Guidelines to Use During Completion of Animal Care and Use Forms

Copies of the forms and Guidelines may be obtained from the UCAR Office or downloaded from this site. These Guidelines and forms must be used; no others will be accepted. According to NIH and University Policy, a new and full review and approval of UCAR protocols is required every three years (36 months).

The University requires that all laboratory animal use be reviewed and approved by UCAR regardless of funding source.

General

Please submit the original and 7 copies of Section A, and only if applicable, B and C. Provide an electronic version as well, prefferably MSWord document.

Please comply with our request that all forms be typed and, if using word processors or computers, the answers be printed in regular font to readily contrast them from the questions. Use Times font, 10 to 12 point.

NOTE: UCAR will not review any forms that are not typed or produced by letter quality printers, do not have pages consecutively numbered or that deviate from the UCAR format. Contact the UCAR Office Staff for information about borrowing a diskette containing the UCAR forms.

Investigators are encouraged to contact the UCAR Office Staff at extension 5-1693 or DLAM veterinary staff at extension 5-2651 for assistance in development of animal protocols. It is required that veterinary consultations be made for procedures that "cause more than momentary or slight pain or distress to the animals" as defined in the Animal Welfare Act regulations. There is no charge for such consultations. Animal Care and Use forms must be received in the UCAR office prior to or at the time of submission of grants for sign-off. New grants may be sent to funding agencies with either a "pending" or an approval letter.

Section A: Animal Care and Use Summary

1. Only University faculty members are eligible to submit protocols to UCAR. In the case of students, e.g. undergraduate, graduate or postdoctoral, or other, the application must have the name of the faculty sponsor followed by the name of the student and signatures of both. The P.I., i.e., faculty member, is responsible to the University for the conduct of research under his/her supervision. The Department in which the P.I. has his/her primary appointment; department, University mailing address, and University telephone number are self explanatory.

2. Title of Proposal: Title of protocol.

3. Under Type of UCAR Approval, check the appropriate line. Indicate the Course Number and Title if the category is Teaching. If the proposal is either new or a competing renewal, a new set of forms must be submitted to UCAR. Modification of an approved protocol is for any significant changes in an approved protocol that may affect the animal's welfare and hence must be prospectively reviewed and approved before it is performed. Failure to do so is a violation of the University's Animal Welfare Assurance to the Public Health Service and the Federal Animal Welfare Act. Proposed changes must be clearly identified and contrasted with the previously approved protocol. A pilot project is for a relatively short duration (usually less than one year) for the purpose of acquiring preliminary data for use in a subsequent grant or contract application. The Committee requires approximately 30-60 days to review, revise if necessary, and act on submitted protocols.

4. Under Funding Source and Deadline for Submission, check the appropriate line and indicate the deadline for your submission submission is to NIH, NSF, or any of a number of the other funding sources. Contact the UCAR Executive Secretary if you have any questions about whether peer review is provided by a funding source or about securingpeer review within the University that will satisfy the University's Public Health Service Animal Welfare Assurance and USDA Regulations.

   For New Applications the Public Health Service and most other agencies give the applicant a sixty-day period following the submission deadline for UCAR review and necessary approval. Since the UCAR review process may take up to 60 days, it is advisable to submit one's UCAR proposal as early as possible. Modification of approved protocols must be reviewed and approved prior to initiation. Any substantive changes in an approved protocol that may affect the animal's welfare must be prospectively reviewed and approved before it is performed. Not to do so is a violation of the University's Animal Welfare Assurance to the Public Health Service and the Federal Animal Welfare Act. Proposed changes must be clearly identified and contrasted with the previously approved protocol. For modifications of an approved protocol, the Committee requires 30-60 days to review and approve the modification. In addition to completing the form, provide a memo that explains the change from the original protocol.

   In all cases, whether New, Competing Renewal, or a Modification of an Approved Protocol, it is important to note that the Principal Investigator is responsible for sending a copy of the UCAR Approval Letter to the appropriate funding agency and official. New Applications for Public Health Service proposals are generally all sent to the Division of Research Grants for initial processing and assignment to an appropriate review body. You must send the UCAR Approval Verification Letter to the administrative officer of the assigned review body. If it is sent directly to the Division of Research Grants, unnecessary delays in the review process may occur.

5. Provide a description of the specific aims of the research in language that a member of the general public can understand. It is important to indicate how the results of the research are likely to benefit humans and/or animals. There is a lay person on UCAR who must understand your objectives and methods. These lay summaries may also be used by the University's Public Relations and Communications Personnel. It is particularly important to have an excellent layman's summary for those kinds of studies which may involve the potential for pain or for studies involving certain species, such as dogs, cats, and nonhuman primates. All lay summaries should be carefully and thoughtfully written. Please do so in the space provided in the form. A protocol with an inadequate lay summary will be returned to the P.I., which may lead to unnecessary delays.

6. Provide an abstract of the proposed application so that scientifically trained members of the Committee can understand your general methods and objectives. If an abstract has already been prepared for NIH or another application, you may use it here.

7. Under Use of Vertebrate Animals, sections a. and b. are identical to those required in the Section of grants that discusses animal use and are necessary to clearly justify the rationale for animal use. Although one's application may be for greater than three years of support, UCAR approval will be for three years only. Federal regulations require full review and approval every three years.

8. Animal Numbers and Use Category. For each species to be used, provide a yearly account of the estimated animal numbers and their use categories for the duration of your proposal. This information is required for the U.S.D.A. each year.

