BROOKHAVEN NATIONAL LABORATORY

**INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE (IACUC)**

**ANIMAL USE PROTOCOL**

*The protocol must be submitted in typed form and all applicable items
must be answered. Answers must be written in English and in terms
understandable to all IACUC members*.

> Protocol \#

Title:

Principal Investigator\*:

Address:

Phone Number: Fax: E-Mail:

Other Investigators\*:

Funding Source: BNL Account Number:

Start Date: Length of Project:

Protocol Type (*e.g. Research, Teaching, Other*):

\* Note - if no investigators are BNL employees, please list a BNL
employee contact:

**[A. OVERVIEW]{.underline}**

A.1. **State the hypothesis and/or describe both general and specific
aims of the project**. *This should be a short (1-2 paragraphs) summary
in lay terms.*

######## **B. ANIMAL DESCRIPTION**

**B.1. Species**:

**B.2. Strain/Breed**:

**B.3. Sex**:

**B.4. Age/Weight**:

**B.5. Supplier**:

> *If not a commercial vendor, a recent health report (no older than
> three months) from the animal facility must be submitted to the BLAF
> Manager at least six weeks before the planned experiment or shipment
> of animals. Please contact the BLAF Manager at 631 344-3620 to make
> arrangements for the receipt of the animals.*

**B.6. Animal Numbers**

a\. Total for first three years:

b\. Maximum housed at one time:

**B.7. List total number of animals at applicable levels of
stress/discomfort**

> ***LEVEL A:** No Pain or Distress: e.g., animals will be euthanized
> without any treatments or manipulations, or irradiation with
> unrestricted movement and without anesthesia and without anticipated
> subsequent effects at BNL.*
>
> ***LEVEL B:** Relieved or Momentary Pain or Distress: Momentary pain
> or potential pain or distress relieved by pharmacologic, behavioral or
> other means**.** e.g., injection of any substance including
> anesthetics, post-procedural analgesics, behavioral conditioning,
> restraint or minor pain/distress and medical treatment of disease
> states.*
>
> ***LEVEL C:** Unrelieved or Sustained Pain or Distress: Any procedure
> that would cause more than momentary or slight pain or distress. e.g.,
> chronic untreated disease states, pain research.*

.

+---------------+-----------------+------------------+-----------------+
| # Species     | # A             | # B              | # C             |
+---------------+-----------------+------------------+-----------------+
|               |                 |                  |                 |
+---------------+-----------------+------------------+-----------------+
|               |                 |                  |                 |
+---------------+-----------------+------------------+-----------------+
|               |                 |                  |                 |
+---------------+-----------------+------------------+-----------------+

**B.8. Justify number of animals.** Indicate design of study groups and
statistical methods and include power calculations. Include steps taken
to minimize the number of animals required.

B.9. Justify species to be used.

B.10. Justify that the work is appropriate to be done in this animal
model and why a lower phylogenetic species or non-animal model cannot be
used.

###### [C. ANIMAL CARE]{.underline}

**C.1. Please indicate if animals will be housed at BNL in other than in
the Brookhaven Laboratory Animal Facility (BLAF).** *All singly-housed
rodents will be provided with environmental enrichment unless
scientifically justified.*

**C.2. Describe additional requirements for other than routine animal
care (e.g. housing, feeding)**:

> *Investigative staff must be responsible for feeding all animals,
> weighing the correct amount of food, logging each feeding, and
> adjusting the ration as needed to maintain the rodent at the desired
> weight. If food, equipment and/or other supplies are to be shipped
> from another institution's animal facility, a recent health report
> from the facility must be submitted to the BLAF Manager at least six
> weeks before the planned experiment.*

# [D. EXPERIMENTAL PROCEDURES]{.underline}

**D.1. List the building and room number(s) in which experimental
procedures, surgery, and/or postoperative recovery, will be performed on
live animals (if known)**.

