STUDY PROTOCOL FOR AN AQUACULTURE INVESTIGATIONAL NEW

ANIMAL DRUG (INAD) EXEMPTION FOR OXYTETRACYCLINE

(TERRAMYCIN 343®) FOR IMMERSION THERAPY (INAD #9033)

Sponsor:
U.S. Fish and Wildlife Service, Fisheries and Habitat Conservation

______________________ ___________________
Sponsor Signature Date Approved

Manufacturer:

Pfizer Animal Health

700 Portage Road, RIC-190-43
Kalamazoo, MI 49001-0199

Facility for Coordination of Terramycin 343® INAD:

USFWS’s Aquatic Animal Drug Approval Partnership Program

4050 Bridger Canyon Road
Bozeman, Mt 59715

Proposed Starting Date: July1, 2007

Proposed Ending Date: June 30, 2012

Study Director: Mr. Jim Bowker

_________________________ ________________
Study Director Signature Date

Clinical Field Trial Location and Trial Number:

Facility ______________________________________________________________
Type or Print Name

Investigator____________________________________________________________
Type or Print Name

__________________________________________ ________________________
Investigator Signature Date
I. STUDY ID AND TITLE: ................................................................................................................................. 3
II. SPONSOR: ................................................................................................................................................... 3
III. INVESTIGATORS/FACILITIES: ................................................................................................................... 3
IV. PROPOSED STARTING AND COMPLETION DATES:......................................................................... 3
V. BACKGROUND/PURPOSE: ...................................................................................................................... 4
VI. SPECIFIC OBJECTIVES: .......................................................................................................................... 5
VII. MATERIALS:.............................................................................................................................................. 6
VIII. EXPERIMENTAL UNIT ............................................................................................................................ 9
IX. ENTRANCE CRITERIA .............................................................................................................................. 9
X. TREATMENT GROUPS ............................................................................................................................ 11
XI. TREATMENT SCHEDULES .................................................................................................................... 12
XII. TREATMENT RESPONSE PARAMETERS .......................................................................................... 14
XIII. FORMS FOR DATA COLLECTION......................................................................................................... 15
XIV. RECORD KEEPING PROCEDURES ................................................................................................... 16
XV. DISPOSITION OF INVESTIGATIONAL ANIMALS .............................................................................. 16
XVI. DISPOSITION OF INVESTIGATIONAL DRUG ................................................................................... 17
XVII. DATA HANDLING, QUALITY CONTROL, MONITORING, ADMINISTRATIVE
RESPONSIBILITIES ......................................................................................................................................... 17
XVIII. PLANS FOR DATA ANALYSIS .......................................................................................................... 18
XIX. PROTOCOL AND PROTOCOL AMENDMENTS ................................................................................ 19
XX. PROTOCOL DEVIATIONS ..................................................................................................................... 19
APPENDIX IV. SAFETY DATA SHEET (SDS) FOR OXYTETRACYCLINE HYDROCHLORIDE ....... 20
FORM OTIMM-W: WORKSHEET ................................................................................................................. 21
FORM OTIMM-1. REPORT ON RECEIPT OF DRUG .................................................................................. 23
FORM OTIMM-2: CHEMICAL USE LOG ................................................................................................. 23
FORM OTIMM-3: RESULTS REPORT FORM ..................................................................................... 24

2 Revised 6/07
STUDY PROTOCOL FOR AN AQUACULTURE INVESTIGATIONAL NEW ANIMAL
DRUG (INAD) EXEMPTION FOR TERRAMYCIN 343® IMMERSION THERAPY UNDER
INAD #9033

I. STUDY ID AND TITLE:

Clinical field trials to determine the efficacy of Terramycin 343® immersion therapy to
control mortality caused by certain bacterial diseases in cultured fish.

II. SPONSOR:

Dr. David Erdahl, U.S. Fish and Wildlife Service, Branch Chief, Aquatic Animal Drug
Approval Partnership (AADAP) Program, 4050 Bridger Canyon Road, Bozeman, MT
59715; Phone: 406-994-9904; Fax: 406-582-0242; Email: [email protected]

Manufacturer: Pfizer Animal Health

700 Portage Road
RIC-190-43
Kalamazoo, MI 49001-0199

Contact: Mark Subramanyam

Phone: 269-833-3388
Fax: 269-833-2707
Email: [email protected]

Study Director: Mr. Jim Bowker, U.S. Fish and Wildlife Service, Aquatic Animal Drug
Approval Partnership (AADAP) Program, 4050 Bridger Canyon Road,
Bozeman, MT 59715; Phone: 406-994-9910; Fax: 406-582-0242;
Email: [email protected]

Field Trial Coordinator: Bonnie Johnson, USFWS - AADAP

Study Monitors: See Appendix II for names and addresses.

III. INVESTIGATORS/FACILITIES:

See Appendix IIIa for names and addresses.

IV. PROPOSED STARTING AND COMPLETION DATES:

Reauthorization Starting Date: July 1, 2007

3 Revised 6/07
 Reauthorization Expiration Date: June 30, 2012

Proposed Termination Date: To be determined by research progress.

V. BACKGROUND/PURPOSE:

Oxytetracycline has historically been the drug of choice when diagnostic evidence shows
salmonids to have furunculosis, caused by Aeromonas salmonicida; bacterial
hemorrhagic septicemia, caused by Aeromonas (liquefaciens) hydrophila and other
closely related bacteria; pseudomonas disease, caused by Pseudomonas sp.; enteric
redmouth, caused by Yersinia ruckeri; flexibacteriosis, caused by Flexibacter
(Cytophaga) columnaris, Flexibacter (Cytophaga) psychrophilus, or closely related yellow
pigmented gliding bacteria as described in U. S. Food and Drug Administration (FDA)
Public Master File #5456; or, vibriosis caused by Vibrio anguillarum, Vibrio ordalli or other
closely related bacteria.