9. Justification of the number of animals to be used for each species. According to federal regulations the UCAR needs assurance that: 1) a sufficient number of animals is used to satisfactorily answer the scientific questions posed, and 2) an excessive number of animals are not used. When appropriate, provide an estimation of the sample size (power analysis) needed to reach an appropriate level of statistical significance for your study. In calculating your sample size provide information about the known variability in the control population of your measured variable as well as the minimum statistically significant change you plan to determine in your experimental population. Consultation with the Department of Biostatistics may be helpful in addressing this section.

10. Alternatives and Literature Searches. The intent of the question is to determine whether the P.I. has adequately considered in vitro techniques, and also techniques using either less sentient animals, or procedure that produce the least amount of pain or discomfort. This question is an attempt to have the P.I. utilize, what has become to many, the 3-Rs - Replacement, Reduction and Refinement, that was attributed to an often quoted text by Russell and Birch (Russell, W.M.S. and Birch, R.L., The Principles of Humane Experimental Technique, Methuen, London, 1954). The term Replacement is intended to see if, or how, living animals can be replaced by other non-animal or less sentient animal models. Reduction refers to the use of the fewest animals necessary to achieve experimental objectives by applying the most appropriate experimental design including the prospective consideration of statistical considerations. The term "Refinement" refers to the use of more accurate and sensitive technical methods of laboratory analyses that may lead to the use of fewer animals if possible. It refers to both the use of the most appropriate animals based upon, not only the model itself, but also the genetic and microbiological quality of the animals that, if well defined, and maintained free from adventitial microbiological agents, may result in more reproducible and valid research results. It means the close control of non-experimental variables, such as, the maintenance of proper environmental controls and animal care and veterinary practices.

    The Federal Regulations state that "the principal investigator" has "considered alternatives to procedures that cause more than momentary or slight pain or distress to the animals and has provided a written narrative description of the methods and sources used to determine that alternatives," (to these painful procedures) "are not available." (Reference: The Federal Register, Thursday, August 31, 1989, Vol. 54, No. 168, page 36152, entitled "9 CFR Parts 1, 2, and 3, Animal Welfare; Final Rules."

    The method that is recommended is to conduct an up to date search of the literature specifically related to the proposed research program and specific protocol under consideration. This search would attempt to identify either less sentient animal species, techniques and/or procedures that would be intrinsically less invasive or painful, or in vitro techniques that could be considered.

    The source(s) used for the literature search should be databases such as Medline, PubMed, Web of Science, TOXNET, AGRICOLA, or others that are relevant to the protocol topic. Information about Miner Library assistance with animal testing alternatives literature searches can be found at http://www.urmc.rochester.edu/hslt/miner/resources/researchers/Animal_Testing_Alternatives.cfm . The Animal Welfare Center (AWIC) at the National Agricultural Library also provides assistance with alternatives literature searches. AWIC can be contacted by telephone at (301) 505-6212, FAX (301) 504-7125, or via e-mail at awic@nal.usda.

11. If recovery surgery is to be performed, please check yes, and complete Section B - Recovery Surgical Procedures. If you check no, you do not propose to do recovery surgery, go on to question 10.

12. Will infectious agents, radionuclides, hazardous substances or drugs that may be shed into the animal environment be used? Examples include HIV., MPTP., DMBA, BRDU., cyclophosphamide, cyclosporin, and human cells, tissue, blood, or viruses. If no, please indicate and go on to item 11. If yes, complete all parts of Section C and send the complete UCAR form (Sections A, C, and if applicable, Section B) to Environmental Health and Safety for review.

13. Pain or distress to animals.
    
    
    1. Describe specific procedures that will be employed to minimize or limit pain or distress. Describe the use of anesthetics, analgesics or other drugs that will prevent or reduce perception of painful stimuli. Describe any physical or environmental changes which may also reduce discomfort or injury. Animals in which pain or discomfort may occur must be observed daily by research staff. Where pain or distress is produced and cannot be relieved, scientifically justify. What endpoints or conditions would be used to determine the time of euthanasia?

    2. Describe any pain or distress the animals may ultimately experience. If none is expected, so state. If pain, stress, distress, or injury or permanent physical, physiological, or pathological impairment is anticipated, please describe. Based upon your experience, that of colleagues, or the literature, what adverse physiological, pathological, or behavioral sequelae may probably occur as a result of the proposed study? What complications may be associated with this type of intervention? What could and/or would be done to alleviate discomfort or injury?

    
    

14. Describe the euthanasia method(s) to be used. Specify the agent (proprietary and generic name), dosage (mg/kg of body weight), and route of administration. If physical methods are to be used, describe the technique. The American Veterinary Medical Association's 2000 Report of the Panel on Euthanasia, as published in J.A.V.M.A. Vol. 218, No. 5, Pages 669-696 (2001) must be consulted and deviations from recommended methods must be scientifically justified. For example, decapitation or cervical dislocation of rodents without prior sedation must be scientifically justified. Copies of this report are available from the UCAR Office.

    What measures are taken to insure that the laboratory animal is dead before being taken to the Vivarium animal morgue? Measures include, opening the chest and severing major vessels, exsanguination, decapitation, and harvesting of vital organs. For animals that euthanasia does not include physical methods, it is recommended that the chest be opened or major vessels severed.

15. Describe all other manipulations (other than recovery surgery) not already described above that any animal or experimental group may undergo. The manipulations and procedures listed below are not all-inclusive. They are examples of the information required by UCAR so that the humane aspects of the proposed study are appropriately considered. Guidelines specific to monoclonal antibody production, polyclonal production, and tumor "endpoints", are available from the UCAR Office.