D.2. In each box, list all personnel working directly with animals and
indicate number of years of experience for each procedure for each
species. All BNL personnel will be put on the appropriate Occupational
Medicine Protocol. Non-BNL employees working with primates will be put
on the appropriate Occupational Medicine Protocol.

+----------------------+-------+-------+-------+-------+------+-------+
| > !                  |       |       |       |       |      |       |
| [](media/image1.wmf) |       |       |       |       |      |       |
+----------------------+-------+-------+-------+-------+------+-------+
|                      |       |       |       |       |      |       |
+----------------------+-------+-------+-------+-------+------+-------+
|                      |       |       |       |       |      |       |
+----------------------+-------+-------+-------+-------+------+-------+
|                      |       |       |       |       |      |       |
+----------------------+-------+-------+-------+-------+------+-------+
|                      |       |       |       |       |      |       |
+----------------------+-------+-------+-------+-------+------+-------+
|                      |       |       |       |       |      |       |
+----------------------+-------+-------+-------+-------+------+-------+
|                      |       |       |       |       |      |       |
+----------------------+-------+-------+-------+-------+------+-------+
|                      |       |       |       |       |      |       |
+----------------------+-------+-------+-------+-------+------+-------+

> Note: Any personnel with less than one year experience in any of the
> above categories must take the applicable training listed below.

D.3. Indicate which training courses apply to this protocol.

All courses may be found at <http://www.bnl.gov/training>

+------+------------------------------------+-------------------------+
| > [C | > [Course Title]{.underline}       | > [Procedures covered   |
| heck |                                    | > by                    |
| > if |                                    | > Module]{.underline}   |
| > r  |                                    |                         |
| equi |                                    |                         |
| red] |                                    |                         |
| {.un |                                    |                         |
| derl |                                    |                         |
| ine} |                                    |                         |
+------+------------------------------------+-------------------------+
| > [  | > Basic Overview of Laboratory     | > Overview required by  |
| Requ | > Animal Care and Use              | > all animal users      |
| ired |                                    |                         |
| >    |                                    |                         |
|  for |                                    |                         |
| >    |                                    |                         |
| all] |                                    |                         |
| {.un |                                    |                         |
| derl |                                    |                         |
| ine} |                                    |                         |
+------+------------------------------------+-------------------------+
|      | > Biomethodology of the Mouse      | Restraint, handling,    |
|      |                                    | identification,         |
|      |                                    |                         |
|      |                                    | sexing, husbandry,      |
|      |                                    | behavior of mice        |
+------+------------------------------------+-------------------------+
|      | > Biomethodology of the Rat        | Restraint, handling,    |
|      |                                    | identification,         |
|      |                                    |                         |
|      |                                    | sexing, husbandry,      |
|      |                                    | behavior of rats        |
+------+------------------------------------+-------------------------+
|      | > Experimental Techniques in       | Injections, blood       |
|      | > Rodents                          | sampling, oral          |
|      |                                    |                         |
|      |                                    | > gavage, euthanasia    |
+------+------------------------------------+-------------------------+
|      | > Post-Procedure Care of Mice and  | Analgesia, pain and     |
|      | > Rats: Reducing Pain and Distress | distress recognition    |
|      |                                    |                         |
|      |                                    | > and alleviation,      |
|      |                                    | > post-operative care   |
+------+------------------------------------+-------------------------+
|      | > Survival Surgery in Rodents      | > Anesthesia, aseptic   |
|      |                                    | > surgical technique    |
+------+------------------------------------+-------------------------+
|      | > Primate Safety                   | > Covers the safe       |
|      |                                    | > handling of non-human |
|      |                                    | > primates              |
+------+------------------------------------+-------------------------+
|      | > Controlled Substance Awareness   | > Required if any       |
|      | > and DEA background Check         | > controlled substances |
|      |                                    | > will be used          |
+------+------------------------------------+-------------------------+
|      | > Regulated Medical Waste          | > Required if any       |
|      | > Management                       | > regulated medical     |
|      |                                    | > wastes (animal        |
|      |                                    | > carcasses, needles,   |
|      |                                    | > syringes) will be     |
|      |                                    | > generated as a result |
|      |                                    | > of the work           |
+------+------------------------------------+-------------------------+

