17MT TAS Protocol Revision 4

TARGET ANIMAL SAFETY STUDY PROTOCOL INAD #11-236

The Safety of 17α-Methyltestosterone Administered in the Feed for Induction of Sex Inversion in Tilapia

Study Protocol Number SIUC-05-MT-04

Study Director: Anita M. Kelly, Ph.D.

Fisheries and Illinois Aquaculture Center Southern Illinois University-Carbondale

Mailcode 6511 Carbondale, IL 62901-6511

Study Sponsor: Dave Erdahl, Ph.D.

U.S. Fish & Wildlife Service Aquatic Animal Drug Approval Partnership

4050 Bridger Canyon Rd. Bozeman, MT 59715

January 22, 2007

TABLE OF CONTENTS Title................................................................................................................................................. 1 Purpose and Objective.................................................................................................................. 1 GLP Compliance. .......................................................................................................................... 1 Sponsor........................................................................................................................................... 1 Study Director ............................................................................................................................... 1

Study Personnel................................................................................................................. 1 Feed Preparation............................................................................................................... 2 Methyltestosterone Analysis (feed).................................................................................. 2

Quality Assurance Officer............................................................................................................ 3 Test Facilities ................................................................................................................................. 3

Test Animals .......................................................................................................................... 3 Justification ........................................................................................................................... 4 Procurement .......................................................................................................................... 4

Test Article .................................................................................................................................... 4 Verification of Methyltestosterone in the Feed .............................................................. 4 MSDS ................................................................................................................................. 5 Dosage ................................................................................................................................ 5 Duration of treatment....................................................................................................... 5 Concomitant therapy ........................................................................................................ 5 Justification ....................................................................................................................... 6 Inventory............................................................................................................................ 6

Placebo: .......................................................................................................................................... 6 Source................................................................................................................................. 6 Dosage ................................................................................................................................ 6 Justification ....................................................................................................................... 6 Inventory............................................................................................................................ 6

Experimental design ..................................................................................................................... 6 Procedure........................................................................................................................... 7 Pathology ......................................................................................................................... 13 Adverse Events ................................................................................................................ 14

Data Analysis ............................................................................................................................... 14 Response Variables ......................................................................................................... 14 Conclusions about the study’s objectives...................................................................... 16 Statistical software .......................................................................................................... 16

Study Records and Archiving .................................................................................................... 16 Attachements and Appendices................................................................................................... 17 LITERATURE CITED .............................................................................................................. 17

Study Number: SIUC-05-MT-04 Target Animal Safety Study of MT in Tilapia

1 Title: The Safety of 17α-Methyltestosterone Administered in the Feed for Induction of Sex Inversion in Tilapia

2 Purpose and Objective: To assess the safety of 17α-methyltestosterone (MT) fed to

tilapia (family Cichlidae) fry for induction of sex inversion to produce all male populations. The results of the safety study, if accepted by CVM, will be used to complete the TAS technical section data requirements and support a New Animal Drug Application to the U.S. Food and Drug Administration to approve the use of MT for this indication for the family Cichlidae.

3 GLP Compliance: The study will be conducted in compliance with the Good

Laboratory practices (GLP) for nonclinical laboratory studies at 21 CFR Part 58. All study personnel will receive GLP training annually or before the start of a study. Copies of SOPs can be found in Appendix C.

4 Sponsor

Dave Erdahl, Ph.D. USFWS 4050 Bridger Canyon Rd. Bozeman, MT 59715 Phone: (406) 857-9265 Fax: (406) 582-0242

5 Study Director

Anita M. Kelly, Ph.D. Fisheries and Illinois Aquaculture Center Southern Illinois University-Carbondale Mailcode 6511 Carbondale, IL 62901-6511 Phone: (618) 453-6099 Fax: (618) 453-6095

5.1 Study Personnel 5.1.1 Christopher C. Green, M.S.

Fisheries Research Laboratory Southern Illinois University-Carbondale Mailcode 6511 Carbondale, IL 62901-6511 Phone: (618) 536-7761 Fax: (618) 453-6095 Richard E. Echols, B.S. Fisheries Research Laboratory

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Southern Illinois University-Carbondale Mailcode 6511 Carbondale, IL 62901-6511 Phone: (618) 536-7761 Fax: (618) 453-6095

5.1.2 Joe Simeca, DVM, Ph.D.

Vivarium Attending Veterinarian Southern Illinois University-Carbondale Mailcode 6506 Carbondale, IL 62901-6506 Phone: (618) 457-4343

5.1.3 Maureen L. Doran, B.A., HTL (ASCP) Histology Preparation Life Science III School of Medicine Mailcode 6503 Southern Illinois University Carbondale, IL 62901-6503 Phone: 618-453-1584 [email protected]

