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| "Why don't we also have each faculty member bring in a note from mother" |
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| "Why don't we also have each faculty member bring in a note from mother" |
General Instructions
These instructions are my unofficial attempt to answer commonly asked questions and to avoid common problems encountered by faculty when completing the "Animal Use Protocol Review Form". The "official" form is available as a paper document from ORSP and LAS, on disk from ORSP and can be downloaded from the ORSP site in WordPerfect 6.0, MS Word 6.0a and Word for Macintosh 4.2.1. These instructions may be read as normal text, but are designed to be used with Dr. Bob’s Word97 version of the protocol review form. Dr. Bob’s Word97 version contains hyperlinks to the corresponding instructions in this document.
In addition to the many helpful suggestions found in this document, you will find links to other documents including:
As always, if you have questions about completing the Animal Use Protocol form, feel free to call Karen Janes, or the LAS veterinarians, Dr. Robert L. Harris or Dr. Stephen K. Curtis.
Disclaimer: The protocol review process is a committee process and no one member can anticipate what the committee will officially decide as to the adequacy of any response.
Animal Research Regulations and Guidelines
Animal Welfare Act
Rutgers Links
LAS Home Cage
Protocol Instructions Mar 98.doc [Instructions for Rutgers Protocol Form]
Rutgers Policies:
Rutgers Animal Welfare Policy
Adoption Policy
Bleeding Techniques in Rabbits
Disposition of Farm Animals
Food and Water Restriction
Guidelines for Producing Monoclonal Antibodies in Mice
Housing animals outside animal facilities
Pithing Frogs
Rodent Euthanasia Policy
Surgery Policies
Rodent Surgery Policy
Non-rodent Survival Surgery
Video Taping Policy
INVESTIGATOR INFORMATION: Principal Investigator (Question I.A.1.)
Generally, you should be a faculty member to be the PI of record for an animal use protocol. Graduate students, post-docs, technicians, and non-Rutgers personnel may not be the PI on an animal use protocol. If you have a question as to who should be the PI for a protocol, contact the
Animal Care and Facilities Committee (ACFC) and ask.GRANT INFORMATION: Project Sponsor or Prospective Sponsor: (Question I.C.1)
Rutgers’ animal use protocol approval is not tied to specific grants. That is, there need not be a one-to-one correspondence between grants and protocols. Any and every use of animals in a grant, however, must be included in at least one animal use protocol.
Similarly, animal care charges need not be grant specific or protocol specific. With advance notice, Laboratory Animal Services will assign animal care charges to any one or more accounts that you specify.
If this protocol has already been approved by the ACFC for a grant proposal submitted to another funding agency and all aspects of animal use are otherwise identical, provide the title and number of the approved protocol. In such cases the completion of the remainder of this form is not required. This does not apply if your are submitting the required rewrite to a previously approved protocol. Please sign at - Section D. (Question I.C.7)
"The required rewrite to a previously approved protocol" refers to the protocol review committee’s policy that now requires that every protocol be rewritten from scratch "denovo" every 3 years. Ignore question 7 if you are submitting a "denovo" protocol. If you are submitting a grant for which the animal use procedures are identical to a previously approved Rutgers animal use protocol, I suggest you contact the committee administrator. It may not be necessary for you to submit a protocol form at all.
INVESTIGATOR'S STATEMENT (Question I.D.)
An investigator statement regarding "unnecessary duplication of research" is required by USDA.
Signature: Don’t forget to sign your protocol form! This is a common mistake.
Course Information: Your department chair’s signature is required if this is for a course.
ANIMAL SPECIES TABLE: Vendor/Source (Question II.A.)
Links to many animal vendors can be found in the
Las HomeCage.For vendors with more than one facility (e.g. Charles River), please indicate the facility (i.e. city) you will order from.
If different from animal facility where housed, are animals housed overnight in this experiment area?
Vertebrate animals are generally housed in centralized animal facilities. Housing animals in labs or other locations is sometimes allowed when scientifically justified, but only by approval of the ACFC. These areas are subject to inspection by the ACFC, USDA, AAALAC and other regulatory agencies.
III. PROJECTS INVOLVING ONLY EUTHANIZED ANIMALS
If no manipulations are to be made other than euthanasia for purposes of, for example, tissue collection, check box and proceed to item 16. (Question III.1.)
