General Information:

- The information requested must be typed. This form is available on disk in WordPerfect. Copies of the form or disk are available in the Division of Theoretical Surgery office in room 905 Hektoen.

- Six (6) copies of this application must be submitted to the Institutional Animal Care and Use Committee (IACUC).

- When submitting applications or project proposals to funding sources, one (1) complete copy of the final funding agency application must also be submitted with the six (6) copies of this form. If this application is submitted without the final completed funding agency application, the IACUC review and/or approval will be delayed. The Animal Facilities cannot sign off without the IACUC application and grant application, if applicable.
 

- The applications and forms must be submitted to the IACUC through the Theoretical Surgery office. A schedule of the IACUC meetings and submission deadlines may be obtained from this office (Debra X8717). Please allow sufficient time for the review and approval process. Submit protocols at least 1 week before the scheduled IACUC meeting for full consideration. Although the National Institutes of Health allow 60 days from the time of submission of an application for verification of IACUC approval, Committee concerns sometimes take several weeks to resolve. When IACUC verification of approval is completed, it is the responsibility of the applicant to notify the funding agency.
 

- Principal Investigators (PI) may be contacted after submission of their application to the Theoretical Surgery office but before the full IACUC review. Questions or concerns regarding the protocol must be addressed by the PI at that time. Any delay in PI response will ultimately delay action taken by the Committee.
 

- Full Committee review of a protocol requires a quorum of the Committee members at the meeting. A quorum is defined as a majority of the Committee members. If a quorum is not present, protocols may not be reviewed until the next regularly scheduled meeting.
 

- All questions must be answered. If all questions in the "Application For Animal Use Protocol Review" are not answered, the form will be returned to the applicant for completion prior to Committee review. If a question is not applicable, enter not applicable or N/A, do not leave blank.
 

- IACUC approval is required for all projects using live or dead vertebrate animals on and off campus. The only exception to this is when the animal use is not funded through CCH or Hektoen and is being conducted totally independent of any association with this Animal Facility. If a project is to be conducted at a facility other than a CCH facility, the IACUC will require approval by the other institution's IACUC prior to final approval of the protocol. If this is applicable, the review and approval process can be expedited by submitting the protocol for review at both institutions at the same time.
 

- This approval is for the entire project period (generally a maximum of three years). Reports must be made annually to confirm that significant changes have not been made without IACUC approval.
 

- If a project is submitted with significant changes to more than one funding agency or is resubmitted to the same funding agency again, it may have more than one IACUC approval. If there are no significant changes upon resubmission, a one-page letter can be submitted to the IACUC rather than the full application. The IACUC approval is for the project to be conducted one time. The IACUC approval numbers will be cross-referenced to ensure that the number of animals used is the number required to do the project once.
 

Specific Instructions by Item:
 

1. If an application is being submitted to a funding agency, the title of the application must be the same as that used on the "Application For Animal Use Protocol Review." Abbreviate the words in the title if necessary, to 80 spaces.
 

2. Self-explanatory. Use campus address.
 

3. When the funding agency is the National Institutes of Health, please include the specific institute. If a commercial company please give the company name. If the funding is departmental, enter departmental. If from private funds other than departmental, please indicate the specific fund or enter private source. The information on this form must agree with the information in a grant application.
 

Note: Don't forget to fill out Institutional Approval Forms for your grant application and submit them to Karen Smith, PhD in the Office of Research Development (X4940) prior to sending out your grant proposal to a funding agency.
 

4. Enter the date when the project is expected to begin and end. This may be one year or several years.
 

5. New indicates that this is a new project or application that has not been submitted to the Committee previously. The AWA and PHS require three-year resubmission of projects.
 

6. Indicate whether collaborative projects, which include all or part of the animal use, are to be conducted somewhere other than Hektoen-CCH. 
 

7. All individuals working on the project should be listed. The IACUC must be notified of all new personnel that will be working with animals. Use campus address. If additional spaces are needed, duplicate page and list additional investigators on second page.
 

8. Provide sufficient information so the scientific committee members can understand what questions are to be answered and how you propose to answer them. If in preparing a grant application, you already provided this information on one page you can attach this page as part of your Animal Use application. The Committee will not accept more than one page with the aims and objectives.
 

9. Non-technical language for a non-scientist is self-explanatory. If in doubt, use language that you would to explain this project to a seventh grade junior high class.