5.1.4 Andrew E. Goodwin, Ph.D.

Certified Fish Pathologist (AFS-FHS) Aquaculture/Fisheries Center University of Arkansas at Pine Bluff 1200 N. University Drive Mail Slot 4912 Pine Bluff, AR 71601 Phone: (870) 575-8137 Fax: (870) 575-4638

5.2 Feed Preparation Rangen, Inc David Brock, Ph.D. 115 13 Ave. South Buhl, ID 83316 Phone: 208-543-6421 Fax: 208-543-6090 5.3 Methyltestosterone Analysis (feed)

CanTest Ltd. Brent Coates

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4606 Canada Way Burnaby, BC Canada V5G 1K5 Phone: 604-734-7276

6 Quality Assurance Officer 6.1 William L. Muhlach, Ph.D.

Department of Zoology Southern Illinois University-Carbondale Mailcode 6501 Carbondale, IL 62901-6501

7 Test Facilities 7.1 Fisheries Research Laboratory

Southern Illinois University-Carbondale Mailcode 6511 Carbondale, IL 62901-6501

7.2 Vivarium

Southern Illinois University-Carbondale Mailcode 6506 Carbondale, IL 62901

7.2.1 All fish will be maintained in a single room of the Vivarium solely dedicated to this

study. 7.2.2 The Southern Illinois University-Carbondale facilities above are fully accredited by the

American Association for Accreditation of Laboratory Animal Care. 8 Test Animals

Tilapia (Oreochromis niloticus) fry from the family Cichlidae will be used in this study. Eggs will be removed from the mouth of the female and placed into hatching containers. Once the fry have absorbed their yolk sac (approximately 3 days post hatch) the fry will be fed a commercial tilapia diet for 2-3 days prior to initiation of the diets that will be used in this study. The treatment period will not start until the fry have reached 6 days of age. Only fish that are actively feeding prior to the initiation of the study will be used. Actively feeding fish will swim toward the surface and actively intake feed, resulting in less than or equal to half of the food remaining on the bottom of the tank. Fish that are not actively feeding will not be included in the study. Other exclusion criteria include fish that have external signs of disease or are deformed. Since tilapia have not phenotypically sexually differentiated at 10 days of age, all fish will be used regardless of genotypic gender. No attempt will be made to determine the genotypic sex of the fish.

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8.1 Justification

Oreochromis species are characterized by a precocious sexual maturity and a high reproductive efficacy, resulting in overpopulation in ponds. A monosex male population is a solution to control reproductive activity and also to increase production because the male grows faster than the female in these species (Ruwet et al. 1976; Hanson et al. 1983). Sex reversal with synthetic androgens is one of the most frequently applied techniques to produce monosex male populations in tilapia. This involves the administration of synthetic androgens or estrogens to differentiating fry either by injection, immersion, or via the feed. Use of steroid-treated feeds for the production of all-male populations is widespread in tilapia aquaculture (MacIntosh and Little 1995). This technique results generally in 100% masculinization both at experimental and commercial stages (Guerrero 1975; Berger and Rothbard 1987; Mc Geachin et al. 1987; Guerrero and Guerrero 1988; Vera-Cruz and Mair 1994; Melard et al. 1995). However, the FDA has not approved the use of MT for sex inversion in fish, which will eventually be consumed by humans. Therefore the objective of this study is to conduct a target animal safety study to determine if MT is harmful to tilapia when provided in the feed.

8.4 Procurement

Tilapia fry will be obtained from brood stock maintained at Southern Illinois University-Carbondale or from a commercial vendor.

9 Test Article

17α-methyltestosterone (MT), CAS number 58-18-14 will be incorporated into #0 and #1 tilapia starter diet, Batch Numbers #tba by Rangen Inc, Buhl, ID (Batch numbers will be described in the Final Study Report (FSR)). A 5-g sample of each batch of medicated feed will be collected and archived as per 21 CFR Part 58.

9.1 Source: Control and all hormone treated feed (2.3 kg of each) will be supplied by Rangen, Inc., Buhl, Idaho. Treated feeds will be mixed according to the manufacturer’s standard operating procedures. Briefly, treated feeds will have methyltestosterone (MT) added by mixing MT with ethanol and while stirring the dry diet in a mixing bowl, the MT ethanol mixture will be sprayed onto the feed. The hormone-ethanol solution will be thoroughly mixed with a numerically equal volume of the feed (e.g. 100 mL hormone solution with 100 g of feed). Uniform distribution of the hormone will be accomplished by thoroughly mixing the solution with the feed. The wet diet will be dried by allowing the ethanol to evaporate. Appropriate amounts of MT will be added to the dry diets to achieve MT concentrations of 0, 60, 180, and 300-mg/kg of feed. Note that separate hormone-ethanol solutions will be prepared for each treatment dose.

