective measures when handling the animals, their feed/water and their wastes (including carcasses, if applicable). Existing SOPs or IACUC Research Materials Hazard Classification Forms supplementing this Application for Vertebrate Animal Use (AVAU) document required protective equipment and measures by specific substance name.

NOTE: If a designated staff member submits this form on behalf of the PI, we require that the staff copy the PI on the email during submission of this document.

I have read, understand, and will comply with the above assurance statements.

Principal Investigator: CHECK HERE TO CERTIFY YOUR COMPLIANCE WITH THE STATEMENTS ABOVE.

Date of submission:

Any deviation from an approved protocol or violations of pertinent policies, guidelines or laws may result in immediate suspension of this project.

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To the extent that it is possible, provide responses to all questions in wording and terminology that would be understandable to members of the public. Both scientist and non-scientist members of the IACUC will review this application.

SECTION A. Animal Care and Use

1. Describe in non-scientific terms the purpose and importance of this animal use activity.

2. Describe the proposed animal use in non-scientific terms. Include all manipulations and procedures. This description should provide an understanding of what happens to an animal from the time of acquisition to the endpoint of the activity. If planning surgery, list the surgical procedure here, then describe in detail in Section E below.

3. What is the rationale for using animals for this activity? Is it possible to accomplish your objectives without using animals?

4. Describe the appropriateness of the species.

5. Provide the following information for all animals. List no animal more than once; count each in the highest proposed category of use. See http://research.ncsu.edu/sparcs/compliance/iacuc/iacuc-forms/pain-and-distress-categories/NOTE: For breeding colonies, herds or flocks, project/count any animals euthanized, sampled for phenotyping or testing in the Category C level of pain and/or distress

Species (common name)* age and/or weight**

Source*** Pain and/or distress category (B,C,D, or E)

total number requested for 3 years

*For wildlife field studies involving capture methods, anticipated non-target (by catch) species should also be indicated by species or in aggregate as “miscellaneous.”

**Give ranges if the specific information is unknown.

***Please choose from the following sources: commercial vendor, client-owned (teaching hospital, non-university farms), university-owned teaching and research herds or flocks, rental or stock animals, purpose-bred, collected from wild, animals in natural habitat, other (define).

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4. Provide responses to items a through c below to demonstrate how you determined the total number of animals requested:

a. Is this a laboratory exercise for purposes of teaching students?

Yes No

b. Do you have data from prior studies that is sufficient to calculate the sample size?

Yes No not applicable

c. How did you determine the number of animals for use in this study or activity? (Respond below*)

A response to this item is required for ALL applications.

Guidance: Using the specifics of your experimental plan (or demonstration or course syllabus, as applicable), demonstrate how the numbers of animals required to achieve your scientific (or teaching) objectives for this project (i.e., the numbers given in Sec. A.3) were calculated. If this is a research or testing protocol, you are required to include results of a power analysis used to determine the total number of animals you will require. For guidance, please see information provided at these sites:

http://research.ncsu.edu/sparcs-docs/iacuc/power_analysis.pdf

http://www.3rs-reduction.co.uk/html/6__power_and_sample_size.html

Include details of numbers of animals per group for control groups and treatment groups. Add 10% additional animals for potential experimental failure. You may provide information in the form of a table or flow chart.

Note: After approval of your protocol, you must submit an amendment to exceed the allotment of animals described here.

*Start your response here:

SECTION B. Invasive sample collection from live animals (identify the samples as blood/urine/feces/tissue/other [define])

Species sample site(s) of sample collection

method(s) volume(s) frequency of collection

Provide details for any sample collection procedures that may not be clear from the table in Section B or Item A.2. Respond below*

Note: As a general guideline for blood collection in animals, we recommend a one-week recovery period when

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removing 7.5% of circulating blood volume and a two-week recovery period when removing 10% of the circulating blood volume. Source: “A Good Practice Guide to the Administration of Substances and Removal of Blood, Including Routes and Volumes.” Journal of Applied Toxicology.21, 15-23 (2001).

