Boston University Charles River Campus

BOSTON UNIVERSITY ANIMAL USE APPLICATION

Institutional Animal Care and Use Committee VERTEBRATE ANIMAL USE ONLY IACUC USE ONLY Project Number Renewal of - USDA Category Approval Date 1a. Principal Investigator Principal Investigator(s): Degree(s): Academic Title: College/Department: Department Address: Phone Contacts: Office: Lab: Cell: Email: Fax:

1b. Emergency Contact Please list additional personnel to be contacted in case of an emergency (or clinical deterioration of your animal subjects) outside of normal working hours. Note: These must be updated as the contacts and/or information changes. Name: Contact Information: Phone: Cell: Pager: Name: Contact Information: Phone: Cell: Pager: Name: Contact Information: Phone: Cell: Pager:

1c. Access to Animal Space The veterinarian is mandated to have access to all live animal use spaces.  Are you planning to work with animals in the field? No Yes If yes, skip to Section 2.

 Are you planning to work with live animals outside the Boston University Animal Science Center (BU ASC)? No Yes If YES, have you provided your access plan (keys or card access) to the BU ASC office? Yes No If no, please contact the BU ASC office.

2. Project/Course and Title A. New Proposal: Title: or B. 3-year Renewal: Renewal of protocol number: Title:

Form 7/9/13, Rev. 12/16/13; 3/20/14, 5/19/14, 10/18/14 BOSTON UNIVERSITY ANIMAL USE APPLICATION

Provide a brief summary of the major findings from this project over the last three years and any publications arising from this work:

3. Scientific Objectives Please describe the scientific objectives of this research.

4. Benefits to Society Describe for the non-scientist the potential benefits of this research to society.

5. Funding One of the responsibilities of the IACUC, which is mandated by the NIH and other funding agencies, is to correlate animal use protocol applications with the description of animal work as outlined in the grant proposal. The purpose of this mandate is to assure that funded animal work is included in the referenced IACUC protocol.

Please provide the Vertebrate Animal Section for each grant indicated below with the submission of this IACUC Protocol Application.

Please provide the following information regarding any Grant(s) and its/their status: Funding Source Status/ Grant Number-if funded. BU account # if no grant number. Date of Award

6. Procedures performed on live animals (check all that apply): All procedures must be described in detail in Question 10.

Observation Only of Laboratory Animals Transportation of Animals from/to BU ASC (No other procedures performed) Blood/Tissue Sampling Field Research Photo/Video Recording Live Capture of Wild Animals Non-Recovery Surgery Teaching/Instructional (In-Class Recovery Surgery Instruction) Major Surgery Restraint of Movement while Awake Minor Surgery Fasting and/or Water Deprivation Multiple Survival Surgeries* Special Diets Immunization ** Breeding Inoculation of Biologicals **

Tumor Growth Administration of Compounds Euthanasia Other:

*Multiple Survival Surgeries. Multiple procedures that may induce substantial post-procedural pain or impairment may be conducted on a single animal only if justified by the PI, and reviewed and approved by the IACUC. ** All materials used for hybridoma propagation, spontaneous neoplasia, etc. must be tested for murine pathogens. Certification must be provided.

7. Species Information • For Vendor/Source, list the name of the company or institution supplying the animals. • Please duplicate columns as necessary for additional species.

Category Species A: Species B: Species C: Species D: Species

Strain

Age/Weight

Vendor/Source

Estimate maximum number of cages per day for BU ASC planning purposes

8. Justification for Each Species Listed above: Complete all questions (check all that apply): A. Rationale for Use of Animals Species: A B C D Studies evaluate a biological process / mechanism that cannot be evaluated in vitro.

Methods have been tested in vitro and now must be performed in vivo.

Animal tissues are required for development of an in vitro system. Other:

B. Appropriateness of the Species Species: A B C D Accepted animal model for testing purposes

Large amounts of relevant data have been derived from this species.

Proposed research requires the physiology, anatomy or size of this species.

Other:

C. Narrative: Please explain why animals, and the particular species indicated, are needed for this study. For example, could the same information be obtained by experiments using tissue culture or computer models? Identify the specific alternative models / methods you have explored that do not involve animals. Make clear why this project cannot be done without animal subjects. This narrative must include adequate information to assess that a reasonable and good faith effort was made to determine the availability of alternatives or alternative methods (see IACUC’s Policy on Search for Alternatives).