9.1.1 Verification of Methyltestosterone in the Feed

Each batch of feed will be analyzed for MT concentration prior to and upon completion of the feeding trials. Certification of MT content in feed will be provided by CanTest

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Ltd. Feeds that are within + 10% of the nominal will be utilized in this study. If analysis shows that MT concentration falls outside + 10% of the target dose, a new batch of medicated feed will be prepared. Un-medicated feed will be analyzed upon completion of the feeding trials only.

Homogeneity- Three samples will be removed from each bag of feed by the Study Director, given an identification number and analyzed for homogeneity confirmation. Homogeneity will be confirmed by separately placing each “bag” of feed into a Hobart mixer mixing the feed for 5 minutes and removing a 50-gram sample of feed from the top. The mixing protocol will be repeated twice more for a total of three 50 g samples per bag. Analysis- MT concentration in the feed will be determined by CanTest, Ltd. Determination of MT in the feed will follow the procedure developed by Marwah et al. (2005). For feed having MT concentrations above 120 mg/kg, appropriate methods will be used to dilute the sample so that the concentrations are within the validated range of the assay (e.g., dilute with a pre-determined volume/weight of non-medicated feed). Dilution factors will then be used to determine actual MT concentrations in the feed. Procedures used to dilute such feed samples and dilution factors used to determine actual MT concentrations in feed will be fully described in the FSR.

9.1.2 MSDS All study personnel will be required to review the methyltestosterone MSDS (Appendix A). The Study Director or designee will ensure that all study participants are familiar with this MSDS, as well as with the MSDS of all other chemicals used during the course of this study.

9.2 Dosage

Feeds will contain 0, 60, 180 and 300 mg/kg MT. Fish will be fed 15% of their wet body weight daily. MT will be administered at a dosage of 0, 9, 27, or 45 mg/kg of fish biomass.

9.2.1 Duration of treatment

MT medicated feed will be fed to tilapia fry for 28 consecutive days. 9.2.2 Concomitant therapy

Because this is a target animal safety study in which data on the effects of MT are being collected, there should be little or no concomitant therapy. Preferably no treatment should occur in the six days prior to the initiation of the feeding trials or during the feeding trials.

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9.2.3 Justification Tilapia aquaculture in the United States has grown dramatically since 1991. In 2003, domestic tilapia production was 9200 mt, which is less than half of the 100,000 mt of tilapia that was imported in the same year (American Tilapia Association). The market for tilapias in the United States, more so than in developing countries, demands the production of larger fish. However, excessive reproduction is a problem in tilapia culture where overcrowding of young causes competition for the available food and represses the growth of the entire population.

Many approaches for controlling reproduction have been tried and one of the more promising techniques is hormone induction of monosex populations (Guerrero 1975). In addition to eliminating the problem of unwanted reproduction, all-male populations have a greater growth potential (Kirk 1972). One method of producing all male populations is to treat young fish with androgens to induce phenotypic sex inversion resulting in 100% male offspring.

Various steroids are differentially potent and can be influenced by the mode of administration (Yamamoto 1969). It is considered more efficient to administer sex hormones orally than by injection or immersion. The addition of the hormones to the feed is thus a convenient mode of presentation. The androgen dosage that has been consistently effective for sex inversion in tilapia is methyltestosterone at 60 mg/kg of feed (Guerrero 1975; Sanico 1975; Tayamen 1977). Lower levels have been less effective, or ineffective. Although initial studies fed the hormone for 60-120 days, the typical hormone treatment period is 28 days.

The dosages to be used in this safety study are 0, 1, 3, and 5X the proposed standard dosage.

9.4 Inventory: Appendix B. 10 Placebo: #0 or #1 tilapia starter diet, which has been mixed with ethanol that does not contain

MT. 10.1 Source: Rangen, Inc. 10.2 Dosage

Fish will be fed a feed that does not contain MT at 15% of their body weight per day in a manner identical to that used to feed medicated feed to fish.