*Insert any additional information here:

SECTION C. Health and Safety Information

1. Safety plan for storage and use of chemical substances approved by NCSU Environmental Health and Safety? 

Yes No Pending not applicable

If yes, Approval/plan# 

If no, contact NCSU Environmental Health and Safety; phone 919-515-1282. See: https://ehs.ncsu.edu/safety-plan/

2. Answer the following questions to determine the need for Institutional Biosafety Committee (IBC) approval.

Does this animal protocol involve?

a. Introduction of rDNA by any means into animals including, but not limited to, transfer of genetically engineered cells (transfected by any means), cells that come from a transgenic animal, or using viral vectors? Recombinant DNA (rDNA) is the joining of naturally occurring or synthetic DNA sequences (insert) to host DNA such that the resulting joined sequences are capable of replicating in a host cell (vector).

a. Yes    No Unsure

b. Creation, use, or crossbreeding of transgenic or knockout animals except for breeding one line of transgenic or knockout rodents for colony maintenance?

b. Yes   No Unsure

c. Use of organisms or viruses known to be pathogenic to humans, plants or animals? (e.g., parasites, viruses, bacteria, fungi, prions, rickettsia) or biological materials that may contain these microorganisms?

c. Yes   No Unsure

d. Use of CDC/USDA regulated Select Agents or Toxins (human, animal, or plant). Refer to this list at http://www.selectagents.gov/SelectAgentsandToxinsList.html

d. Yes   No Unsure

e. Use of human or other primate tissues, blood or body fluids or materials derived from them, including established cell lines (e.g. HeLa, HEK 293, etc.).

e. Yes No Unsure

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f. Use of disease carrying vectors (such as arthropods) or other biologically active agents (e.g., toxins, venoms) that may cause disease in humans or cause significant impact if released to the environment?

f. Yes   No Unsure

If you responded with “Yes” to any question above, then apply for IBC Approval. IACUC will withhold approval of this application until completion of IBC approval.

If you responded “No” to all questions, then you do not require IBC approval.

***If you responded “Unsure” about any question above, contact the Biosafety Officer at 515-6858 to discuss IBC review***.

For guidance on IBC forms and requirements, refer to the chapter "Procedures Governing the Use of Biohazardous Agents" in the Biosafety Manual at https://drive.google.com/file/d/0Bwfv9WVwZC73R3lRclA1RTlPSjQ/view

g. I have received IBC approval and my Biological Use Authorization specifically details the use of animals as indicated above and in this AVAU.

g. Yes*   No    Pending *If yes, provide the IBC approval #:

Note: Items submitted for IBC Approval should not be included in the IACUC Research Materials Hazard Classification form.

3. Transplantable tumors (see definition below).

Yes No List/dosage:

Definition: Tumor cell lines or other biologics that originate in rodents, passage through rodents or maintained with rodent serum may support the transmission of rodent diseases in target animals. These diseases can be a serious threat to rodent colony health. We require testing of any biologics that qualify in this category prior to use. Contact a laboratory animal veterinarian for the facility where you propose to house animals to initiate a plan of action in advance of your animal work. Direct your inquiries to CVM-LAR @ laroffice@ncsu.edu or CoS BRF/Tox @ https://brf.sciences.ncsu.edu/. For other administrative units housing rodents, direct inquires to the University Attending Veterinarian. See contact information here: http://www.ncsu.edu/iacuc/index.php

4. Does this animal protocol involve use of radioisotopes/radiation producing devices, including X-ray?

Yes No

If yes, then you require Radiation Safety Approval.  Have you obtained this approval?

Yes No Pending

If yes, Approval #

Note: Items submitted for Radiation Safety Approval should not be included in the IACUC Research Materials Hazard Classification form.

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5. Do you propose experimental use of chemicals, pharmaceuticals (drugs), nanomaterials, food additives or other items (including implants or devices) in animals? See guidance below.

Yes No

NOTE: Describe dietary manipulations in detail in Section A.2, and Section D.1, if applicable.

Substances Administration: Please list ALL substances planned for use in live animals (with the exception of substances administered for anesthesia and immobilization; identify those in Section D). See Guidance below chart. You may also be required to complete a separate form for EHS review.

Substance Route Dose ConcentrationMaximum Volume/Site

Frequency

Guidance: If you selected “Yes” for item 5 (above), you are required to include the IACUC Research Materials Hazard Classification (RMHC) as an attachment along with the AVAU. The RMHC form is NOT required for the routine use of medications for veterinary care (routine anesthesia, pain management, vaccinations, etc.) Follow instructions on the Classification form.  This form is located on the IACUC forms page (https://research.ncsu.edu/sparcs/compliance/iacuc/iacuc-forms/). If you have questions about the Research Materials Hazard Classification form, please call Environmental Health and Safety at 919-515-6862

SECTION D. Potential Pain and Distress

Use the section to discuss all procedures or conditions possibly accompanied by pain, distress, or discomfort. Include discussion of infectious or spontaneous disease studies (including potential detrimental phenotypes, if applicable).