In warmwater fish culture, oxytetracycline also has been useful in the control of enteric
septicemia of catfish, caused by Edwardsiella ictaluri and bacterial hemorrhagic
septicemia, pseudomonas disease, and flexibacteriosis in catfish, sturgeon, temperate
basses, sunfishes, and other fish species including several listed as threatened or
endangered under the Endangered Species Act.

Integrated fish health management practices usually prevent the occurrence of these
diseases. However, adverse environmental conditions, uncontrollable water supplies
and unforeseen factors can lead to severe disease outbreaks requiring prompt treatment
in order to prevent significant losses of fish valuable to natural resource stewardship.
Such treatment also reduces the discharge of infectious agents into the natural
environment thereby reducing the spread of disease. Treatment strategies for the use of
oxytetracycline (Terramycin 343®) in fish shall be designed to meet the needs of each
species or lot, the size and numbers of fish to be treated, the layout of the facility, and
environmental conditions. In all cases the objective shall be to minimize the impacts of
disease on fish health, fish quality and survival, and to fully meet fishery management
needs.

The purpose of this INAD is to develop clinical field trial data that will demonstrate the
efficacy and safety of Terramycin 343® to control mortality caused by certain bacterial
diseases in cultured fish under a variety of environmental conditions, at a wide range of
temperatures, and in a variety of fish species. These data will be used to support a new
animal drug application (NADA) for Terramycin 343®. Because there are many factors
that can affect the success or failure of Terramycin 343® immersion therapy, data is
needed that will determine the best ways to use the drug. Drug dosages, treatment
schedules, fish handling methods and other variables should be tested. Complete
documentation of studies that are well conceived and well carried out will be of great
value.

4 Revised 6/07
The U.S. Fish and Wildlife Service (USFWS) anticipates that it may require several years
to carry out all clinical field trials and laboratory studies required to extend and expand the
current label for Terramycin 343® to cover major aquaculture needs. Therefore, the
USFWS may request that the U. S. Food and Drug Administration (FDA) grant
re-authorization of this Terramycin 343® INAD sometime in the future. In the interim, the
USFWS will continue to work closely with the sponsor, the National Coordinator for
Aquaculture New Animal Drug Applications, and other research and conservation
agencies to develop other required New Animal Drug Application (NADA) research data
to support expanded labels claims for Terramycin 343®. Therefore, clinical field trials
planned under this particular INAD are but one part of a larger coordinated and diligent
inter-agency effort that will eventually meet all Terramycin 343® NADA data requirements

VI. SPECIFIC OBJECTIVES:

The two major objectives of this study protocol are as follows:

1. Collect scientific data necessary to support pivotal efficacy trials to further

establish the effectiveness of Terramycin 343® immersion therapy to control
mortality caused by certain bacterial diseases in cultured fish under a variety of
environmental conditions, at a wide range of temperatures, and in a variety of
fish species.

2. Provide an opportunity for fish culturists to legally use Terramycin 343®

immersion therapy to control certain bacterial diseases in cultured fish that
occur under a variety of environmental conditions, at a wide range of
temperatures, and in a variety of fish species so that they can maintain healthy
stocks of fish during the period of time necessary for collection of data that will
be used to support expanded label claims for the use of Terramycin 343®.

Within these two relatively broad objective areas, there are also four more specific study
protocol objectives. The four specific study objectives are described below:

Objective A

Determine the efficacy of a single bath treatment of 20 mg oxytetracycline per liter of

water for 1 hour for the control of furunculosis, bacterial hemorrhagic septicemia,
enteric redmouth, flexibacteriosis, and vibriosis in a variety of salmonid fish species
under a variety of rearing or environmental conditions. Fish species that may be
treated are listed in Appendix VIa. Treated fish must be maintained in culture
facilities for a minimum of 21 days before they can be released or harvested.

5 Revised 6/07
 Objective B

Determine the efficacy of a single bath treatment of 20 mg of oxytetracycline per liter

of water for 1 hour for the control of enteric septicemia in catfish, and bacterial
hemorrhagic septicemia, pseudomonas disease, and flexibacteriosis in catfish,
sturgeon, temperate bass, and other cool and warmwater fish species listed in
Appendix VIa. Treated fish must be maintained in culture facilities for a minimum of 21
days before they can be released or harvested.

Objective C

Determine the efficacy of bath treatment at 20 mg of oxytetracycline per liter of water

for 1 hour administered on 1 to 4 consecutive days for the control of furunculosis,
bacterial hemorrhagic septicemia, enteric redmouth, flexibacteriosis, and vibriosis in a
variety of salmonid fish species under a variety of rearing or environmental conditions.
Fish species that may be treated are listed in Appendix VIa. Treated fish must be
maintained in culture facilities for a minimum of 60 days before they can be released
or harvested.

Objective D

Determine the efficacy of bath treatment at 20 mg of oxytetracycline per liter of water

for 1 hour administered on 1 to 4 consecutive days for the control of enteric septicemia
in catfish, and bacterial hemorrhagic septicemia, pseudomonas disease, and
flexibacteriosis in catfish, sturgeon, temperate bass, and other cool and warmwater
fish species listed in Appendix VIa. Treated fish must be maintained in culture
facilities for a minimum of 60 days before they can be released or harvested.