Note: For the next three questions #10,11,and 12, please ensure that the total animal numbers are the same for each question.

10. Use the common name for each species. Enter the total number of animals that will be required for the entire project period under the total column for each of the respective species. Include any additional animals in this total that you estimate may be required to allow for technique development or unexpected losses and explain under Question #12. The Theoretical Surgery office will maintain a record of the number of animals ordered and/or used for a project, and will withhold additional purchases when the total number of animals ordered and/or used equals the total number requested and approved. If during the conduct of the project, additional animals are required, a request in letter form with adequate justification should be submitted to the IACUC for review.
 

11. Animal use is categorized according to the degree of pain and/or distress to which the animals may be subjected. The categorization listed is the one currently used by the United States Department of Agriculture (USDA). In designing experiments, consideration should be given to methods that result in a lesser degree of unavoidable pain or discomfort, and the smallest number of animals consistent with accomplishing the scientific or educational objectives.
 

The USDA is responsible for enforcing the regulations under the Animal Welfare Act of 1966 and all subsequent amendments. Under these regulations, all research facilities are required to file annual reports. For this report and the IACUC review, the use of animals is to be categorized as either A, B, or C. Animals are to be listed only in the column under which they will be subjected to the most pain. Examples of procedures under the respective category are:
 

Category A (Little or no pain or distress)

- All procedures that if carried out on human subjects would not require anesthesia or analgesia, such as subcutaneous or intramuscular injections or withdrawal of blood (periorbital bleeding of rodents and intracardiac punctures of all species are in Category B);

- Behavioral testing that does not involve restraint;

- Exposure to noxious stimuli from which escape is possible;

- Standard methods of euthanasia that result in rapid loss of consciousness, such as anesthetic overdose or decapitation preceded by sedation or light anesthesia (e.g. animals euthanatized for tissues only).
 

If a procedure is in category A, but could be interpreted as inducing pain or distress by someone unfamiliar with the area of research, provide under Question #16b an explanation as to why you believe that it will not cause any pain or distress. An example of an explanation would be that similar procedures in humans cause no pain or distress.
 

Category B (Pain or distress with appropriate anesthetics, analgesics or tranquilizers)

- Procedures that can be performed humanely under local anesthesia, such as exposure of superficial vessels or implantation of chronic catheters;

- Surgical procedures;

- Multiple intradermal injections as is sometimes used in immunization with complete Freund's adjuvant (CFA) [intradermal administration of CFA is not recommended-special justification required under 16b.];

- Periorbital bleeding;

- Intracardiac bleeding or injections;

- Prolonged physical restraint;

- Induction of radiation sickness;

- Stress and shock studies;

- Exposure to noxious stimuli from which escape is impossible;

- Burn or trauma studies.
 

Category C (Pain or distress without the use of appropriate anesthetics, analgesics or tranquilizers)

- All procedures listed under category B that must be accomplished without benefit of anesthetics, analgesics, or tranquilizers;

- Cervical dislocation without prior sedation or light anesthesia.
 

All procedures that are under category C require justification under Question #16b. The annual USDA report requires that when animals are listed under this category, a statement must be attached to the report explaining the reasons that anesthetics, analgesics or tranquilizers cannot be used. The annual USDA report, including the list of studies under category C, are covered under the Freedom of Information Act and as such are available to the public. Animal welfare groups often request these reports.
 

12. It must be clearly evident that the number of animals justified in this section agrees with the total number of animals requested. If additional animals are being requested for development of the procedures, training or estimated attrition due to the nature of the experiment, give the numbers of additional animals requested and how they were determined. It is important that the smallest number of animals be used, but at the same time the number of animals used must be large enough to allow for proper statistical analysis.
 

13. a. Self-explanatory

b. An explanation is required. The use of animal cells or tissues in culture is an in vitro system. If cultures are used, but the cells have to be fresh rather than from a continuous culture explain why.

c. Explain why this species was chosen. A one line answer will usually not provide sufficient information. If it might appear that human volunteers could be used, provide a description of why they cannot be used instead of animals
 

14. a. Special animals include those surgically altered prior to arrival, nude mice, pregnant, neonates, etc.

b. Individual housing, suspended floors, special bedding, etc.

c. If multiple rooms are used, indicate what procedures are done in the different areas.

d. Special feed, special water, etc.
 