**D.4. Concisely describe all manipulations and experimental procedures,
including surgeries, performed on the animals.** Everything done to the
**live animal** at BNL must be detailed here. A short description of
experimental procedures done elsewhere should be included. Include the
end point of the experiment and timing of euthanasia, if applicable.
Flow diagrams or charts are helpful. Materials and methods portion of
grant applications or other detailed descriptions may be attached.

**D.5.** **Describe if animals are subjected to food/water deprivation
or prolonged and/or unusual restraint and provide justification.**
Describe how animal health is monitored during deprivation.

**D.6. For animals in other than Level A (above), perform a literature
search for alternatives to pain/distress.** Procedures that have pain
eliminated by the use of anesthetics and/or analgesics are still
considered painful even though the animal is not expected to experience
any pain or distress. If alternative procedures are discovered, you must
identify them and justify why those procedures are not being considered.
*Please note the Research Library Staff is available to assist with
literature searches.*

Date of Search:

Database(s) Searched:

Keywords Searched (**Must include the word "alternatives" and exact
procedures being performed)**:

Years Included in Search:

Narrative of Search Results (If alternative procedures are discovered,
you must identify them and justify why those procedures are not being
considered) :

D.7. Indicate how procedures have been refined to reduce the amount of
potential pain, distress or morbidity.

**D.8. Is death used as a study endpoint wherein animals must die
without intervention such as pain relief and/or euthanasia?** If yes,
explain why an earlier end point is not acceptable.

**D.9. List all chemical agents (sedatives, analgesics, anesthetics,
paralytics, euthanasia, study drugs, radiotracers) administered to the
animals.** For euthanasia involving CO~2~, please use 100% CO~2~ at a
20% air replacement per minute rate. For ketamine anesthesia, please use
intraperitoneal (ip) injections, not intramuscular (im).
Ketamine/xylazine may be stored for up to 28 days after mixture.

  -------------- -------------- ---------- ----------- --------------- --------------
  **Type**       **Agent**      **Dose**   **Route**   **Frequency**   **Controlled
                                                                       Substance
                                                                       (Y/N)**

                                                                       

                                                                       

                                                                       

                                                                       

                                                                       
  -------------- -------------- ---------- ----------- --------------- --------------

> *Please note: If Schedule I controlled substances are listed, this
> protocol must be reviewed and approved by the BNL Pharmacist to ensure
> that the substances are included in BNL's DEA license.*
>
> a\. List the name(s) of the individual(s) administering each/all of
> the above agents:
>
> b\. Indicate building and room numbers where stored and security
> procedures for controlled substance(s)
>
> c\. If paralytic agents are used in conjunction with surgical
> manipulations, indicate the means by which absence of pain is
> monitored and/or determined, and who is responsible

**D.10. Is surgery involved?** If yes, indicate whether surgery is
survival or non-survival.

> a\. Describe monitoring and supportive care provided during surgery
> (who, what, and how will this be done?)
>
> b\. Describe indications for analgesic therapy to be administered
> before, during, and/or following surgery
>
> c\. Describe post-operative and/or anesthetic monitoring and
> supportive care (who, what and how often)
>
> d\. Who will maintain surgical and post-operative records and where
> will they maintained?

**D.11. Is anesthesia involved?**

> a\. Describe monitoring and supportive care provided during anesthesia
> (who, what, and how will this be done?)
>
> b\. Who will maintain anesthetic records and where will they
> maintained?

**D.12. Are animals to be used in more than one major surgical procedure
from which they are allowed to recover?** If yes, please describe and
justify.