VII. MATERIALS:

A. Test and Control Articles:

1. Drug Identity

a. Active ingredient

Common Name: Oxytetracycline hydrochloride

Product Name:: Terramycin 343® Soluble Powder

Chemical Family: Tetracycline derivative

CAS Number: 2058-46-0

6 Revised 6/07
 Appearance: Yellow powder

Odor: None

b. Strength and dosage form

Terramycin 343® is a broad-spectrum, highly concentrated antibiotic

powder intended for administration in the drinking water of swine for the
control of specific diseases. It is also approved for use as an
immersion treatment to mark the skeletal tissue of finfish fry and
fingerlings (NADA 008-622). Terramycin 343® contains 343g of
active oxytetracycline hydrochloride per pound of product.

c. Manufacturer, source of supply

Pfizer Animal Health

700 Portage Road, RIC-190-43
Kalamazoo, MI 49001-0199

Contact: Mark Subramanyam

Phone: 269-833-3388
Fax: 269-833-2707
Email: [email protected].

2. Verification of drug integrity/strength:

The Manufacturer, Pfizer Animal Health, will provide the analytical data necessary
to establish the purity of each lot of Terramycin 343® supplied. The lot number
and date of manufacture for each batch of Terramycin 343® will be placed on the
label of each container. The form "Report on Receipt of Drug - Guide for
Reporting Investigational New Animal Drug Shipments for Poikilothermic Food
Animals" (Form OTIMM-1) will clearly identify the lot number and date of
manufacture of Terramycin 343® shipments. If the integrity of the Terramycin
343® is compromised (i.e., by spilling or contamination of the stock container or
feed bags) the event will be carefully recorded, dated, and signed in the Chemical
Use Log (Form OTIMM-2). The Study Monitor assigned to the Investigator
involved will be immediately notified.

3. Storage Conditions

Terramycin 343® must be stored in the original container supplied by the

Manufacturer with the appropriate investigational label attached. The container
should be stored out of direct sunlight in a well ventilated area at room
temperature. The storage unit for Terramycin 343® must be labeled to indicate

7 Revised 6/07
that it contains hazardous material and that "NO Food or Drink is to be Stored in
this unit".

4. Handling Procedures

Each Study Monitor and Investigator will be required to have a current copy of the
Material Safety Data Sheet (MSDS) for Terramycin 343® (see Appendix IV).
Each person involved with the study and each person who may be present during
the use of Terramycin 343® shall be required to read the MSDS. Safety
precautions as outlined in the MSDS will be followed at all times when working with
Terramycin 343® .

5. Investigational labeling

Copies of the labels to be attached to each container of Terramycin 343® are

provided in Appendix V. It is the responsibility of the Investigator to ensure proper
labeling of all containers of Terramycin 343®.

6. Accountability

Pfizer Animal Health will be the sole supplier of Terramycin 343® to all
Investigators under INAD 9033.

1. USFWS and Non-USFWS Facilities

Immediately upon receiving an order/shipment of Terramycin 343®, the

Investigator will complete Form OTIMM-1 “Report on Receipt of Drug -
Guide for Reporting Investigational New Animal Drug Shipments for
Poikilothermic Food Animals". The investigator will archive the original in
the facilities INAD file, and send a copy to his/her Study Monitor. Both the
Investigator and the Study Monitor are required to sign Form OTIMM-1.
The Study Monitor will then forward a copy to the Study Director at the
AADAP Office. The Study Director will archive one copy, and send two
copies of Form OTIMM-1 to FDA. Arrangements should be made between
Investigators and Study Monitors to insure completed Form OTIMM-1s are
received by the Study Director in a timely manner.

All Investigators are also responsible for maintaining an accurate inventory

of Terramycin 343® on-hand. A Chemical Use Log (Forms OTIMM-2) will
be supplied to each Investigator. Each time Terramycin 343® is used, it
must be recorded by the Investigator on Form OTIMM-2.

7. Preparation Procedures

Oxytetracycline will be supplied to Investigators as Terramycin 343® containing

343g of active oxytetracycline hydrochloride per pound of product. Hence, when

8 Revised 6/07
 calculating desired treatment concentration, Investigators should consider
Terramycin 343® to be 75.6% active oxytetracycline. Terramycin 343® should not
be adulterated in any manner prior to use.

B. Items Needed for Treatment, Data Collection, Etc.:

Sampling techniques and diagnostic equipment will most likely be provided by trained fish
health biologists serving as Study Monitors or their designee(s). Equipment and
supplies needed would include items to sample, culture, grow and identify culture growths
microscopically. Standard fish culture supplies and equipment would also be required.

When the Study Protocol has been approved and treatments are scheduled, the
Investigator at each facility covered by Terramycin 343® INAD 9033 will need to complete
several forms. These forms are described in Section XIII (p. 12). Copies of these forms
are attached to this Study Protocol.

VIII. EXPERIMENTAL UNIT

The experimental unit in these clinical field trials will consist of contained or isolated
groups of fish. This will generally be a groups of fish contained in tanks, raceways, or
ponds. However, the experimental unit in clinical field trials may also be individual
animals. If individual animals are considered to be the experimental unit, treatment
response parameters for each animal must be evaluated separately.

IX. ENTRANCE CRITERIA

A. Facilities/Investigators

The proposed facility and the Investigator must be listed in Appendix IIIa of the
Study Protocol before Terramycin 343® can be ordered and dispensed under
this INAD. Last minute deviations can be requested by the Sponsor, Study
Director, or by an Investigator in case emergency use-pattern needs should
arise (See Section XX).

B. The characteristics of the study animals (species, number, etc.) is presented

in Appendix VIb.

C. Environmental conditions

Environmental conditions will be variable and include a broad spectrum of

water temperatures and water quality parameters. Environmental conditions
will be reported on a Form OTIMM-3.