9. Transgenic/Knockout Animals A. Will genetically modified animals be used? No If no, please proceed to #10 Yes If yes, please identify the specific modification (modified gene):

B. Describe any phenotypic consequences of the genetic manipulations to the animals affecting their clinical presentation:

C. Describe any special care or monitoring that the animals will require (Such as soft food for malocclusion, sterile cages for immunosuppression, late weaning due to small size):

10. Description of Procedural Steps A. Include a detailed description of all the procedural steps, including surgery, performed upon each animal.

B. If an individual animal undergoes more than one procedure please provide a step-by-step flow chart.

C. If applicable, please describe the breeding protocol. Please include the fate of the offspring, genotyping, identification and all other details as applicable:

D. List potential adverse effects that may occur from the procedures, both non-surgical and surgical, and describe the actions that will be taken by the research staff (for instance, analgesia, supportive care, and consultation with the veterinarian).

E. Exogenous Substances: List all compounds (drug, experimental agent, cells, DNA, other) given to animals.

Route Pharma- Known or (IV, ceutical Dose suspected Compound Volume IP, Frequency Duration Grade: (mg/kg) deleterious IM, Yes or effects SC) No*

*Exogenous substances in section E must be pharmaceutical grade unless you can justify using non- pharmaceutical grade materials. For all non-pharmaceutical grade materials, please provide a scientific justification as well as a brief description of substance quality and preparation, for instance, “it will be prepared using sterile technique, diluted with sterile saline and sterile filtered with a 0.22uM syringe filter before use.”

F. Will you follow the IACUC’s Policy for Use of Pharmaceutical-Grade Chemicals and Other Substances? Yes No If no, please justify:

G. Will you follow the IACUC’s Policy for Humane Endpoints? Yes No If no, please justify: 11. Surgical Procedures A. Will surgery be performed? No If no, please proceed to #12. 5

Yes Note: Tissue collection from euthanized animals, including perfusion, is not considered surgery.

B. For survival surgeries, will you follow the IACUC’s Aseptic Surgery Policy ? N/A (non-survival) Yes No If no, please justify:

C. For rodent survival surgeries, will you follow the IACUC’s Rodent Surgery Guidelines ? (An IACUC Anesthesia & Surgical Monitoring Form is available.) N/A Yes No If no, please justify:

D. Will multiple survival surgeries be performed on the same animal? N/A No Yes If yes, please justify and include the time interval between surgeries:

E. Provide a description of the post-operative care including frequency of observation, continued analgesics, monitoring health status, and end points. (An IACUC Post-Operative Monitoring Form is available.)

F. Identify the individuals who will provide post-operative care. Note: Please submit an amendment to update personnel whenever changes occur. Name: Name: Name: Name: Name: Name:

12. Assessment of Animal Pain and/or Distress A. Classification: Please provide the total number of animals needed in each category for each species over the 3-year protocol approval period.

Species & Animal Numbers per 3 Year Approval USDA Pain / Stress Category A B C D USDA Category C: Animals upon which teaching, research, experiments, or tests were conducted involving minimal, momentary, or no pain/distress. Also, pain-relieving drugs are not used. Examples include: injections, tattooing, blood sampling, non-surgical collection of body fluids, euthanasia & tissue harvest, behavioral observation, food/water deprivation.

USDA Category D: Animals upon which experiments, teaching, research, surgery, or tests were conducted involving accompanying pain/distress to the animals and for which appropriate anesthetic, analgesic, or tranquillizing drugs are used. USDA Category E: Animals upon which teaching, experiments, research, surgery or tests were conducted involving accompanying pain or distress to the animals and for which the use of appropriate anesthetic, analgesic, or tranquilizing drugs would have adversely affected the procedures, research or interpretation of the teaching, research, experiments, surgery, or tests. An explanation of the procedures producing pain/distress in these animals and the reasons such

drugs were not used must be fully justified below.

B. Justification for the proposed animal numbers: Please account for the total number of animals required over the 3-year protocol approval period. If applicable, please include animals that are bred but not used experimentally. Note: The recommended way to justify animal numbers is to list each experimental group, the number of animals per group, and the reason this number was chosen for each group. For example, there may be an expected failure rate for a given procedure that requires additional animal numbers per treatment group, or there may be an inherent variability among animals that requires sufficient sample size for valid statistical comparisons to be made. Power calculations may be requested for clarification by the IACUC (see IACUC Policy on Sample Size Calculations).