**D.13. By what method and by whom will animals be euthanized and how
will death be confirmed?** *If a chemical agent is used, please list in
Section D7.* For euthanasia involving CO~2~, please use 100% CO~2~ at a
20% air replacement per minute rate. Justification must be provided for
any physical method, such as decapitation or cervical dislocation,
without anesthesia.

**D.14. List criteria for intervention and/or removal of animals from
study or early euthanasia.**

-   *Examples are severe ataxia; rapidly increased heartrate or
    respiratory rates; oral, nasal or vaginal discharge such as pus or
    blood; wound dehiscense; marked swelling, tumor(s) greater than 2 cm
    or ulcerating, ulcer greater than 10% of body surface area,
    inability to eat or drink, loss of weight, great discoloration in an
    appendage or surgical area; immobility.*

-   *Unless otherwise noted 100% CO~2~ at a 20% air replacement per
    minute rate will be used for early euthanasia for rodents.*

#### [E. SPECIAL CONSIDERATIONS]{.underline}

**E.1. Will tumor cell lines or other materials potentially transmitting
natural pathogens be administered to the animals? If yes,** describe
blood or serum tests that have been conducted proving the
lines/materials are non-infectious regarding natural pathogens. Please
contact the BLAF Manager for assistance with this process.

**E.2. List hazardous materials being used in this study.**

a\. Radioactive:

b\. Infectious/rDNA:

c\. Toxic/Carcinogenic:

**E.3. Does this work involve exposing animals to external radiation?**
If yes, indicate source.

**E.4. Does this work duplicate previous experiments/activities?** If
yes, justify.

**E.5. Indicate if animals will be shipped from BNL. If yes, indicate
that BNL's preferred shipping procedures will be followed.** If other
arrangements are necessary, please describe.

####### 

**E.6. Indicate if animals will be transported to BNL. If yes, detail
transportation.**

####### [F. INVESTIGATOR ASSURANCE]{.underline}

I affirm to the best of my knowledge that all the above information is
complete and accurate and agree to accept responsibility for this
project in accordance with applicable Federal and State of New York
regulations, USDA guidelines, and established BLAF policies and
procedures. No changes will be made without prior approval from the
IACUC.

In order to reduce risk to all personnel and laboratory animals, I agree
to:

a.  Follow BNL procedures for aspects of the animal care and use such as
    preoperative care, anesthesia, surgical technique, postoperative
    care, sampling techniques, euthanasia, and disposal of contaminated
    carcasses and waste.

b.  Ensure that my instructions to laboratory personnel are implemented.

c.  Ensure that all project personnel comply with the required
    Occupational Health Program before handling animals.

d.  Instruct all personnel in my laboratory that they should inform me
    if they believe that the treatment of any research animal is
    inappropriate. If the situation is not resolved, the employee should
    contact the Attending Veterinarian, or the IACUC Chair and/or
    Institutional Official.

I am aware that all research outlined under this protocol must be
carried out under approved Experimental Safety Review(s) (ESR). I am
aware that it is my responsibility to ensure that all individuals
working on this protocol have been listed on an appropriate ESR and that
their training is up to date. I am aware that work cannot proceed
without an approved ESR.

Principal Investigator: [\_\_\_\_\_\_]{.underline} Date: [.]{.underline}

*Your Safety Coordinator will be notified of your IACUC approval.*

##### 

##### [G. APPROVALS]{.underline}

I attest that the following issues have been appropriately addressed:
Scientific merit of project; Appropriateness of conducting the project
at BNL; Adequacy of funding for the project; Appropriateness of the
expertise and experience of the PI and project personnel;
Appropriateness of training for the PI and project personnel, and;
Adequacy of department resources to support this protocol.

BNL Department Chair [\_\_\_\_\_\_]{.underline} Date: [.]{.underline}

Pharmacist (or designee): [\_\_\_\_\_]{.underline} Date: [.]{.underline}
(Required for Schedule I controlled substances)