D. Ability of Investigator to fulfill all the requirements of the Study Protocol

9 Revised 6/07
 See Appendix IIIb for example of knowledge required of hatchery managers
(i.e., Investigators).

V. Pathogen/disease considerations

A. Bacterial fish pathogens should be presumptively identified by

procedures described in Section 3 of the "Blue Book" (Procedures for
the Detection and Identification of Certain Fish Pathogens, Third
Edition, Fish Health Section/American Fisheries Society, 1985). Other,
more sensitive methods described elsewhere in peer-reviewed
references, or as mutually determined by the local fish health biologist,
in consultation with the Study Monitor, also may be used. (Note:
Diagnostic methods other than those in the Third Edition of the
"Blue Book" should be described on a separate sheet attached to
a Form OTIMM-3 “Results Report Form”).

B. There should be increased mortality rates among fish in a rearing unit(s)

for three or more consecutive days. (Note: Station history and the
experience of the investigator, monitor, or the fish health biologist may
over-ride this criterion to halt potentially explosive disease outbreaks.
In such cases, however, careful diagnostic surveillance should be
carried out in all rearing units proposed for treatment and controlled
tests should be carried out if at all possible.)

C. Typical disease signs should be detectable in at least a few fish and the
causative pathogen should be identified.

D. Since the efficacy of Terramycin 343® immersion therapy for the control
of mortality caused by bacterial diseases is being tested, investigators
must be prepared to make no changes in the fish cultural procedures or
environmental conditions and apply no other treatments once a decision
has been made to conduct Terramycin 343® therapy. Complicating
bacterial or other aquatic pathogens should be carefully documented.
If necessary, these infections can be treated once Terramycin 343®
response (efficacy) data has been collected. However, it may require
as long as 10 days after the completion of Terramycin 343 ® therapy to
determine differences between test and control groups and to complete
post-treatment evaluations.

Prior to initiating each treatment event, the Investigator must first complete
Form OTIMM-W. “Worksheet for Designing Individual Field Trials” that pertains to
each specific treatment event. The worksheet should be filled out, signed, and
sent by Fax to the Study Monitor. The Study Monitor will review the planned
treatment (worksheet), sign it, and forward (Fax) the paperwork to the AADAP

10 Revised 6/07
 Office. The AADAP Office will then review the worksheet, assign the approved
treatment a Study Number, and then notify both the Investigator and the Study
Monitor of the assigned number and approval to proceed. In most cases, this
entire process should be able to be accomplished within a single working day.
After initiation of the field trial, the Investigator should also record the assigned
study number on Form OTIMM-2 and Form OTIMM-3, as well as on any additional
correspondence regarding that specific treatment event. If for some reason the
Investigator is unable to reach his/her Study Monitor with regards to worksheet
approval, and infection/disease/treatment need is rapidly escalating, the
Investigator should contact the AADAP Office for a study number and permission
to proceed.

X. TREATMENT GROUPS

A. A treatment group or experimental unit may be an entire tank, pond,

raceway, or group of fish, or it may be individual animals.

B. Separately confined, untreated control fish will not be required in

supplementary field studies conducted to determine the effectiveness of
Terramycin 343® treatment. Fish from a group or lot will first be examined to
determine if treatment with Terramycin 343® is required. When treatment is
underway or has been completed, fish from the same group will be examined to
determine the effect of treatment on the parameters used to initially sanction the
treatment. Evaluation will in all cases consist of determining fish mortality,
although in some cases degree or severity of bacterial infestation may also be
quantified.

Although untreated control groups are not a required element of treatment under
this INAD exemption and are at the discretion of the Investigator, they are strongly
encouraged whenever circumstances permit. Control groups are extremely
important to not only document response to treatment, but also to validate
potential adverse reactions in treated animals. Use of control groups will ensure
that results of efficacy studies provide useful information that will support an
NADA.

It is important that all fish are treated in a similar fashion. If fish are physically
moved into separate test groups or different rearing units, caution should be used
so that handling and rearing conditions are as similar as possible. Control fish
should be kept under conditions as similar as possible to treated fish for valid
comparison. Although not required, replicate treatment groups are strongly
encouraged in both treated and control groups. Assignment to control and
treatment groups should be random and designed to avoid bias.

Blinded studies can reduce bias in data collection. Whenever possible,

investigators should consider methods by which treatment response observations

11 Revised 6/07
 are recorded by individuals who are unaware which fish have been treated and
which fish are controls.

The designation of specific treatment groups often depends upon the number of
affected treatment units, the nature and severity of the disease being treated, and
the variables being tested. Two or three different treatment groups are generally
anticipated.

1. Spotty, low level, or chronic disease patterns:

A number of facilities participating in this INAD are doing so as a means of

being prepared, in advance, to use Terramycin 343® in the event a bacterial
disease outbreak occurs. If management practices have been good,
disease occurrences often result in low morbidity and mortality rates.
Terramycin 343® therapy may be occasionally required as a part of the
process of a comprehensive fish health management program. These
situations are the most typical. Even though there may be too few units
involved to allow for treatment replication, careful record keeping is
important so that useful data can be collected. Handling of clinically ill fish
should be kept to a minimum until they have been successfully treated.
Even the careful separation of diseased fish into new groups for treatment
may alter environmental conditions present during disease initiation,
thereby potentially rendering the Terramycin 343®therapy trial meaningless.

2. Epizootics:

At some participating facilities disease outbreaks may be more widespread,

more severe, and occur more regularly. Sufficient fish and test units at
these facilities may be available to conduct higher quality studies (i.e.
studies that include replication, randomization, blinding, etc.). Such
situations are suitable for the conduct of pivotal, carefully designed and
controlled studies. Investigators at these facilities are encouraged to
contact the AADAP Office for assistance with study design and completion.
These facilities will be given top priority for assistance in study design,
on-site assistance from the AADAP Office, and diagnostic support from fish
health biologists.

XI. TREATMENT SCHEDULES

A. Route of administration

Terramycin343® will be administered only as an immersion treatment.