Check this box if the only thing you are going to do the animals is to euthanize them by an accepted method. If you will anesthetize an animal that will not recover (e.g. for perfusion fixation), but will doing procedures while the animal is unconscious but still alive, you should not check this box. In such a case, you need to at least complete section IV.A., "Acute Survival Procedures".
Do not check off question III.1. if your project will involve the following procedures, in a conscious or anesthetized animal:
IV. PROCEDURES ON ANESTHETIZED ANIMALS
Examples in this category include
Recommended anesthetics and dosages are given in the LAS HomeCage. Please use generic drug names (e.g. "Pentobarbital", not "Nembutal"). Dose should be mg/kg of body weight, not volume (e.g. not ml/kg).
LAS Manual, Anesthesia chapter
SURGERY (SURVIVAL) (Section IV.B., Questions 5 – 10)
The
Animal Care and Facilities Committee (ACFC) has established Standard Operating Procedures for surgery performed on rodents and non-rodents. Following these SOP’s will assure that investigators comply with established guidelines. While it doesn’t make sense phylogenetically, procedures on birds, amphibians and reptiles should follow the rodent surgery SOP’s.LAS Manual, Surgery chapter
Multiple surgery - Have the animals undergone previous surgery here or elsewhere, or will they undergo multiple surgeries in this protocol? (Question IV.B.6)
Multiple surgeries on a single animal are sometimes scientifically necessary when studying multiple systems. Re-using animals, unless they are endangered or otherwise limited in availability, is not sufficient justification. Cost savings alone are not sufficient justification to submit an animal to multiple survival surgeries.
Guide, "Multiple
Major Surgical Procedures", p. 11-12
Anesthesia for survival surgery: (Question IV.B.8.)
Guide, "Pain,
Analgesia and Anesthesia, pp. 64-65
Post-operative Care: (Question IV.B.9.)
Guide, in "Surgery", p. 63
Post-operative analgesia: (Question IV.B.9.c.)
An analgesic is a "pain killer" for pain after the surgical anesthesia has ended. Post-operative pain relief can be more effective if the analgesic is started prior to anesthetic recovery ("pre-emptive analgesia").
If you are not using analgesia provide a justification: (Question IV.B.9.d.) No help for this item at this time. Survival Surgery being performed on non-rodent mammals (e.g. rabbits, dogs, cats, primates, etc.), must be done in an approved facility. Name the facility: (Question IV.B.10.) Rabbits are not rodents and rabbit surgery must meet all the procedural and facility requirements as used for other non-rodent species.Rutgers Policy: Non-rodent Surgery, "Post-operative care"
Rutgers Policy: Rodent Surgery, "Post-operative care"
Guide, "Pain, Analgesia and Anesthesia, pp. 64-65
AWA, "Need for analgesia"
Rutgers Policy: Non-Rodent
Surgery Policy
Guide,
"Facilities for Aseptic Surgery", pp. 78-79
AWA,
"Surgery facility requirements"
C. NON-SURGICAL SURVIVAL PROCEDURES ON ANESTHETIZED ANIMALS
Coming soon: references for limits on volumes for blood collection.
Anesthesia for non-surgical survival procedures: (Question IV.B.C.12)
Guide, "Pain,
Analgesia and Anesthesia, pp. 64-65
V. PROCEDURES ON CONSCIOUS-UNANESTHETIZED ANIMALS
Procedures on conscious unanesthetized animals: (Question V. 13-14)
Procedures to describe here would include: ascites production; injections; other routes of dosing such as gavage and skin painting; breeding colonies, etc. Details on blood collection and immunization are requested in questions 14.a. and 15.
Ascites production should be carefully justified with respect to alternative in vitro methods.Rutgers Policy: Guidelines for Producing Monoclonal Antibodies in Mice
OPRR "Dear colleague letter: Sources of Custom Antibody
OPRR "Dear colleague letter: Production of Monoclonal Antibodies Using Mouse Ascites Method
Information Resources for Adjuvants and Antibody Production
If collecting blood or other tissue/fluid provide the following information: (Question V.14.a.)
Rutgers Policy: Bleeding Techniques in Rabbits
Coming soon: references for limits on volumes for blood collection.
Will animals be immunized? (Question V. 15)
Freund's complete adjuvant may only be used for initial injections. Freund’s incomplete adjuvant may be used repeatedly.