1. List each procedure or condition with potential for pain, distress, or discomfort, and give the species and number of animals affected. Describe the clinical signs or abnormalities that are expected or possible. Guidance: Please do not repeat the information provided in Section A.2. Instead, provide a list of the procedures or conditions.

2. Describe the monitoring plan for pain and distress, including frequency and duration of checking for health or behavioral abnormalities. Identify the personnel responsible for monitoring the animals.

3. Describe minimization of pain, distress, and discomfort. (Use Section D.6 to describe use of anesthetics, analgesics, tranquilizers, or other palliative therapies.) Include description of palliative interventions, and the specific criteria/endpoints for euthanasia, if applicable. (Examples of criteria/endpoints include not eating for >24

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hours, loss of >15% of normal body weight, self-mutilation, non-weight bearing for >24 hours, etc. In some cases, it may be appropriate to euthanize animals at the first sign of clinical abnormality).

4. If painful or distressful procedures or conditions will NOT be relieved with anesthesia, analgesia, tranquilization, other palliative therapies or humane endpoints (i.e. any category E animals listed in the table in section A), provide scientific justification. Include citation(s) to published studies if applicable.

5. If death is intended to serve as an experimental endpoint (i.e., if animals must be allowed to die from an experimental condition or procedure), provide scientific justification.

6. If painful or distressful procedures or conditions are relieved with anesthesia, analgesia, tranquilization, or other therapies:

a) For each species, list procedure or condition including use of anesthesia, analgesia, tranquilization or other palliative therapies. Provide drug, dose, route, frequency of administration, and anticipated duration of therapeutic effect. Include all medications, such as pre- and post-anesthetics, antibiotics, paralytics, etc. (If applicable, describe surgery in next section).

Species Procedure or condition

Agent* Dosage, route Frequency Duration

*Please note – the use of non-pharmaceutical grade compounds must be justified scientifically in section D. 10 below. For details, please visit (http://research.ncsu.edu/sparcs/compliance/iacuc/iacuc-policies/iacuc-non-pharm-grade_compounds/).

b) Describe monitoring procedures to ensure adequacy (i.e. appropriate depth) and safety of anesthesia or tranquilization.

c) Describe monitoring procedures for recovery from anesthesia or tranquilization.

d) How will you ensure adequacy of post-operative/post-procedural analgesia or other pain-relieving therapies? (Include monitoring and techniques utilized to determine level of pain/distress in animals)

7. Physical restraint (more than one continuous hour): Describe physical restraint methods. How will potential distress be minimized (e.g. sedation, acclimation/training)?

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8. Describe trapping or other capture methods used in wildlife field studies, unless discussed in Section A.2. Explain/describe minimization of pain, distress, and discomfort.

9. Exceptions to standards: Describe and justify any exceptions to federal regulations or standards (USDA Animal Welfare Regulations, The Guide for the Care and Use of Laboratory Animals, The Guide for the Care and Use of Agricultural Animals in Research). Give the species of animal and total number.

Examples of exceptions include:

Multiple major survival surgeries Food or water restriction

Maintaining animals at temperatures and/or humidity levels outside the ranges specified by the standards

Not cleaning and/or sanitizing at required frequencies

Altered light cycles

Not meeting space requirements

Exceptions from the exercise plan for dogs

Use of paralytic agents

Individual housing of animals usually maintained in social housing

Describe exceptions here:

10. The IACUC expects investigators to use pharmaceutical-grade medications whenever they are available, even in acute procedures. Non-pharmaceutical- grade chemical compounds should only be used in live vertebrate animals after specific review and approval by the IACUC for reasons such as scientific necessity or non-availability of an acceptable veterinary or human pharmaceutical-grade product. Cost savings alone is not an adequate justification for using non-pharmaceutical-grade compounds in live vertebrate animals.