B. Dosage, treatment duration, and dosing interval/repetition

12 Revised 6/07
 Objective A [For the control of furunculosis, bacterial hemorrhagic
septicemia, enteric redmouth, flexibacteriosis, and vibriosis in a
variety of salmonid fish species]

Treatment at 20 mg oxytetracycline per liter of water for 1 hour.

A single treatment event with no repetition.

Objective B [For the control of enteric septicemia in catfish, and bacterial

hemorrhagic septicemia, pseudomonas disease, and
flexibacteriosis in catfish, sturgeon, temperate bass, and other
cool and warmwater fish species]

Treatment at 20 mg oxytetracycline per liter of water for 1 hour.

A single treatment event with no repetition.

Objective C [For the control of furunculosis, bacterial hemorrhagic

septicemia, enteric redmouth, flexibacteriosis, and vibriosis in a
variety of salmonid fish species]

Treatment at 20 mg oxytetracycline per liter of water for 1 hour.

One to four treatments administered on consecutive days.

Objective D [For the control of enteric septicemia in catfish, and bacterial

hemorrhagic septicemia, pseudomonas disease, and
flexibacteriosis in catfish, sturgeon, temperate bass, and other
cool and warmwater fish species]

Treatment at 20 mg oxytetracycline per liter of water for 1 hour.

One to four treatments administered on consecutive days.

C. Drug preparation and administration procedures

Oxytetracycline will be supplied to Investigators as Terramycin 343®

containing 343g of active oxytetracycline hydrochloride per pound of product.
Hence, when calculating desired treatment concentration, Investigators
should consider Terramycin 343® to be 75.6% active oxytetracycline.
Terramycin 343® should be thoroughly mixed in rearing unit water.
Terramycin 343® should not be adulterated in any manner prior to use.

13 Revised 6/07
 D. Permissible concomitant therapy

Since efficacy data are being collected during the INAD process, there should
be little or no concomitant therapy. Preferably, there should be no other
therapy during a period extending from 2 weeks prior to treatment to 2 weeks
after treatment. Investigators must be prepared to make no changes in fish
cultural procedures or environmental conditions, and apply no other drug
therapy once a decision has been made to conduct Terramycin 343®
treatment. However, if concomitant therapy is required in order to protect
valuable fish stocks, it should be fully documented and the efficacy data from
the Terramycin 343® treatment involved should be appropriately labeled.

XII. TREATMENT RESPONSE PARAMETERS

The collection and reporting of source data begins with the decision to treat valuable fish
based on hatchery records or other pertinent species information indicating treatment is
warranted. Daily morbidity and mortality records, case history records, as well as any
extenuating or mitigating circumstances that may affect treatment response need to be
documented. All pertinent treatment response parameters should be reported on Form
OTIMM-3. Treatment response parameters that should be addressed include the
following:

1. Primary Parameters

Morbidity and mortality data, coupled with case history and analyses of
bacterial load, usually indicate when Terramycin 343® treatment is needed.
Typically, source data must be collected for 5 days before treatment,
during treatment, and for 10 days after the treatment period has ended.
Collection of this data is critically important. Samples of kidney or other tissue
should be removed from groups of representative fish and tested by
bacteriological, serological, or other methods to determine the presence of
target pathogens.

2. Secondary Parameters

Secondary parameters may also include general observations on fish behavior

and response to routine culture/handling activities. This would include such
responses as feeding activity, feed consumption, apparent level of stress,
negative fish behavior, etc.

14 Revised 6/07
 3. Adverse Reactions

Any adverse reaction to treatment should be reported immediately to the Study

Monitor, who will in turn notify the Study Director. Such responses might
include extremely negative responses/behavior by the fish or hazards to the
applicator. Although Terramycin 343® immersion therapy has been used
extensively for many years with beneficial effect in fish culture, it is possible
adverse reactions may occur under certain environmental conditions or with
respect to specific species/strains of fish. Carefully observe all treated fish for
any signs of any adverse reaction to treatment. The Investigator should
carefully document all observations of adverse reactions. If any signs of drug
toxicity are detected, they should also be documented and immediately
reported to the Study Monitor, who will in turn notify the Study Director.

Note: Investigators are strongly encouraged to record observations/comments

with respect to all phases of treatment. This may include a description
of events before, during, and post-treatment. All extenuating or
mitigating treatment circumstances need to be described in detail.
Such information is imperative so that accurate study/data analysis
can be performed.

XIII. FORMS FOR DATA COLLECTION

When the Study Protocol has been approved and treatments are scheduled, the
Investigator at each facility covered by Terramycin 343® INAD 9033 will need to complete
the following forms:

Form OTIMM-W. Worksheet for Designing Individual Field Trials under INAD
9033

Form OTIMM-1. Report on Receipt of Drug - Guide for Reporting

Investigational New Animal Drug Shipments for
Poikilothermic Food Animals

Form OTIMM-2. Chemical Use Log for Clinical Field Trials Using Terramycin
343® under INAD 9033

Form OTIMM-3. Results Report Form for use of Terramycin 343® under INAD
9033

Copies of these forms are attached to this Study Protocol.

15 Revised 6/07
XIV. RECORD KEEPING PROCEDURES

The data should be recorded in permanent ink (preferably black). The data should be
recorded on the official data record forms at the time the observations are made. The
raw data should be original, i.e., they should be the first recording of the observations,
rather than a transcription of original observations to another data sheet. Each original
data sheet should be legibly signed and dated by the person making the observation and
recording the entry. If more than one person makes and records the observations,
entries should be properly attributed to each person. The data should be accurate and
legible. If a mistake is made, it should be crossed out using a single strike-through and
the correct data should be recorded next to it. Each change to the raw data should be
initialed and dated by the person making the change, and a statement should be provided
explaining why the change was made. If the data sheet needs to be copied, all data
should be transferred, including the properly noted changes. The original record should
be retained and submitted with the revised copy, along with a memo explaining the
reason for the copying.