    UCAR understands that some experimental plans are simple, others are complex, and involve many different experimental and control groups that receive different treatments or procedures. Please provide for each given group of animals a summary of the specific procedures to be performed. In the description please include the temporal relationship of each procedure when appropriate.

    For example:

    Group A: (10 rats) acclimatization, baseline bleed and immunization begun on 8th day; boosters on days 21 and 42; bleed on days 60, 90, 120, on day 120 anesthetize, exsanguinate and harvest of the spleen.

    Group B: (10 rats) acclimatization, baseline bleed begins on 8th day, mini-osmotic pump containing X surgically implanted under general anesthesia on day 15; boosters on day.....harvest of the spleen.

    Group C: (10 rats) acclimatization, anesthesia, terminal bleed and harvest of spleen on day 8.

    Descriptions of other manipulations:

    1. Substance administration. Describe materials to be administered, dosage (mg/kg); (please note that a drug concentration, e.g. mg/ml or xml of a y% solution is not a dose!) total volume, volume per site, if applicable; dosing regimen (frequency and total number of doses), gauge of needle to be used, specific adjuvants used, if any; and any other information relative to the administration of substance to living animals.

    2. Specimen withdrawal. Describe what specimens will be taken from living animals (e.g., blood, urine, feces, bile, expired gases, cerebrospinal fluid and salivary gland secretions). Specifically describe the technique used, including, e.g., site of collection, preparation of the site if drawing blood or CSF, gauge of needle, volume of specimen, frequency of specimen collection, approximate range of weight of animals and species used.

    3. Physical restraint. If manual restraint only is used, please state. If physical restraint requiring the use of any device is to be used, please describe the device (dimensions and materials), duration and frequency of physical restraint and animal conditioning procedures. Also describe the frequency and method of cleaning and sanitizing the device. Provide scientific justification for the use of physical restraint if it is longer than a few minutes duration to facilitate either substance administration or specimen collection.

    4. Use of chemical restraint. Describe in detail the use of tranquilizers, anesthetics and analgesics not related to recovery surgical procedures, which need only be described in Section B. Give the proprietary and/or generic name of the drug, dosage (mg/kg of body weight). (Note that a concentration is not a dose!) Provide the route, gauge of needle, and method of evaluating the initial desired effect, e.g., anesthesia or tranquilization, method of monitoring for continuation of desired effect for whatever duration the effect is required, duration of procedure requiring chemical restraint, and planned schedule for supplementing the animal, if needed. For "acute" experiments, deep surgical anesthesia is required prior to organ or other specimen collection, which is then followed by the death of the animal. Describe the means by which one will establish and/or maintain an adequate plane of anesthesia.

       If muscle paralysants are necessary for the planned studies, give the generic name and dose of the product. Justify its use and describe in detail the methods to be used to monitor the animal for pain or distress. Muscle paralysants may not be used without adequate scientific justification, a well-defined and acceptable anesthetic regimen and an effective method to assess the adequacy of the accompanying anesthesia. Consultation with the veterinary staff in DLAM during protocol development and before submission to UCAR is essential.

       If you do not have personal experience with the specific use of any of the chemicals or pharmaceuticals you plan to use and have described on the UCAR forms, include a copy of a reference concerning the use of the chemical or pharmaceutical substances in the specie(s) in question for the purpose you have described, e.g. as a general anesthetic for rodent recovery surgery or a substance that reportedly increases heart rate. If such a specific reference is unavailable, describe how the chemical agent was selected, any previous experience while at another institution or information communicated from outside colleagues. UCAR may require that one or more of its members and/or veterinarians from DLAM be present to determine, if possible, the physiological or pathological effects.

    5. Aversive Conditioning, Food or Water Scheduling, or Sensory Deprivation. If any form of aversive conditioning (e.g., footshock) is proposed, scientifically justify and both qualitate and quantify, as best you can, the aversive condition. If access to food or water is restricted in any manner, scientifically justify and describe in detail the criteria which you propose to use to determine that each given animal, so restricted, receives an adequate diet and fluid intake. DLAM veterinarians have developed an acceptable recordkeeping procedure that places the responsibility on the PI or on Vivarium animal care technicians A feeding or watering log for each individually-housed animal must be kept. Please work with a DLAM clinical veterinarian and Vivarium Supervisory personnel in developing and implementing any restricted or scheduled food or water regimen. If the creation of caloric or other specific deficiency is an intrinsic part of the proposed study, please describe.

    6. If any form of sensory deprivation is proposed, scientifically justify and describe in detail the duration, extent and known or anticipated effect of the sensory deprivation.

    7. Administration of any harmful or potentially harmful physical agents (e.g., irradiation, microwaves, radio frequency waves, thermal injury, physical injury, environmental injury, such as high levels of sound, high or low temperature, barometric pressure, ultrasound, high intensity light or exposure to potentially damaging wavelengths). Please scientifically justify the use of such physical agents and describe the conditions of exposure, the "quantity", duration and frequency of exposure, and the expected effects of such exposure.

    8. Nonrecovery or acute surgical procedures. Describe briefly any nonrecovery procedure, e.g. tissue or fluid harvesting, or nonrecovery or acute surgical procedure immediately following which the animal is euthanatized without regaining consciousness. Be sure to give a detailed description of the anesthetic regimen and methods for both establishing and maintaining a surgical plane of anesthesia.