USDA’s Policy Manual states, "Freund's Complete Adjuvant used for antibody production may cause results ranging from momentary or slight pain to severe pain depending on the product, procedure, and species."
Rutgers Policy: Guidelines for Producing Monoclonal Antibodies in Mice
OPRR "Dear colleague letter: Sources of Custom Antibody"
OPRR "Dear colleague letter: Production of Monoclonal Antibodies Using Mouse Ascites Method"
Information Resources for Adjuvants and Antibody Production
USDA Animal Welfare Policy Manual, Policy 11: Painful/Distressful Procedures
VI. OTHER PROCEDURES
Prolonged Restraint: (Question VI.16)
Procedures which formerly required prolonged restraint in a chair, sling or other device, can often now be accomplished in freely moving animals using tethers, telemetry, or implantable pumps.
Guide, "Physical Restraint", p. 11
Disease or Pathological Condition: (Question VI.17)
Will this protocol require the use of animals that have a disease or pathological condition (including those experimentally induced)? (Question VI.17.a.)
Examples of pathological conditions include:
If you are using an animal with a special genetic trait, such as a mutant strain, a transgenic or knockout mouse, explain whether there is a phenotypic expression of the trait that will adversely affect the animals.
Rutgers Policy: Guidelines for Producing Monoclonal Antibodies in Mice
AWA, "Death as an endpoint and requirements for euthanasia"
What criteria will be used to identify animals that may be in pain and/or distress? (Question VI.17.d.)
Where consistent with experimental goals, death as an endpoint should be avoided. Describe the endpoint? What criteria will be used to identify the endpoint? (Question VI.17.g.)
Guide, in "Animal Care and Use Protocols, p. 10
Does the study design require that animals be allowed to die from the disease and/or pathological condition ("death as an endpoint")? (Question VI.17.h.)
Guide, in "Animal Care and Use Protocols, p. 10
Dietary Manipulation, or food and/or water restriction: (Question VI.18.)
This section of the protocol form was expanded in the March 1998 protocol form revision in response to language in the 1996 Guide for the Care and Use of Laboratory Animals. The intent of these questions is to document the steps taken to assure that dietary manipulation and food or water restriction are done appropriately.
Guide, "Food or Fluid Restriction", p.12
c. Water Restriction: Will you restrict the amount of or access to water? (Question VI.18.c.)
This section of the protocol form was expanded in the March 1998 protocol form revision in response to language in the 1996 Guide for the Care and Use of Laboratory Animals. The intent of these questions is to document the steps taken to assure that dietary manipulation and food or water restriction are done appropriately.
Question 18.c.5 need not be answered if the answer to 18.c. is "No".
Guide, "Food or Fluid Restriction", p.12
The final disposition of the animals is: (Question VI.20)
Rutgers Policy: Adoption Policy
Rutgers Policy: Disposition of Farm Animals
Final Disposition: Euthanasia: (Question VI.20)
The animal use sections of many grant applications (e.g. PHS Form 398, Section F) require you to describe the method of euthanasia and to state whether the method is in compliance with AVMA Guidelines.
Rutgers Policy: Rodent Euthanasia Policy
Guide, "Euthanasia, p. 65-66
LAS Manual, "Euthanasia"
AVMA Panel on Euthanasia
CO2 Inhalation (compressed gas and dry ice): (Question VI.20)
Dry ice may be used to generate CO2 and may be more convenient in labs where dry ice is generally available. The committee is concerned that animals’ skin not be exposed directly to the solid dry ice or other cold surfaces. Describe the how you will avoid this.
Rutgers Policy: Rodent Euthanasia Policy
AVMA Panel, "Carbon Dioxide"
Decapitation - Provide justification: (Question VI.20)
Rutgers Policy: Rodent Euthanasia Policy
AVMA Panel, "Physical Methods"
LAS Manual, "The Controversy Concerning Decapitation and Cervical Dislocation"
Cervical Dislocation - Provide justification: (Question VI.20)
Rutgers Policy: Rodent Euthanasia Policy
AVMA Panel, "Physical Methods"
LAS Manual, "The Controversy Concerning Decapitation and Cervical Dislocation"
Pentobarbital - Specify route and dose: (see table below) (Question VI.20)
AVMA Panel, "Barbituric Acid Derivatives"
Exsanguination under anesthesia - Specify anesthesia: (Question VI.20)
AVMA Panel, "Physical Methods"
Other - Specify dose and route: (Question VI.20)
Note: Ketamine is not an acceptable method of euthanasia.