XV. DISPOSITION OF INVESTIGATIONAL ANIMALS

Animals that die during treatment should be disposed of by burial or incineration. All fish
treated with Terramycin 343® must be maintained in culture facilities for a specified
withdrawal time following completion of therapy before stocking/release or harvest.
Specific withdrawal time is based upon treatment objective as defined in Section XI.B of
this study protocol and are as follows:

Objective A: 21 days
Objective B: 21 days
Objective C: 60 days
Objective D: 60 days

No withdrawal period will be required for stocked fish that will not be
harvestable/catchable after release, or are illegal for harvest, during the withdrawal times
specified above (e.g., if fish are treated under Objective C and are not susceptible to
harvest for a minimum of 60 days following completion of treatment, they may be
stocked/released immediately). No withdrawal period shall be required for dead fish that
will be buried or rendered into non-edible products.

The Investigator must verify compliance with requirements regarding the disposition of all
treated fish on Form OTIMM-3.

16 Revised 6/07
XVI. DISPOSITION OF INVESTIGATIONAL DRUG

Terramycin 343® will be used only in the manner and by the individuals specified in the
Study Protocol. If any unused Terramycin 343® remains at the end of the study period,
Investigators should contact Study Monitors for instructions regarding drug disposal.
Spoiled or out-dated Terramycin 343® should be disposed of in a landfill. The
investigational drug may not be redistributed to others not specified in the Study Protocol.

XVII. DATA HANDLING, QUALITY CONTROL, MONITORING, ADMINISTRATIVE

RESPONSIBILITIES

A. Drug distribution

See Section VII.A.6. Accountability (page 5) for information and details.

B. Study Monitors

Study Monitors are generally fish health professionals with experience in

diagnosing and treating fish diseases, and the ability to monitor overall fish health
with respect to ongoing fish culture practices. A study monitor should be
assigned to each facility that is authorized to treat fish with Terramycin 343®. A
list of Study Monitors, along with addresses and phone numbers, can be found in
Appendix II. Study Monitors are responsible for supervision of the trials,
adherence of the Investigator to the Study Protocol, and inspection of the site.

C. Special equipment and materials

Most of the equipment and materials required for this study (with the exception of
the Terramycin 343® itself are already available at each participating fish hatchery.
The use of various drugs, chemicals, and therapeutants to meet management
and/or production goals is a common occurrence at most fish hatcheries. Fish
hatchery managers (i.e., Investigators) are well trained and well equipped to
handle these situations (see Appendix IIIb). If any additional equipment or
materials are required, they will be provided by the Study Monitors (See Section
VII.B. Items needed for sample collection, observations, etc., page 6).

D. Administrator of the drug

Terramycin 343® will be administered directly by the assigned Investigator (fish

hatchery manager) or under the Investigator's direct supervision (see Appendix
IIIa for names). Terramycin 343® will be maintained in a secure location, and only
the Investigator or persons under his/her direct supervision will have access.

17 Revised 6/07
 E. Drug accountability records

See Section VII.A.6. Accountability (page 5) for details and Forms OTIMM-W,
OTIMM-1, OTIMM-2, and OTIMM-3 (page 12) for actual forms to be used in the
study.

F. Recording observations

The Investigator or a person under his/her direct supervision will be responsible for
implementing the Study Protocol, making observations, collecting samples, and
recording data during the clinical field trials. After the data have been collected
and recorded on the forms, the Investigator will send the data to the Study
Monitors who will review the information and ensure that all required data is
provided. The Study Monitors will in turn send the data to the Study Director.
The Study Director will analyze and summarize the data and prepare an annual
report that will be submitted to the FDA.

G. Data storage

The Investigator is responsible for complete and accurate data collection. The
Investigator is also responsible for archiving a complete set of all original data. A
copy of Form OTIMM-1 should be sent immediately to the Study Monitor, who will
in turn forward a copy to the Study Director. A copy of Form OTIMM-2 should be
sent to Study Monitors with the corresponding Form OTIMM-3. A copy of Form
OTIMM-3 should be sent to the Study Monitor after completion of the entire
treatment period, which includes the post-treatment observation period. All
forms must be submitted by the end of the calendar year. Study Monitors
should carefully check each set of data for accuracy and completeness. If there
are any discrepancies in the data, the Study Monitor should contact the
Investigator immediately to rectify the problem. After review, Study Monitors
should forward all data to the Study Director. As stated above, a complete set of
raw data should be archived by the Investigator. All data should be stored in a
secure place. Another complete data set (copies) will be archived by the Study
Director.

XVIII. PLANS FOR DATA ANALYSIS

Data analysis will be completed by the Study Director located at the AADAP Office. Data
from the treatment year will be summarized through tabulation and appropriate statistical
analysis. An annual report will be prepared and submitted to the FDA. This submission
will probably include a request for an extension of the INAD based on the data collected
during that year. When sufficient data are collected, the entire INAD data set will be
summarized in a final report for submission to support a full NADA.

18 Revised 6/07
XIX. PROTOCOL AND PROTOCOL AMENDMENTS

A signed copy of the Study Protocol must be retained by each Investigator. At any time
before the study begins, desired changes in the Study Protocol should be brought to the
attention of the Study Director. The desired changes will be fully described in the form of
an amendment along with the reason for the change. The amendment will be signed by
the Sponsor (or its representative)and forwarder to the FDA for review. Copies of the
signed amendment will be attached to each copy of the Study Protocol. Investigators
will be liable for non-compliance violation if drugs are used without a Study
Protocol or in a manner different than specified in the Study Protocol, if forms are
not filed on time, or if the study data are not properly collected, maintained, and
reported. The Study Monitor is responsible for ensuring that all INAD procedures are
being followed as defined by the Study Protocol.