    9. Specific use of adjuvants to produce polyclonal antibodies in mammals and birds. UCAR has literature available that provides information on newer, more refined adjuvants and those that minimize the possibility of excessive inflammation leading to tissue necrosis, than Complete (CFA) or Incomplete Freund's adjuvant (IFA) as well as a method for immunizing domestic fowl and harvesting antibody from their eggs. Seek UCAR assistance for materials on alternative methods used for polyclonal antibody stimulation. Use of these alternate species and adjuvants are encouraged; however, if Complete Freund's Adjuvant is used, it may only be administered once. Subsequent injections aimed at developing an anamnestic response can use Incomplete Freund's Adjuvant. Other procedures aimed at reducing the incidence of tissue necrosis and unnecessary pain or discomfort are available from the UCAR Office.

    
    

16. Will living animals be removed from the Vivarium? The term Vivarium means any UCAR authorized animal housing facility at the University. If no, please indicate and go on to item 17. If yes, animals must be taken to a laboratory, please give the specific laboratory or room location, the maximum duration that animals will be kept in the laboratory, and a brief description of the kinds of procedures to be performed in that location. For example, "Rats will be taken to 3-xxxx (location) in compliance with UCAR "Animal Transport Policy" where they will be anesthetized with pentobarbital, euthanized by exsanguination, and tissue or organ samples collected (within several hours). It is important to indicate when individual animals are taken to the laboratory on multiple occasions because of disease prevention, public health and security considerations.

    The USDA requires that if a regulated animal (e.g., dog, cat, monkey, guinea pig, hamster, ferret or rabbit) is out of its authorized housing room for more than 12 hours, that area must be designated as a "Study Area" and must be inspected semiannually by UCAR. University policy restricts laboratory rats and mice (presently non-regulated animals) from being kept outside the Vivarium for greater than 24 hours unless an application is made and approved by UCAR for a satellite facility. This requires the demonstration of the scientific necessity for such a facility that cannot be met in existing Vivarium space. A satellite must meet all governmental and University regulations and standards. All satellites are subject to the "University Policy on Satellite Animal Care and Use Facilities" in effect at the time. For further information on either satellite facilities or study areas, please contact the UCAR office.

17. Identify all personnel working with living animals. Starting with the PI, give the name, University location (office or laboratory), University telephone number, and at least two non-University telephone numbers to be used in a case of an emergency. The PI must update this information when staff or staff responsibilities change. The PI can also prioritize the persons listed as to order one may attempt to locate in case of an emergency. Someone in the investigative group must always be available. If a veterinarian is unable to reach an emergency contact to seek guidance and assistance in determining a course of action, they may have to treat an animal or euthanitize it.

18. Are the persons identified in item 17 enrolled in the Occupational Health Program for Persons with "substantial" animal contact? If no, please contact the UCAR Executive Secretary at extension 5-1693, for information and guidance.

    All personnel with "substantial animal contact" are required to be evaluated in light of their occupational exposure to certain laboratory animals and enrolled in an occupational health program appropriate for their situation. Anyone working directly with nonhuman primates, sheep, and wild caught animals (bats, squirrels) or having even minimal exposure to them, or their tissues and fluids, are considered to have "substantial" animal contact and must be evaluated by the University Health Service. Serum banking may be required in some cases. Semiannual tuberculosis testing, baseline CBC's or clinical chemistry determinations may be performed under certain situations. The need for a specific program will be evaluated by University Health Service staff. For workers with exposure to specific pathogen free (SPF) rodents and rabbits in programs that do not involve any specific hazardous substance or infectious agent, all that may be required is a pre-employment assessment, health history, and periodic tetanus immunization update. Personnel working with hazardous substances as part of the experimental protocol (eg. radiation hazard, chemical hazard, carcinogen, mutagen, neurotoxin, biohazard) must be evaluated by the Environmental Health & Safety Division as part of Section C of the UCAR form.

19. & 20. Have all of the personnel listed above completed the Responsible Care and Use of Laboratory Animals Certification Program? If no, please contact the UCAR Executive Secretary at Extension 5-1693 for information about the program. It is required by federal law that anyone working with laboratory animals have appropriate training and/or experience with the species and specific procedures to be performed. This information should be listed in Question 20.

Carefully read the certification and assurances you are giving the University when you sign the completed Section A of the protocol.

Section B: - Recovery Surgical Procedures

1. Species and approximate number of animals to be used per year. Provide a separate Section B for each species. This is especially important when both a USDA regulated species, e.g., rabbit, cat, nonhuman primate or dog and a USDA unregulated species, e.g., a laboratory bred rat (Rattus norwegicus) or mouse (Mus musculus) is used. The species and number on which surgical procedures are to be performed may be different from the species and numbers given in Section A because perhaps not all animals will undergo such procedures. A separate Section B facilitates the review.

2. Specific surgical facility or laboratory to be used. Regulations promulgated by the USDA state that, "All survival surgery" on all mammalian and avian species "must be performed using aseptic procedures, including sterile surgical gloves, masks, caps, sterile surgical gloves and aseptic techniques". In the case of "major" operative procedures on non-rodents, these will be conducted only in facilities intended and dedicated to that purpose. The definition accepted by the USDA and NIH for "major surgery" or "major operative procedure" is: "any operative procedure that enters or opens a body cavity or any procedure that produces permanent handicap in an animal that is expected to recover".

   The performance of minor survival surgery on regulated species and survival surgery on Rattus norwegicus and Mus musculus usually do not require the use of a dedicated surgical suite, approved for major surgery. However, recovery surgery on all species must be done aseptically. UCAR will decide, based on the description of the recovery surgical procedure whether or not it is to be considered "major" or "minor." In general, UCAR has accepted as "minor" those procedures commonly performed on an outpatient basis in human medicine, provided, of course, that the investigator describes appropriate aseptic procedures that will be used. Please review your copy of UCAR "Guidelines for Rodent Survival Surgery". If you do not have one, contact the UCAR Executive Secretary.