15. Yes or No must be checked for each class of hazardous agent. The use of any tissues or fluids from humans may constitute a possible biohazard. The use of these agents should be discussed with the Division prior to submitting to determine if the use of these agents is possible in the Facility. 
 

N.B. A copy of the user's radioisotope permit must be appended to assure the Facility that the user is licensed to use the isotope involved and that the user is aware of the need for students, residents, and technicians to be properly educated with respect to the hazards of isotope use, proper handling of isotopes, and disposal methods of radioactive waste and isotope-contaminated material. 
 

Fill out pages 9 and 10 to provide further details about hazardous materials.
 

16. a. Each procedure to be performed on each species used in the study should be described in the order it is performed, giving the time frame and intervals for performing the procedures and between procedures. A brief example is:
 
 

After a five day period of acclimatizaton to the research facility, all animals will be anesthetized with pentobarbital 55 mg/kg IP and 200 ul blood drawn from the periorbital sinus for baseline CBC and serum chemistries. On day 2, splenectomy will be performed under the same anesthesia described above using sterile instruments, sterile surgical gloves, and aseptic procedures. On day 3, animals in group 1 will receive two intramuscular injections of 0.1 ml lysine, group 2 will receive IM two injections of 0.1 ml taurine, and the control animals will receive two IM injections of 0.1 ml of saline. On day 12, all animals will be anesthetized as described above and blood will be drawn by cardiac puncture. The animals will then be euthanatized with an overdose of pentobarbital 90 mg/kg IP.

b. It may be apparent to someone working in your field of research that pain or distress will not occur, but it may not be apparent to a non-scientist or someone unfamiliar with the research. If there is any question that pain or distress could occur, describe or provide evidence indicating why pain or distress would not occur. 
 

c. Chronic (survival) major surgery on all species must be done using sterile instruments, sterile surgical gloves, and aseptic techniques. Rodent surgery does not require a special facility, but should be done in an area prepared and set aside for this purpose during the surgery. If rodent surgery is being performed, it must be stated that the surgery will be performed using sterile instruments, sterile surgical gloves, and aseptic techniques. If unfamiliar with these terms or procedures contact the Facility for instruction. By submitting this IACUC form and signing it the PI is assuring that all individuals are and/or will be properly experienced and trained in the procedures that they are proposing to conduct and with the proposed species. Failure to ensure this will result in suspension of the PI's privileges to use animals.
 

For all other species, including guinea pigs and hamsters, chronic (survival) major surgery must be conducted only in facilities intended for that purpose and approved by the Facility Supervisor or Division Head and the IACUC. Standard aseptic techniques must be followed, which includes, but not limited to, wearing of sterile surgical gloves, gowns, caps, and face masks; use of sterile instruments; and aseptic preparation of the surgical field. The techniques to be used must be described.
 

Minor surgical procedures, such as wound suturing and peripheral vessel cannulation, can be performed under less stringent conditions if they are performed in accordance with standard veterinary practices.
 

Closure techniques and types of suture should be described. Post surgical care must be described to include observing the animal to ensure uneventful recovery from anesthesia and surgery; administration of supportive fluids, analgesics, and other drugs as required; providing adequate care for surgical incisions; and maintaining appropriate medical records.
 

d. Multiple major survival surgical procedures on a single animal will not be approved unless the surgeries are related components of a research project. Cost alone is not an adequate reason. If multiple major survival surgery is indicated, please provide a detailed justification on a separate page. Major survival surgery is defined as any surgical intervention that penetrates a body cavity or has the potential for producing a permanent handicap in an animal that is expected to recover.
 

e. Drugs, dosages and/or routes of administration that differ significantly from those customarily used must be justified. When two or more drugs are combined, the dosage must be reported in a manner that the actual amount of each drug to be given is stated (i.e. Ketamine/Xylazine 35/5 mg/kg BW IM; Ketamine 35 mg/kg BW IM and Xylazine 5 mg/kg BW IM; or Ketamine/Xylazine 0.35 ml/kg BW IM mixture of 100 mg Ketamine and 14 mg Xylazine per ml).
 

If the dosage indicated is at the lower end of a recommended range, please include a statement that the drug will be given to effect and additional amounts will be given as required to maintain the appropriate level of anesthesia, analgesia, or tranquilization to relieve any unnecessary pain or distress.
 