XX. PROTOCOL DEVIATIONS

Deviations from the established Study Protocol occasionally cannot be avoided. If

deviations occur, the Study Monitor should be notified immediately. Protocol
deviations should be fully documented and should be accompanied by a written
explanation of what happened, why, and what steps were taken to mitigate the
deviation. Deviation statements should be signed and dated. These statements
should be forwarded to the Study Monitor along with Form OTIMM-3, and ultimately be
submitted to the Study Director.

19 Revised 6/07
Appendix IV. Safety Data Sheet (SDS) for Oxytetracycline hydrochloride
The SDS for Oxytetracycline hydrochloride can be found at the drug sponsors website:
http://pharmgateah.com/pdf/msds/Pennox%20343_(USA)_EN_sds%20rev2.pdf

20 Revised 6/07
 Form OTIMM-W: Worksheet for Designing Individual Field Trials under Terramycin 3437
INAD 9033

INSTRUCTIONS
1. Investigator must fill out Form OTIMM-W for each trial conducted under this INAD before actual use
of Oxytetracycline for Immersion. The Investigator is responsible that Form OTIMM-W is completed
accurately.
2. Investigator should keep the original on file, and fax a copy to the Study Monitor for review.
3. After review, the Study Monitor will fax a copy to the AADAP Office for assignment of the Study Number.
4. The AADAP Office will review the worksheet, and then fax the assigned trial Study Number to both the
Investigator and Study Monitor, at which time the trial may be initiated.
5. Note: Both Investigator and Study Monitor should sign and date Form OTIMM-W.

SITE INFORMATION
Facility
Address

Investigator
Reporting Individual (if not Investigator
Phone Fax

FISH CULTURE AND DRUG TREATMENT INFORMATION
Fish species to be treated Disease to be treated
Average fish weight (gm) Average fish length (in)
No. of fish per unit (e.g. 10,000 fish/raceway)
Number of treated units Number of treated fish
Number of untreated Number of control fish
control units
Anticipated date treatment Anticipated number of treat-
will be initiated ment
Intended drug target Estimated total weight of fish
20
dosage (mg/L) treated (lbs)
Estimated total amount of Planned duration of drug
drug needed for proposed treatment (hours) 1
treatment (gm)
Drug manufacturer Drug lot number

Form OTIMM-W Worksheet for Designing Individual Field Trials under INAD 9033 Revised 06/07
 STUDY DESIGN: Describe in detail the purpose of the clinical trial. Study design must be carefully fo-
cused and lend itself to rigorous evaluation. If more space is required to describe study details, title additional
page(s) “Study Design” and attach them to this Worksheet.

Study designed by; __________________________________________________________

DISPOSITION OF TREATED FISH (Human Food Safety Considerations):

____ Estimated time (days, months) from last treatment day to first possible harvest for human
consumption

Check applicable box(es):

____ Study Objective A - Withdrawal period of 21 days for variety of salmonid fish.
____ Study Objective B - Withdrawal period of 21 days for non-salmonid fish.
____ Study Objective C - Withdrawal period of 60 days for variety of salmonid fish.
____ Study Objective D - Withdrawal period of 60 days for non-salmonid fish.
____ Investigator should initial here to indicate awareness that fish disposition must be in compliance
with FDA-mandated withdrawal times as described in Section VI of the Study Protocol.

WORKER SAFETY CONSIDERATIONS:

Investigator should initial here to indicate that all personnel handling drug have read Material
Safety Data Sheet for Terramycin 3437 and have been provided protective equipment, in good
working condition, as described in the MSDS

Date Prepared: _______________ Investigator: __________________________________

Date Reviewed: _______________ Study Monitor: _________________________________

Form OTIMM-W Worksheet for Designing Individual Field Trials under INAD 9033 Revised 06/07
FORM OTIMM-1. Report on Receipt of Drug - Guide for Reporting Investigational New
Animal Drug Shipments for Poikilothermic Food Animals

INSTRUCTIONS
1. Investigator must fill out Form OTIMM-1 immediately upon receipt of oxytetracycline.
2. Investigator should keep the original on file, and send one copy to the Study Monitor for review.
3. Within 10 days of receipt, the Study Monitor should send a copy to the AADAP Office.
4. Note: Both Investigator and Study Monitor should sign and date Form OTIMM-1.

The sponsor, U.S. Fish and Wildlife Service, submits a notice of claimed investigational exemption for the
shipment or delivery of a new animal drug under the provisions of Section 512 of the Federal Food, Drug,
and Cosmetics Act.
Name of Drug Terramycin-343 INAD Number 9033
Proposed Use of Drug Treatment of certain bacterial diseases that occur in a variety of fish species
Date of CVM Authorization Letter October 1, 2007
Date of Drug Receipt Amount of Drug Received
Drug Lot Number Trial Number
Name of Investigator
Address of Investigator
Location of Trial
Pivotal Study Non-pivotal Study (yes/no) ----
Approximate Number of Treated Approximate Number of
Animals Control Animals
Number of Animals Used Previously1
Study Protocol Number 9033
Approximate dates of trial (start/end)
Species, Size, and Type of Animals
Maximum daily dose and duration 20 mg/L for 1hour
Methods(s) of Administration Immersion (static bath treatment 1 - 4 days)
Withdrawal Period 21 days for 1 day treatment; 60 days for 2 - 4 day treatment
1
To be filled out by the NIO

Date Prepared: ________________ Investigator: _______________________________________

Date Reviewed: ________________ Study Monitor: _____________________________________

Date Reviewed: ________________ Sponsor: __________________________________________

Revised 8/07
Terramycin 3437under INAD 9033

INSTRUCTIONS
1. Investigator should initiate a new form OTIMM-2 immediately upon receipt of each shipment of Oxytetra-
cycline for Immersion.
2. Form OTIMM-2 should be updated whenever drug is used, transferred, or discarded.
3. Investigator should save all copies of this form until the end of the calendar year, at which time they should
maintain all originals on file and send one copy of the completed form(s) to their Study Monitor. Within 10
days of receipt, the Study Monitor will ensure accuracy and send a copy to the AADAP Office for inclusion
in the permanent file.
4. Note: Both Investigator and Study Monitor should sign and date Form OTIMM-2.