AVMA Panel, "Unacceptable Injectable Agents"
Method of Euthanasia: (Question VI.20.a.)
Dose should be expressed as mg/kg of body weight. Pentobarbital is commonly available in forms intended for anesthesia (~65 mg/ml) and for euthanasia (~385 mg/ml). The euthanasia form is not suitable for anesthesia. Both products are controlled substances.
VII. PROCEDURES INVOLVING HAZARDOUS AGENTS
Volatile Anesthetic: (Question VII.21.)
Use of ether as an anesthetic is strongly discouraged. Ether is flammable can be explosive. Ether must be used in an REHS approved fume hood. Class II biosafety cabinets filter particulates, not gases, and a biosafety cabinet is not appropriate for use with ether unless its exhaust is ducted to the outside, and the cabinet is approved by REHS for use with flammable/explosive material.
Metofane® (methoxyflurane) may be used in an anesthetic chamber or with a nose cone, but there must be provisions for venting (e.g. a fume hood) the metofane. Metofane has become increasingly expensive as its use in general veterinary practice declines.
Halothane and isoflurane must be used with a precision anesthetic vaporizer. Vaporizers are designed for a specific gas. These anesthetics achieve lethal concentrations when used without a vaporizer. Isoflurane and halothane are both available now as generic drugs and are much less expensive than they used to be. Anesthetic systems must scavenge all anesthetic waste gases given off by the machine or patient.
Hazardous Agents: (Question VII.22.):
Rutgers Policy: Use of hazardous substances in animals
Rutgers Policy: Use of transplantable tumors and cell lines in animals
LAS Manual, "Hazardous Agents"
Are permits required for field studies, holding exotic animals, etc.? (Question VIII.23.)
A number of professional societies have developed guidelines for various species (e.g. birds, fish, mammals, reptiles and amphibians) less commonly used as research animals.
"Personnel Qualifications and Training", pp. 13-18
The narrative is very important. It is where you can explain your research in a logical, step-by-step manner. It is OK to plagiarize from your own grant application(s), but it is rarely appropriate to do so with significant editing.
The audiences for a grant application and the protocol narrative are very different. In the first, you are trying to impress experts in your field. Most people reading your narrative are not experts in your field. Some are non-scientists. Then are the veterinarians! You can use technical terms and jargon, but the terms should be defined in a way that a lay person will understand.
The committee is most interested in what happens TO the animals. Brief descriptions of non-animal procedures and handling of animal tissues in vitro may be needed to put the animal procedures in context, but the committee is not interested in the same level of detail about these procedures as are grant reviewers. One page is a suggested limit for the narrative.
AWA, "Information required in animal use protocols"
Sample protocol narratives
XI. JUSTIFICATION FOR ANIMAL USE (Questions XI. 27-30)
Answers to these questions are required by very specific language in the federal Animal Welfare Act (AWA) and the AWA regulations. The committee is very picky about these questions because they are routinely reviewed by USDA inspectors and AAALAC site visitors.
Guide, "Animal Care and Use Protocols", p. 10
Justify the appropriateness of the number of animals to be used. Check all that apply: (Questions XI. 29.)
If you are using large numbers of animals (e.g. 100 or more mice, 2 or more Giant Pandas!), it is recommended that you provide a breakdown of how the animals will be used. If large numbers are to be used as part of a breeding colony, indicate this. If some procedures involve a "high ethical cost" (unrelieved pain, use of paralytics, death as an endpoint, prolonged restraint, etc.) make it clear how many animals fall into this category. Although not asked for by the committtee (yet!), it may be helpful to briefly outline the study design , showing the number of animals per group, the number of groups per experiment, and the number of experiments to be performed each year.
AWA, "Rationale for species and justification of animal numbers"
If the proposed procedures might cause more than momentary pain or distress, (It is the USDA's position that terminal surgery is a potentially painful procedure with the pain alleviated by anesthesia [depending on the plane of anesthesia] and that it is considered a painful procedure which is alleviated by drugs.) list the sources that were used to determine that non-painful alternatives are not available. Check all that apply: (Questions XI. 30.)