   The Division of Laboratory Animal Medicine's Experimental Surgical Facility is available to any investigator at the University and has been developed as a resource by the School of Medicine and Dentistry. It is well-equipped, provides many anesthetic regimen options, including a number of very safe and effective anesthetic gases, and sophisticated equipment to aid the surgeon and anesthetist in monitoring the depth of anesthesia. The surgery is staffed by well-trained personnel and is in very close proximity to the offices and laboratories of the veterinary staff of the Division. The technical supervisor and other technical staff are well-trained and equipped to handle most anesthetic-related problems.

   The regulations promulgated by the USDA state that, "non-major recovery surgery and all surgery on rodents do not require a dedicated surgical suite, but must be conducted aseptically". UCAR, under its Public Health Service Animal Welfare Assurance, determines whether or not a given procedure is major or minor and whether or not the procedure must be conducted in an approved dedicated surgical suite or aseptically within a specifically designated and maintained area within a laboratory. However, such surgery MUST be performed using sterile instruments, surgical gloves, and aseptic technique. The laboratory or facility in which survival surgery is conducted on rodents should contain an area that is readily sanitizable, free from extraneous equipment and supplies that are designated for this purpose. It is suggested that a laminar flow cabinet, similar to that used for tissue culture work, is an excellent device in which aseptic surgery can be performed on rodents.

   Section B is primarily reviewed by a Division veterinarian(s) and then by the "attending veterinarian," the term used by regulatory bodies for the University "Veterinarian-in-Chief".

3. Type or kind of operative procedure. Give a brief indication of the type or kind of procedure. For example: adrenalectomy, nephrectomy, thoracotomy, cannulation of x vessels, stereotaxic lesioning of the brain, partial hepatectomy.

4. Describe preoperative procedures, including fasting, premedication, and preparation of the surgical site. UCAR has tried, in this revised set of guidelines, to make this section easier to complete by using some check-off options and leading one to better describe what's being done. For example, it is usually recommended that an animal be fasted for 12-18 hours prior to undergoing general anesthesia and surgery. It is advisable to restrict water for only a few hours prior to surgery, if possible. This is often not possible in cases where the surgery is scheduled in the early morning. In this case one may make arrangements for a limited amount of water to be available to the animal through a special-request form, submitted to the Vivarium.

   One may wish to premedicate the animal with a tranquilizer to facilitate a smooth induction and recovery as well as requiring less anesthetic, and/or administer atropine to lessen salivary secretions. The surgical site should be clipped, shaved, or otherwise depilated. So-called nude mice, neonatal rats and mice and/or some other species may not require clipping or shaving but the site must be appropriately cleansed and prepared using an appropriate surgical antiseptic, e.g., an iodine-based surgical detergent-disinfectant. When describing premedications, specify the proprietary and/or generic name, dosage (mg/kg of body weight), and route of administration. Other relevant preoperative procedures should also be described. For guidance on any aspect of this or subsequent items, please contact one of the Division of Laboratory Animal Medicine's veterinarians or the supervisor of the Division's Surgical Facility for assistance.

5. Describe anesthetics and other drugs used. Describe the proprietary and generic name, or only the generic name(s) of drugs to be used and give both approximate dosage (mg/kg body weight) and route of administration. Since most anesthetics administered to small non-rodents need to be given "to effect," to reach a surgical plane of anesthesia, all dosages are considered approximate ones. Indicate the anticipated duration of the surgical procedure and how one would supplement the initial anesthetic dosage, if necessary.

   If the anesthetic product contains more than one agent, please give the dose, (not concentration or milliliters/kilogram of body weight), of each separate agent. Preanesthetic or anesthetics must be described by their generic chemical constituents. Proprietary names may be given in addition to the name of the generic active ingredient(s). For example you may state the proprietary name of the agent as "Surital," an ultra short-acting barbiturate commonly used to induce anesthesia prior to intubation and maintaining the patient on gaseous anesthetics, but you must also give its generic chemical name, in this case, thiamylal sodium.

   If one chooses to use a preanesthetic or anesthetic not contained in Chapter Two of UCAR's Training Manual, please supply a reference about the specific drug's successful use in the species proposed. The reference should describe its efficacy and, if possible, therapeutic ratio or other factors, such as potential toxicity to laboratory animal or staff, e.g., Urethane. If such drug(s) are effective and safe preanesthetic and/or anesthetics, please request that they be added to the UCAR list of preferred drugs used for preanesthesia or anesthesia.

   Please do not describe a "dose" in milliliters/pound or kilo of body weight or give the concentration of the agent (mg/ml or % concentration). Neither of the above two examples are dosages. In some cases, both dosage in mg/kg body weight and concentration as either mg/ml or as a percent solution, should be given. For example, if chloral hydrate is to be used intraperitoneally at a certain mg/kg dose, indicate its concentration in percent, as it is proposed to be used. Chloral hydrate, given intraperitoneally in too high a concentration, may be painful upon induction even if the study is nonrecovery or acute, or it may be painful and cause peritonitis and subsequent adynamic ileus upon recovery.

   If a gaseous anesthetic product is used, please indicate how the animal was induced using x or y preanesthetic or anesthetics by route and dosage in mg/kg, followed by the generic name and concentration of the anesthetic, description of the method of gaseous drug delivery, precautions taken to guard against exposure of personnel to waste anesthetic gases or possible explosion. Chloroform may not be used as an anesthetic because of its hepato- toxicity to man and animal.