Qty of AQUI-S® 20E from

previous page (ml) ________Facility ____________________________Reporting individual______________

Date Amount of Lot Study Amount TM-343 TM-343 TM-343 Inventory

TM-343 number of Number TM-343 transferred discarded remaining by
received TM-343 used in (gm) (gm) on hand (initials)
(gm) received treatment (gm)
(gm)

xxxx xxxx
xxxx xxxx
xxxx xxxx
xxxx xxxx
xxxx xxxx
xxxx xxxx
xxxx xxxx
xxxx xxxx
xxxx xxxx
xxxx xxxx
xxxx xxxx
xxxx xxxx
xxxx xxxx
xxxx xxxx

Date Prepared: ________________ Investigator: ________________________________________

Date Reviewed: ________________ Study Monitor: _____________________________________

Form OTIMM-2 Chemical Use Log Revised 06/07

STUDY NUMBER ___________________________________ Page 1 of 3

Form OTIMM-3: Results Report Form for use of Terramycin 343® under INAD 9033

INSTRUCTIONS
1. Investigator must fill out Form OTIMM-3 no later than 10 days after completion of the 30-day post-
treatment observation period. Study Number must be recorded on all pages of Form OTIMM-3. Attach lab
reports and other information.
2. If Terramycin 343® was not used under the assigned Study Number, fill out only the Site Information
portion on this page, and skip to the end of page 4 and fill out only the “Negative Report” section.
3. Investigator should keep the original on file, and send a copy to the Study Monitor. Within 10 days of
receipt, the Study Monitor should send a copy to the AADAP Office for inclusion in the permanent file.
4. Note: Both Investigator and Study Monitor should sign and date Form OTIMM-3.

SITE INFORMATION
Facility
Reporting Individual

TREATMENT INFORMATION AND SCHEDULE

Drug lot number Total amount drug used (gm)
Fish species treated OTIMM dosage used (mg/L) 20
Duration of drug treatment 1 Number of treatments
(hours)
Disease treated Disease diagnosed by
Average fish weight (gm) Average fish length (in)
Number of fish per unit (e.g. 10,000 fish/raceway)
Number of treated units Total number of treated fish
Number of control units Total number of control fish
Treatment date(s)

WATER QUALITY PARAMETERS

Ave pre-treatment temp (oF) Dissolved Oxygen (mg/L)
Ave treatment temp (oF) pH
Ave post-treatment temp (oF) Hardness - CaCO3 (mg/L)

Form OTIMM-3 Results Report Form Revised 06/07

STUDY NUMBER ___________________________________ Page 2 of 3

Daily Mortality Record

INSTRUCTIONS
1. Investigator should fill out the Daily Mortality Record as completely as possible.
2. Prior to initiation of the trial, fill out Rearing Unit ID, whether a rearing unit is Treated or Control, and the
number of fish in each rearing unit.
3. Water temperature and individual tank mortality should be recorded on a daily basis.
4. Please mark all treatment days with an asterisk.
5. Use additional copies of this form if more than 6 rearing units are involved in the trial.

FACILITY
Rearing Unit ID
Treated or Control
Number of Fish
Water Daily
Day Date Temp Mortality Mortality Mortality Mortality Mortality Mortality Observer
(Fo) Initials
1
Pre-Treatment

2
Period

3
4
5
1
Treatment

2
Period

3
4
1
2
3
Post-treatment Period

4
5
6
7
8
9
10

Form OTIMM-3 Results Report Form Revised 06/07

STUDY NUMBER ___________________________________ Page 3 of 3

RESULTS: Describe in detail treatment results. Was treatment successful? If treatment did not appear to
be successful, explain why not? Were there any mitigating environmental conditions that may have impacted
treatment results? Were there any deviations from the Study Protocol?

PATHOLOGY REPORT: Attach pathology report to this form. Report should include: 1) a description of
how the pathogen(s) was identified; 2) disease identification records that confirm the presence of the pathogen;
and 3) the name and title of the individual performing the diagnosis.

Pathology Report included: ________ pre-treatment ________ post-treatment

TOXICITY OBSERVATIONS: Report any apparent drug toxicity including a description of unusual fish
behavior.

DRUG DISCHARGE RESULTING FROM THIS TREATMENT: Use Addendum 2: Discharge Worksheet
for calculations and attach completed Discharge Worksheet to this form. Enter the value from Addendum 2 step
3 in this space.

OBSERVED WITHDRAWAL PERIOD: (Investigator should initial the appropriate box below)

Observed withdrawal period: _____ 21 days; Objectives A & B

Observed withdrawal period: _____ 60 days; Objectives C & D

Estimated number of days between last treatment and first availability of fish for human consumption
(ensure this time period meets the withdrawal period). _________________

NEGATIVE REPORT Oxytetracycline for Immersion was not used at this facility under this
Study Number during the reporting period. (Investigator should initial for negative reports as
soon as the Study Number is known to be no longer needed or valid.)

Date Prepared: __________________ Investigator: ______________________________________

Date Reviewed: __________________ Study Monitor: ____________________________________

Form OTIMM-3 Results Report Form Revised 06/07