C. If animals are part of Category E above, please describe the painful and/or stressful procedures and provide full justification for not alleviating the pain and/or distress.

D. If animals are part of Pain Category D or E perform a literature search for alternatives for each painful procedure. Include the species as a keyword. Click here for a worksheet and guidance in conducting an alternatives literature search. Make a separate entry for each procedure.

Procedure:

Database years of coverage:

Keywords/concepts:

Database Selection(s): Examples: MEDLINE, EMBASE, BIOSIS, NTIS, FEDRIP, CAB, AGRICOLA,TOXNET, Other

List any other methods used to determine that alternatives are/are not available: E.g. conference attendance, committee membership, professional expertise, training.

Summary of findings:

13. Anesthesia and Analgesia A. Do any of your procedures require Anesthesia and/or Analgesia? No If no, please proceed to #14. Yes

B. If yes, will you follow the IACUC’s Policy on Anesthesia and Analgesia in Research Animals? Yes No If no, please justify:

C. Please complete the Anesthesia/Analgesic Table:

Route Dose Species Species Species Species Category Name (IV, IP, Frequency (mg/kg) A B C D IM, SC)

Anesthetic(s):

Pre-emptive Analgesic(s):

Post-Procedure Analgesic(s):

14. Methods of Euthanasia A. Will you follow the IACUC’s Policy on Euthanasia ? N/A Yes No If no, please justify:

B. Please complete the Euthanasia Table below: Please indicate how each species will be euthanized. If more than one method is used per species please list all methods.

Species Route (IV, Dose Method IP, IM, (mg/kg) A B C D SC)

C. If you are using cervical dislocation or decapitation, as the method of euthanasia will pre- sedation or anesthesia be used? See IACUC’s Policy on Euthanasia. N/A Yes No If no, please justify:

D. Please indicate how animal death is verified (see IACUC Euthanasia Guidelines):

For animals euthanized by overdose of an injectable agent (e.g. pentobarbital, euthasol), the animals must be rechecked for lack of vital signs 10 minutes after the administration of the euthanasia agent. Death will be verified by:

Visually inspecting the animals for the absence of movement and respiration.

Verifying the absence of respiration, cardiac function, corneal reflex, muscle tone, and mucous membrane color. Note that amphibians and reptiles may have reflexive movements and a heartbeat after death.

For animals euthanized by CO2 or inhalation of other gaseous agents, a secondary physical method after an animal is rendered unconscious and insensitive to pain is required. Death will be verified by:

Decapitation

Cervical dislocation

Exsanguination - i.e., great vessels severed, cardiac perfusion, removal of vital organs

Incision of the chest cavity to produce a pneumothorax (collapsed lung) and cessation of respiration.

For animals euthanized by removal of a vital organ (decapitation or open thorax to remove heart), no verification will be needed.

15. Animals Not Euthanized A. If animals are not to be euthanized, please describe their ultimate use.

B. Identify and explain if any individual animal to be used in this project has been or will be used in any other project.

C. I agree to notify the BU ASC Director of the availability of surplus live animals so that other investigators can be notified of their availability. It is understood that re-use of live animals must be reviewed by the IACUC. Yes No If no, please explain:

16. Permits/Licenses A. Are special local, state, federal and/or international, for example, marine mammal permits), required? No If no, please proceed to #17. Yes If yes, please indicate what type:

B. Have they been requested/obtained? No Yes Please attach copies or send to [email protected].

17. Will this project include the in vivo use of any of the following: A. Radioactive Agents (including radioisotopes and ionizing radiation): No If no, please proceed to #18. Yes If yes, please list below:

If yes, have you applied for Radiation Safety approval? If radioisotopes are involved, you must also obtain approval from Radiation Safety through the Office of Environmental Health and Safety. Final IACUC approval is contingent upon approval by the RSC. No Yes If yes, please indicate status and/or approval number:

B. Biological Hazards (including Recombinant DNA): No If no, please proceed to #18. Yes If yes, please list below:

If yes, have you applied for Biosafety approval? If biological hazards and/or recombinant DNA are involved, you must also obtain approval from Biological Safety through the Office of Environmental Health and Safety. Final IACUC approval is contingent upon approval by the IBC. No If no, click here to apply. Yes If yes, please indicate status and/or approval number:

C. Chemical Hazards: No If no, please proceed to #18. Yes If yes, please indicate what type:

Please indicate the Chemical Containment Level (CCL) (see Chemical Containment Level program). CCL-1 CCL-2

CCL-3 CCL-4 N/A

E. Lasers: No Yes If yes, please list below:

F. Other Hazards: No Yes If yes, please list below:

18. Project Location: A. Where will animals be housed? Field: BU ASC Aquarium Facility: BU ASC Barrier Facility: BU ASC Main Facility: Laboratory (Building & Room): Note: Housing is defined as any area where an animal is kept for greater than 12 hours. If housing animals outside of BU ASC please provide scientific justification:

B. Are special housing conditions required? No Yes If yes, please list/describe:

C. Where will the experimentation take place? Field: BU ASC Aquarium Facility: BU ASC Barrier Facility: BU ASC Main Facility: Laboratory (Building & Room):

If you are transporting Rodents, will you follow the IACUC Transportation of Rodents Policy? N/A Yes No If no, please explain:

D. If performing surgery, where will it take place (Recovery & Non-Recovery)? Field: BU ASC Aquarium Facility: BU ASC Barrier Facility: BU ASC Main Facility: Laboratory (Building & Room):

If you are transporting Rodents, will you follow the IACUC Transportation of Rodents Policy? N/A Yes No If no, please explain:

G. Is any animal housing or work being performed at another institution (including BUMC)? No Yes If yes, please contact the IACUC office.

19. Laboratory Personnel and Training: All animal users must comply with applicable animal care and use requirements. Please see training information online. IACUC Animal User Training: Principal Investigators and all personnel working with or supervising research laboratories with live vertebrate animal use must complete an initial IACUC Orientation. Subsequently, IACUC Refresher Training is required annually. Other training modules are required as applicable. Website: Boston University Animal Care Training / Contact: [email protected] Aseptic Surgery Training: Principal Investigators and all personnel conducting survival surgery must complete a one-time Aseptic Surgery Training consisting of online AALAS courses and a Hands-on lab. Click here to submit a Request for Training. Research Occupational Health: Clearance is mandatory for all personnel working with animals. After initial clearance, updates are required annually. If you have not registered with Research Occupational Health, register here. Website: Boston University Research Occupational Health Program Contact: 617-414-ROHP (7647); [email protected] Laboratory Safety Training: Principal Investigators and all personnel working in or supervising research laboratories must complete Laboratory Safety Training annually. If you have not completed Laboratory Safety Training, click here to complete. Website: Boston University Environmental Health & Safety Training Contact: [email protected] Controlled Substances: If you will handle controlled substances and have not registered with the Controlled Substances Program, request registration here.

Please complete the sections below for the PI and each laboratory personnel working with animals under this project. You may duplicate sections as necessary for each person.

Personnel who will perform experimental manipulations on animals. Name: Phone: Email (BU preferred): Yrs. Experience / Species:

Have you registered with Occupational Health (ROHP? Yes No If no, register here. Have you completed Laboratory Safety Training (EH&S)? Yes No If no, click here to complete.

If you will handle controlled substances, have you registered with the Controlled Substances Program (CSP)? Not applicable Yes No If no, Request registration here. 12