Please see NCSU IACUC Guidance here: https://research.ncsu.edu/sparcs/compliance/iacuc/iacuc-policies/iacuc-non-pharm-grade_compounds/

Do you plan to use non-pharmaceutical grade compounds in animals? Yes No

If “yes,” please provide justification here:

SECTION E. Surgery

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Surgery and postoperative monitoring and records must be in accordance with IACUC guidelines. Refer to the Intra- and Postoperative Monitoring and Required Record Keeping for Surgery and Anesthesia and Guidelines for major survival surgery performed on animals used in research, teaching, or testing Contact the University Attending Veterinarian (http://research.ncsu.edu/sparcs/compliance/iacuc/) for additional information. Identify individuals participating in surgical prep, surgery and post-operative care.

1. Will surgery be survival or non-survival?

Survival Non-Survival (animal does not recover from anesthesia prior to euthanasia)

2. Describe in detail the surgical procedure(s) employed for each species to. Include description of pre-surgical preparation and method of closure, if applicable.

3. Describe in detail the postoperative care, including any specialized care, i.e. bandage changes, and suture/staple removal, daily wound care. (Describe use of analgesics in Section D, above).

4. Will individual animals undergo more than one surgical procedure?

Yes No

If “yes,” provide scientific justification:

SECTION F. Euthanasia/Disposition

1. Provide the following information for all planned/experimental euthanasia of animals. Complete Section F.4 if euthanasia is not included in these planned animal use activities.

PLEASE NOTE: If this is a wildlife field study, please provide a contingency plan for the humane euthanasia of any animals found to be seriously injured or morbidly ill at time of capture. Complete the table below, and explain disposition of animals and contingencies for euthanasia in F.4

Species Method/agent Dosage, route

2 Describe confirmation of death

Confirmation of a lack of vital signs (heart rate, respirations, etc.)Bilateral thoracotomy

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Cervical dislocation after administration of euthanasia agent (drug, carbon dioxide, etc.)Other – please describe:

3. If applicable, justify methods that vary from the American Veterinary Medical Association Guidelines on Euthanasia (https://www.avma.org/KB/Policies/Documents/euthanasia.pdf). When used as a sole means of euthanasia, physical methods such as decapitation and cervical dislocation also require scientific justification if used as a sole means of euthanasia.

4. If not euthanized as part of your protocol, what will become of these animals? More information available regarding the NCSU animal disposition policy at NCSU Animal Disposition Policy.

SECTION G. Consideration of Alternatives (this section not required for Category B or C animal use).

COMPLETION OF THIS SECTION IS REQUIRED FOR ALL APPLICATIONS INVOLVING THE USE OF ANIMALS AT THE LEVEL OF CATEGORIES D OR E. For descriptions of animal use categories, see http://research.ncsu.edu/sparcs/compliance/iacuc/iacuc-forms/pain-and-distress-categories/ , “Classification of Animal Use by Pain and Distress Categories”

"Alternative" refers to methods, models, and approaches that:

a) Result in the reduction of the number of animals usedb) Incorporate refinements of procedures which result in the lessening of pain or distress to animalsc) Provide for the replacement of animals with non-whole animal systems or the replacement of one animal

species with another, particularly if the substituted species is non-mammalian or invertebrate.

There must be a written narrative description of the methods and sources consulted to determine the availability of alternatives (reduction, refinements, or replacement). USDA and OLAW suggest the best way to satisfy this requirement is by completion of a literature search.

Sources: NCSU Libraries Research Assistance (http://www.lib.ncsu.edu/researchassistance/) BIOSIS Previews, MEDLINE, Current Research Information System, PubMed, Agricola, Current Contents, NORINA, the Animal Welfare Information Center (AWIC) operated by the National Agricultural Library, professional journals and other sources unique to specific fields of study.

For further guidance, please see information provided by AWIC at: http://research.ncsu.edu/sparcs-docs/iacuc/AWIC_info_requirements.pdf

1. Please respond to items a-d regarding your literature search for alternatives:

a. Identify databases searched. We recommend that you use more than one.

b. Indicate the range of dates searched within the database(s) (i.e. 1900-2009, or 1987-present)

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c. Keywords should include those likely to yield information on alternatives to the potentially painful or distressful procedures or conditions that are part of this protocol. As such, one of the keywords included in your search terms should be either “alternative” or “alternatives.”

List keywords used:

d. Provide the most recent date (month/day/year) that you performed a full search. The search should be completed within three months prior to submission.

2. Identify other sources (consultants, conferences, etc.)

3. Summarize how the above methods and sources identified and described above have helped you identify alternatives to potentially painful or distressful procedures, or determine that alternatives are either not available, or not appropriate for this protocol.