This is the biggie! This is the question that causes more pain and distress for investigators and the committee than any other. This is also the question most closely reviewed by outside reviewers.
"Question 30" must be answered if your project MIGHT involve more than momentary pain or distress. Don’t think your project involves pain? Read on. Among other things, USDA considers surgery performed under general anesthesia to have the POTENTIAL for pain if the anesthesia is not done properly. Likewise, "acute" (i.e. non-survival) surgical or other invasive procedures performed under anesthesia from which the animal does not recover consciousness before anesthesia, is also considered potentially painful. Thus, you must address Question 30, for any surgical procedure, survival or non-survival.
USDA has issued a policy statement to clarify what they feel constitutes a sufficient attempt BY THE INVESTIGATOR (as opposed to the committee) to determine that alternatives to painful procedures are not available. Many committees have in the past interpreted the regulations to require a literature search for every protocol. Some require the search be submitted with the protocol. This has led to a lot of busy work that has very rarely benefited animal welfare or research. USDA’s "Policy 12" makes it clear that a literature search is not required, but that if it is used, the committee must be given enough information so that it can determine that the search was reasonable and sufficient. Items "a" through "d" below are included in the Rutgers form in response to Policy 12.
Note that consideration of non-painful "alternatives" should include not only replacement of animal procedures with non-animal procedures, but also all of the "three-R’s", including replacement with non-animal procedures, reduction of animal numbers, and refinement with better and less painful procedures. It is not sufficient to search solely on the subject of your research (e.g. diabetes). The search must also deal with the procedures involved.If you are like most faculty researchers, you and your lab have been working in your area of interest for many years. Many of the procedures you use have been in use for years and have been constantly refined. The regulations suggest you "defend" your procedures with a single literature search. (In my personal opinion, those who promoted these regulations assumed that all research was painful, irrelevant, unnecessary duplicative, had already been done and that the answers could be found if only you put the right 3 key words into a literature search. We can’t do anything about that.) Instead, I suggest that you proffer yourself as an expert and outline your ongoing efforts to keep up with your field of research.
Suggested language is given below. You are welcome to copy appropriate statements into your protocol.
[ ] I am experienced in this area of research, and I am knowledgeable about alternatives to painful procedures as evidenced by the following:
[ ] I have been involved with research in this area for ______ years, beginning in ____ (year).
[ ] I have kept up with the literature on the subject of this protocol for ___ years, beginning in _____(year).
Subject(s)or keywords relevant to alternative methods: ________________
[ ] I maintain a literature database on the subject of this research, which now contains ____ references. (The committee may ask to examine your database.)
[ ] My lab (research group) has published _____ papers on this subject during the period ____ to ____.
[ ] I have authored ____ papers on this subject during the period _____ to _____.
[ ] I have served on ____ grant review committees for the following funding agencies: _________
[ ] I have served as a reviewer of scientific articles for the following journals for the years indicated.
[ ] I have attended _____ scientific meetings in my field during the last ______ years.
[ ] I have trained ____ doctoral students and _____ post-doctoral students during the period ____ to _____. Staff in my lab currently consists of _____ faculty, _____ post-doctoral fellows, _____ doctoral candidates, _____ undergraduate students and _____ technicians.
[ ] Since beginning work in this field, I have reduced, refined or replaced the use of animals in the following ways: List:
[ ] Other:
LAS Manual, "Alternatives to Animal Research"
USDA "Policy 12 pertaining to requirements of a literature search"
AWA, "Alternatives" Text of the Animal Welfare Act Regulations
Animal Welfare Information Center
AWIC Alternatives Page Includes a sample search strategy for alternatives
AWIC Alternatives Pamphlet Includes a list of suggested keywords for alternatives searches, and a list of databases
National Agricultural Library
The Principles of Humane Experimental Technique Full text of Russel and Burch's classic 1959 book that first proposed the concepts of replacement, refinement and reduction
Hazardous substances in animal research questionnaire: (APPENDIX A.1 and A.2)
Rutgers Policy: Use of hazardous substances in animals
Rutgers Policy: Use of transplantable tumors and cell lines in animals
LAS Manual, "Hazardous Agents"
REQUEST TO USE TRANSPLANTABLE CELL LINES AND TUMORS IN ANIMALS: (
APPENDIX B)Rutgers Policy: Use of transplantable tumors and cell lines in animals
LAS Manual, "Hazardous Agents"