   Ether use as an anesthetic is strongly discouraged. If used, it must be used in an approved fume hood. Ether soaked gauze, cotton, or animal carcasses accidentally soaked in ether should be held in the hood long enough for the ether to evaporate. There have been explosions, fortunately without personal injury, here at the University, because ether-laden materials were placed in the morgue to be cremated.

6. Describe means by which a surgical plane of anesthesia is established, maintained, and monitored. For example, in a nonhuman primate under a surgical plane of anesthesia, there should be little or no deep pain reflexes, elicited by digitally pinching a toepad, little or no corneal or palpebral reflexes, elicited by light digital contact or with a cotton tipped applicator. Any other physiological signs of anesthesia used to assess the depth of anesthesia should be described, such as the rate, depth, and character of respirations, (if the patient is not on a mechanical ventilator), color of mucous membranes, electro-cardiograph, pupillary dilation, and electroencephalograph. Many methods may be used, but UCAR requires that at least two measures be used to assure that the animal is properly induced, then reaches and is maintained at a surgical plane of anesthesia during the procedure. Therefore one must indicate at least two methods used to determine that the appropriate depth is reached.

   Intraoperatively, how is an adequate depth of anesthesia monitored and maintained? For example, an assistant may reconfirm any or all of the clinical signs of anesthesia. The animal may have its blood pressure, heart rate or other vital signs monitored by an assistant or displayed electronically.

   If it is absolutely necessary to use a muscle paralysant, scientifically defend that position and describe, in detail, how an adequate depth of anesthesia will be maintained. A surgical plane of anesthesia must be used for any procedure capable of causing any significant pain in an unanesthetized animal.

7. Describe the surgical procedure, including site of incision, operative manipulations, method of closure, suture material used. The description need not be lengthy but must describe all aspects of the procedure to be performed. If more than one major surgical or operative procedure during one session or during subsequent surgical sessions is anticipated, if not indeed planned, they must be scientifically justified as being essential components of a single research protocol. Economics alone can not be the deciding factor.

   An example:

   A nephrectomy in the dog may be performed by using a flank incision which is made obliquely from a point dorsolateral to the last rib, to a point where the skin fold of the thigh meets the ventral abdominal wall (the site).

   Following blunt dissection through the three muscle layers, the perirenal fat is stripped to expose the kidney. The renal pedicel is exposed and, using three crushing forceps, the pedicel is crushed. The kidney is removed by cutting between the two distal clamps following placement of a ligature (3-0 chromic gut), that is gradually tightened and placed where the proximal clamp was removed. A second ligature is placed for safety and the middle forceps removed (the operative manipulations).

   The peritoneum and muscle layers are separately opposed using 3-0 chromic catgut in an interrupted suture pattern. The skin incision is closed using Dermalon (3-0) in a simple interrupted suture pattern (the method of closure, suture material used).

   
   Silk sutures may not be used to close any layer of the body wall. Silk sutures used in laboratory animals increase the likelihood of postoperative complications due to the material's capillarity, and wick-like effects that frequently result in postoperative infection and possible evisceration and death. Silk sutures buried in the body wall continue to act as focal irritant which stimulates an inflammatory response and a higher than necessary incidence of stitch abscesses. It is recommended that any of the synthetic non-absorbable surgical suture material designed for use in the skin be used. Closure of other layers, i.e., fascia, muscle, and peritoneum must be performed using absorbable suture material, i.e., chromic catgut or other absorbable synthetic suture material. If one believe silk sutures are necessary as a ligature within the body cavity, please justify their usage.

   Describe the suture patterns to be used for closure of x layer with y suture material.

   What, if necessary, will be done to provide fluids or other materials intraoperatively?

   What, if necessary, will be done to maintain the animal's body temperature during prolonged surgery?

8. Indicate and/or describe the precautions taken to minimize wound contamination and postoperative infection, including sterilization of instruments, surgical scrub, surgeon's attire, use of cap, mask, gown, sterile gloves, and drapes.

   In the example of the nephrectomy used above, the following may be stated:

   The dog is anesthetized, intubated, clipped and the site given an initial scrub with BETADINE® surgical scrub in the canine procedure room. The dog is draped and transported to the surgery. It is placed in lateral recumbency, and the site scrubbed two more times using the BETADINE® solution. A final wash with 70% ethanol is used. The surgical site is isolated with four sterile towels fixed with towel clamps. A large sterile fenestrated drape is placed over the entire dog. All instruments are sterilized in a steam autoclave. The surgeon and assistant (after scrubbing, gowning and gloving) with assistance from a circulating staff member, aseptically opens the pack(s) in which instruments and other materials, devices, suture materials, may then be used by the surgeon(s). All suture material, fluids and other materials are sterile. The surgeon and his assistant wear scrub-clothes, surgical masks and caps. They then scrub for 10 minutes using e.g., BETADINE® surgical scrub for 10 minutes in the separate scrub area or room. The surgeon(s) then aseptically put on sterilized gown and gloves.

   The entire procedure is performed aseptically. Other personnel not performing surgery are required to wear scrub clothing, caps, and masks in the operating room at all times. They follow the principles of aseptic technique in assisting the surgeons and in monitoring and maintaining a surgical plane of anesthesia. The surgical facility is wet mopped with an appropriate detergent-disinfectant, e.g., a quaternary ammonium compound, and all surfaces are decontaminated using an appropriate disinfectant prior to all operative procedures.