If the procedure may last longer than the duration of the initial anesthesia, analgesia, or sedation, please indicate that additional anesthetics, analgesics, or tranquilizers will be administered.
 

Ether is not approved for use in the Facility. When the inhalation anesthetics metofane or halothane are used, they must be used in a hood or with a gas scavenging system. Animals that have been euthanatized with ether must have carcasses aerated in a hood for 24 hours prior to placement in the morgue.
 

f. Self-explanatory. Test substances and agents refers to any drug/chemical that is administered to the animal in the conduct of the study. It is not limited to just the drug that is being tested. Maximum volume refers to the highest volume to be used. If the frequency is more than once, should specify the number of times daily and for how many days. 
 

g. Cervical dislocation and decapitation are only conditionally acceptable methods of euthanasia and should be performed only with the animal sedated or under light anesthesia. If these methods have to be used without sedation or light anesthesia, a detailed scientific justification must be provided and approved by the IACUC. The 1993 Report of the AVMA Panel on Euthanasia, appears in JAVMA Vol 202, No. 2, January 15, 1993. Copies can be obtained in the Animal Facility office.
 

Ketamine is not approved for euthanasia. If not sure of the preferred method of euthanasia, discuss with the Veterinarian (x8717). Even though the Facility may actually conduct the euthanasia for you, please give the method they will use.
 

h. Death should not be used as an experimental endpoint. If animals appear to be suffering during an experiment, they should be euthanatized unless this is an integral part of the experiment. A detailed justification should be provided if euthanasia can not be performed. If the animal will not be suffering even though death is an endpoint, please explain.
 

17. To comply with current policies on the humane care and use of laboratory animals, it is mandatory that only trained and qualified individuals perform procedures and observations on laboratory animals and that the IACUC verify the qualifications and training. Qualifications and training can include the number of years working with the specific species, special certification in laboratory animal care, and/or special training for a specific period of time under another qualified individual. A description of the training should be included. Although advanced degrees, residency, and other medical training in non-animal areas may be a qualification, they alone will not be sufficient to satisfy this requirement.
 

18. It is required that when a procedure is used that may involve more than momentary or slight pain or distress to the animal subjects, the investigator must consider alternatives to these procedures and provide a written narrative description of the methods and sources used to determine that alternatives were not available. This is required even though anesthetics, analgesics or tranquilizers will be used to alleviate the pain or distress. Sources may include current literature, Medline search, the Animal Welfare Information Center, etc. This includes pain or distress that may be alleviated with anesthesia, analgesia, or tranquilizers. 

Narrative description of methods and sources does not mean a description of why animals have to be used. In addition, it is required that when procedures are likely to cause more than momentary or slight pain or distress to animal subjects (alleviated or unalleviated by anesthetics, analgesics, or tranquilizers), a veterinarian or his or her designee must be consulted in planning the study.
 

19. Prior to making changes in an approved protocol, approval must be obtained from the IACUC by requesting the change. Changes in anesthesia, analgesia, euthanasia, surgery, species, and some techniques are a few examples. If not sure, please contact the Committee or the Veterinarian.

It is required that the investigator provide written assurance that the activities in this project do not unnecessarily duplicate previous experiments.
 

20. The Chairperson's signature must be obtained to ensure that the Chairperson approves the proposed project.
 

Page 8 According to OPRR regulations, investigators must divulge any personal financial interest that they may have in the proposed research. This financial interest must generally equal or exceed $10,000 in order for it to be considered reportable here. If any of the investigators DO have such interests, they must complete Section D-II of Forms for the Office of Research Development/ Scientific Committee. This document can be obtained from the Office of Research Development in Room 603 Durand or from Sihlali Funeka, IRB Quality Assurence Officer (X4821).
 

Pages 9 and 10 Research Safety Form
 

This form must be filled out if any hazardous agents (e.g., carcinogens, biohazards, infectious agents, rDNA, flammable or corrosive chemicals, radioisotopes, etc.) will be used in animals or in the Animal Facility.
 

The purpose of this information is to help fulfill CCH policy, OSHA requirements regarding workplace exposure to hazardous materials, and CDC requirements for biosafety.
 

Upon approval of the protocol, if hazards are listed here, this form will be copied to the Safety Office to inform them of the potential hazard and the precautions planned for minimizing the associated risks. If there are any questions regarding this form contact the Animal Facilities (x0765) or the CCH Safety Office (x0400).