Have you completed all your IACUC Animal User Training requirements? 1) IACUC Orientation (required one time for new personnel) Yes: Date No If no, click here to complete. 2) IACUC Refresher Training (required annually, beginning one year after IACUC Orientation; not at time of IACUC Orientation.) Yes: Date Date Date No If no, click here to complete. 3) Aseptic Surgery Training (if performing Survival Surgery) Not applicable. Applicable. Click here for guidance on training requirements/registration. Must complete online courses prior to hands-on lab. Access the Rodent Aseptic Survival Surgery Training Track in RIMS from the IACUC Refresher section; select “Take Course”; select Animal Care and Use Courses/BU Tracks) and complete courses listed below: Anesthesia, Analgesia, and Surgery: 1. Aseptic Technique for Rodent Survival Surgery 2. Pain Recognition and Alleviation in Laboratory Animals 3. Post-Procedure Care of Mice and Rats in Research: Minimizing Pain and Distress LAT Courses: 2012 LAT Training Manual: 4. LAT 12: Surgical Instruments and Materials Yes. Date completed all AALAS Courses in the BU Tracks listed above. Yes: Date completed Hands-on “Aseptic Surgical Techniques Lab” Have you completed your Boston University Animal Science Center (BU ASC) Animal User Training? Not applicable 1) Required online Species Specific Module(s) in AALAS Learning Library; accessed through RIMS. (check applicable modules): Bird Fish Ferret Gerbil Mouse Rabbit Rat Other: Other: Other: 2) Indicate any other online module(s) taken: 3) Indicate any hands on training taken with BU veterinary staff: 4) Please provide a synopsis of your training and past experience in these procedures; specify when and where the training for specific activities or procedures was received. 5) Please check the activities and/or procedures to be performed: Animal Care Anesthesia Behavioral Studies Blood Collection Electrophysiology Euthanasia Histological Analysis Imaging Implantations Injections Non-survival Surgery Perfusions Post-Operative Monitoring Survival Surgery Tissue Collection Other: Other: Other: Other: 6) Please provide details about the activities and procedures you will be performing. 7) List the activities or procedures for which additional training will be required prior to carrying out these studies. N/A Please describe your plan to obtain this necessary training.

20. IACUC Policies & Guidelines

The IACUC has many policies and guidelines to ensure the humane care and use of laboratory animals. During the review process the IACUC will assist you in identifying any additional IACUC policies and guidelines that are applicable to this research protocol. Note: This question does not need to be completed prior to submission of the application.

Policy: Will you follow: Yes No If No, please explain: Policy: Will you follow: Yes No If No, please explain: Policy: Will you follow: Yes No If No, please explain:

21. Certification & Signature Please complete, sign below, and return to: [email protected].

I certify that the use of all animals involved in this project will be carried out according to the provisions of the Animal Welfare Act, PHS Animal Welfare Policy, the principles of the NIH Guide for the Care and Use of Laboratory Animals, and the policies and procedures of Boston University.

I certify that my studies do not unnecessarily duplicate previous experiments.

I certify that all components of the grants listed in Question 5 related to the care and use of animals are included in this protocol.

I certify that only procedures that are explicitly covered in my approved protocol will be conducted. I understand that I must notify the IACUC of Boston University through the Amendment process of any changes in the research use of the animals, including the number of animals, species used, or procedures performed, and understand that no additional procedures can be started without express prior approval from the IACUC.

I understand that Boston University and its representatives on the IACUC have the authority to suspend any part of my research, should I not be in compliance at any time with USDA, PHS/NIH, or BU regulations for animal care and use.

I certify that I have provided copies of this protocol to each member of my laboratory and any other personnel that will participate in this research, and that s/he understands the approved procedures and the appropriate methods of care and use of animals as defined by the protocol and the provisions of the USDA, PHS/NIH and Boston University.

I understand that the BU ASC has been made responsible for administering and assigning animal housing space within the central animal facilities. BU ASC staff will make space assignments for the efficient utilization of space, which may result in investigators sharing animal housing space. I further realize that I must notify BU ASC of animal housing needs in order to insure the availability of space before animals are procured.

I will abide by the regulations of the Office of Environmental Health and Safety. I will ensure that 14

Form 7/9/13, Rev. 12/16/13; 3/20/14, 5/19/14, 10/18/14 BOSTON UNIVERSITY ANIMAL USE APPLICATION all laboratory personnel engaged in this project will be informed of any actual or potential hazards (including recombinant DNA, biohazards, and chemical hazards) and that they are adequately trained in procedures of animal experimentation involving hazardous agents.

I understand that approval of this project is valid for the first year of the study only and a protocol Annual Renewal Form must be submitted for projects that continue into a second and a third year. At the end of the third year a new application Protocol Form must be submitted.

I understand that I retain all responsibilities for the protocol. If I delegate certain responsibilities to alternates (e.g. during a sabbatical or extended travel), I am responsible for notifying the IACUC.

Signature of Principal Investigator:

Revised Date: Revised Date: Revised Date:

Form 7/9/13, Rev. 12/16/13; 3/20/14, 5/19/14, 10/18/14