9. Indicate and/or describe postoperative care, including monitoring of recovery from anesthesia, use of analgesics, antibiotics, fluids, wound dressings, suture removal, and monitoring for postoperative complications, including infections (give specific doses, routes and frequency of use, where applicable):

   It is required that the animals recovering from general anesthesia be monitored by either investigative personnel or by members of the Division of Laboratory Animal Medicine (DLAM). Provisions for monitoring for recovery should be described as well as the specific room where the recovery will take place. It is important to note that, in almost all cases where the animal is returned to the basement recovery room, the intensive care unit (ICU) or to the animal's normal housing room after 4 P.M., the investigative group must assume this responsibility. Animals recovering from general anesthesia should not have food or water available to them until they are capable of maintaining sternal recumbency and have a good swallowing reflex. Criteria for assessing when it is safe to remove the endotracheal tube include an easily elicited tracheal cough and an increase in jaw tone. The animal's respiratory rate, mucous membrane color and capillary refill time must be observed closely after extubation.

   The animal must not be left unattended until it is deemed suitably recovered from anesthesia, i.e., the animal is extubated, is capable of maintaining sternal recumbency, vital signs have stabilized and are indicative of an uncompromised recovery.

   During the acute recovery phase it is expected that the recovering patient be examined every 15-20 minutes and observations recorded on the post-op chart. The person making the observation or manipulating the patient in any way must also initial the record and indicate the time that the record was initialed. It is advisable not to offer food or water until the day after surgery in many cases. Specifically state who will be performing these duties. Consultation with the clinical veterinary staff of the DLAM during protocol development, the scheduled day(s) of surgery, and during the immediate and long-term postoperative period is essential.

   Indicate or describe the analgesics, fluids or other medications that may need to be given during the postoperative period. Describe the criteria that will be used to determine if or when, how often, and how much (dose) an animal would be given of a particular analgesic, antibiotic, parenteral fluids, special diet, exercise, or anything that would be contraindicated, due to the experimental design and model. Indicate the proprietary and generic name of the drugs, the dosage (mg/kg body weight), and the route and frequency of administration. Who will be performing these duties?

   If wound dressings are to be used, describe and indicate the frequency of changing and monitoring for possible infection. If indwelling catheters are implanted and exteriorized, describe the frequency of monitoring, flushing, and preventive measures to minimize risk of infection. Who will be performing these duties? How often? What other means will be employed to monitor untoward post-operative complications and, if any are expected, how will they be addressed? Who will be responsible for and at what time postoperatively will skin sutures or wound clips be removed? Under most circumstances, sutures or skin clips may be removed between 7-10 days post-operatively.

10. Describe adverse effects that may be anticipated as a result of the surgical procedure and the steps that will be taken to minimize pain and suffering.

    Describe adverse effects, if any, which are expected as a result of the surgical procedure. Untoward, i.e., planned or unplanned adverse effects and how one plans to deal with them, should have been described earlier. For example, in the case of a unilateral nephrectomy given previously, one would not expect any adverse effect, assuming the health of the remaining kidney. If the purpose was to produce a central nervous system lesion, one might expect, and, indeed want, some specific deficit in central nervous system function

    The steps that will be taken to minimize pain and suffering may include the use of analgesics in the immediate postoperative period, as described above. Additional steps may also involve continuing parenteral or oral supplementation; changing the animal's home cage environment regularly, perhaps even daily; socializing with other dogs or humans at some designated interval and duration; purposeful exercise; evaluation of clinical signs that are unnecessary or extreme, e.g., ascites, that is excessive, or any other measures that can be taken to minimize pain or suffering, but yet not compromise the appropriateness of the animal model.

11. Describe postsurgical manipulations as answered for question 15 of Section A.

12. What criteria would be used to determine that a given animal should or must be euthanatized? These criteria and decision making regarding euthanasia should be discussed fully with the clinical veterinary staff of the DLAM. It is recommended that you seek the assistance of one of the Division of Laboratory Animal Medicine faculty during the development of any recovery surgical protocol. The Committee requires that the attending veterinarian review and approve all recovery surgical protocols for compliance with relevant standards. The regulation promulgated under provisions of the federal Animal Welfare Act stipulates that this be done.

Section C - Use of Infectious Agents or Hazardous Substances

This Section will be reviewed by members of the Environmental Health and Safety Division. Review can require up to thirty days. You should send a copy of the protocol to Environmental Health and Safety for review and a written report. UCAR must receive a written evaluation of the planned use of infectious agents, hazardous agents, and radionuclides, specifying the safety precautions that animal care technicians, veterinarians and other staff and faculty need to take to minimize personal risk or risk to other animals.

There may be some kinds of experiments that require agents or substances that cannot be handled safely and adequately. Therefore, it is important that the question of potential hazards be explored early on in the process of grant preparation or recruitment of new faculty. Discussions must be held between the Environmental Health and Safety consultant and Division of Laboratory Animal Medicine veterinarians.

The UCAR forms do not request all the information detailed below. However, the questions and issues raised should be considered before you seek consultation with a specialist from Environmental Health and Safety and/or UCAR on either infectious, chemical or radionuclide hazards.

It is very important that murine tumor cell lines, hybridomas and other body fluid or tissues that will be inoculated into living animals that are to be housed at the University be screened or MAP tested for murine pathogens as well as well-known human pathogens that can be carried in such living tissue. Arrangements for MAP testing can be made through DLAM.

1. Animals
   1. Identify species and approximate number of animals to be exposed each year of your proposal. It is important to specify the species because different species may respond differently to a challenge from an infectious agent or hazardous substance. For example, one species may become infected yet not shed the agent into the environment. Likewise, the metabolism and possible excretion of a toxic substance or one of its metabolites may be different in different species.

      The numbers of animals to be infected or exposed may have a bearing on the extent of potential risk to personnel or other animals. In this regard, it is important to consider the average and maximum numbers of animals infected or exposed at a single time, in addition to the total number of animals to be used per year.

   2. Will animals exposed or infected be housed? If yes, for how long?

      Consider in quantitative terms, the dose of infectious or hazardous agent that will be administered to a single animal. If known, consider a qualitative estimate of the agent or substances potential effect. For example, is the infectious agent intact, fully virulent, attenuated, or killed? What is known about the toxicity or potential toxicity of a particular compound? What is the quantity of the hazardous substance to be given to an individual animal (mg/kg body weight, if applicable).

      Consider the method of administration - intravenously, intraperitoneally, orally in diet/water, orally by gavage, inhalation, topical application, subcutaneously implanted mini-pump. For inhalation exposures, or any other that may require a period of confinement, consider the exposure chamber, and duration of confinement.

      Consider where the administration of infectious agent or hazardous substance will take place, i.e., the laboratory, animal room or other specialized facility.

      Once infected or administered with a hazardous substance, where will the animals be housed? Is there any specific or designated room in the animal facility, or are animals housed in an unspecified animal room in the animal facility? If you require that the animals be housed in any room or facility not a part of the authorized housing space in the Vivarium, you must scientifically justify this requirement. Consider the facilities, caging and containment equipment as well as procedures to be followed to protect personnel and other animals from inadvertent exposure.

   
   

2. Infectious or Potentially Infectious Agents
   1. The Institutional Biosafety Committee (IBC) must review all infectious or potentially infectious agents and recombinant DNA for use in animals at the University of Rochester. To ensure an expedient review, the Principal Investigator must conduct an initial biosafety assessment and complete the Institutional Biosafety Committee registration forms (the checklists and any applicable on-line forms) for both the animal studies and the laboratory studies prior to and following animal administration. A summary or abstract of the laboratory work must be provided with the registration forms. If the laboratory work prior to animal administration has been previously reviewed and approved, please indicate with the project title and approval date. Instructions and downloadable forms are available on the IBC web site at http://www.rochester.edu/Admin/EHAS/ibcpage.htm. IBC registration submission should accompany UCAR submission to Environmental Safety.

   2. List the agents to be used and the animal biosafety level required. Please refer to the "NIH Guidelines on Recombinant DNA Research" (http://www4.od.nih.gov/oba/guidelines.html) and "Biosafey in Microbiological And Biomedical Laboratories" (http://www.cdc.gov/od/ohs/biosfty/bmbl4/bmbl4toc.htm) CDC 93-8395 (DHHS/CDC Guidelines (version 4). Issued 5/99).

   3. If infectious agents are used in living animals, are they shed into the environment? By what route? Given the agent, dosage, route, age, sex, species and strain of animal to be used, what is known about shedding the agent into the environment?

   4. If blood or blood products, cells or tissue cultures, or transplantable tumors are inoculated into animals, identify the source, type and use of these materials. Since these materials may harbor infectious agents, an evaluation of the source, e.g., human patient, NIH Tumor Repository, naturally occurring tumor from an inhouse colony, and type, e.g., rat plasma protein, cell or tissue culture or transplantable tumor, is important. All such biologically-derived substances must be M.A.P. (Mouse Antibody Protection) tested to exclude adventitial viral agents, including possible zoonotic pathogens, e.g., lymphocytic choriomeningitis virus, which are all too commonly carried in such products. Procedures and costs incident to obtaining M.A.P. testing can be obtained from the Animal Tumor Research Facility of the Cancer Center or from the DLAM office. How will these materials be used? As antigens to produce antibodies? To produce tumors in animals? As cell transplants to correct deficiency states?

   
   

3. Potentially Hazardous Substances

   Individuals working with compounds suspected of being potentially hazardous should work with UCAR and with the Environmental Health and Safety to develop a protocol. Specify in which category above the substance is categorized. Specify the amount to be administered to each animal and/or the dosage on a mg/kg body weight basis. In addition, also give the route of administration, the dosing schedule, i.e., once, twice, three times, at weekly intervals. Consider how much of the substance is known to be eliminated into the environment. What is excreted? Is it the parent compound, element, metabolite? How much of the dosage and over what period of time is it eliminated into the environment? What is its biological half-life? What is the route by which it is excreted? (Feces, urine, expired air, sebaceous or lacrimal secretions.)

4. Radionuclides

   Specify the isotope and amount given per animal. Specify the isotope and provide the dosage e.g., microcurie/animal or per kilogram of body weight. What is the average and maximum number of animals to be administered the isotope at any one time? Has the use of Radionuclides been reviewed and approved? Have you been provided with the safety precautions to be used? If not, submit the protocol for review to Environmental Health and Safety. A copy of the review and approval should be submitted to the "Radiation Safety Officer" extension x5-1473, PO Box HPH, Room G-8842. Please be sure to identify the review by the UCAR Protocol Number, if known.

Consider methods of disposal of infectious agents and hazardous substances, animal waste and carcasses (e.g. cremation, autoclaving, chemical disinfection or destruction)

If chemical disinfection is used, consider the proprietary and/or generic name, the concentration used, and the duration of exposure. If autoclaving is used, how is the autoclave monitored, e.g., spore strips, temperature sensitive device, recording device? What is the temperature achieved and the time held at that temperature?

Manual on the Responsible Care and Use of Laboratory Animals