10.2.1 Justification: The feeding regime for placebo is consistent with the feeding regime for

test article. 10.3 Inventory: Appendix B 11 Experimental design

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Twelve 18.9-L aquaria each containing 50 fish will be utilized in this study. Figure 1 shows the layout of the room. Because the MT may leach into the water and contaminate the water in aquaria containing control fish or fish of different treatment levels, each aquarium will be a stand-alone system equipped with its own filtration system and will not be linked to other aquaria within the system. The water will be recirculated within each tank using an individual AquaClear 20 multistage filtration system (SOP No. 32.0) and water from individual tanks will not mix with any other tank. This system incorporates mechanical, chemical and biological filtration of the water in the tank. The mechanical filtration consists of a foam filter insert for trapping particles and debris. The chemical filtration is composed of an activated carbon insert, which adsorbs odors, discoloration and impurities. The biological filtration consists of an insert containing media to which bacteria can adhere and grow. The biological filtration aids in the breakdown of nitrogenous wastes within the system. The AquaClear 20 filtration system pumps water at 113.6 L per hour, therefore the exchange rate will be 144 times per day or 6 times per hour (1.9 L per minute). Tanks will be siphoned daily (between 700 and 800 hours) to remove debris on the bottom of the tank. No more than 10 % of the tank volume (1.89 L) will be removed and replaced daily.

11.1 Blinding and Randomization

Fish will be randomly assigned to aquaria within a block and aquaria within the block will be randomly assigned to treatment groups. Each diet will be assigned a four digit numeric code by the Study Director. The Study Director will randomly assign treatment groups to tanks without any masked study participants present. Once a tank within a block has been assigned to a treatment group, the Study Director will record the group next to the tank on the Random Assignment of Feed and Tanks form (Appendix B). The Study Director will keep this information in a secure place. The random assignments of treatments to tanks will not be revealed to the study personnel doing the observations and the veterinarian conducting gross necropsies. This will eliminate the potential of data-collection bias based on treatment group. The random assignment of fish to tanks will be conducted with masked investigators present. Feed will be kept in a locked refrigerator for which only the Study Director and one unmasked investigator will have access.

12 Procedure 12.1 Once the fish have hatched, yolk sac fry will be counted, weighed as a group and a

subsample of 10 fish will be measured before being randomly assigned to one of the twelve experimental tanks. In addition, fish will also be assigned to one nonexperimental tank (these fish will serve as extras to replace mortalities that occur prior to the initiation of feeding fish the study diets, i.e. during the acclimation period only). Assignment of fish randomly to tanks will be done by placing 12 pieces of paper numbered 1-12 in a

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box. The masked investigator will pull a number from the box and place 50 fish in that tank. This is repeated until all 12 tanks are stocked with 50 fish. The tanks within each of the three blocks need to be randomly assigned to treatment groups. Four pieces of paper, each containing one of the treatment numbers is placed into a box. In a separate box, four pieces of paper are placed containing the tank numbers within one block of tanks. The Study Director will pick one piece of paper from the treatment numbers box and one piece of paper from the tank number box. The tank within that block is then assigned that treatment number. The assignment of treatment to tank will be recorded on the Tank Treatment Assignment form (Appendix B). Once one block of tanks have been assigned to one of each of the treatment groups, the Study Director will replace the treatment numbers in the box and shake it. The next group of tank numbers is then placed into the second box and the process for random assignment is repeated until all tanks have been assigned a treatment group. Tanks will not be marked in any manner to indicate a treatment assignment. Tanks will be sequentially numbered 1-12 as shown in Figure 1. Assignment of treatments to tanks will be done in the presence of unmasked investigators only. The Study Director will keep the Tank Treatment Assignment Form in a secure place and this form will not be shown to masked investigators or the veterinarian.

12.2 Each group of four tanks will be assigned to the placebo or test article groups following a randomized block design. The tanks will be assigned to blocks for this study (Figure 1). The first four tanks (1-4) closest to the door will assign to block 1. The next four tanks (5-8) will be block 2 and the last four tanks (9-12) will be block 3. Each block of tanks will have one of each treatment group represented.

Once the fish reach 6 days of age, i.e. 6 days after hatch, they will then be weighed,

measured and fed the assigned diets for 28 days. This is the standard duration for administration of the drug.

12.3 Inclusion criteria

The entrance criteria for inclusion in studies conducted under this protocol will include the following: (1) an adequate number of healthy-appearing test fish will be available for the study; (2) test fish will have been acclimated to source water that will be used during treatment for at least 72 h.

12.4 Exclusion criteria

Any event that might occur during the test phase of an experiment to a replicate experimental unit that would negatively impact the outcome of the study would result in exclusion of that replicate from the study. Events such as fish escaping from tanks, feeding the wrong MT dosage to a treatment group, or if excess mortalities not associated with the MT treatment occur (i.e., such as Strep). If a replicate experimental unit is excluded from the study, the study will be stopped and a new study initiated.

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12.5 An analysis of starting water in the aquaria will be conducted to determine water temperature, hardness, alkalinity, dissolved oxygen, nitrate, nitrite, ammonia, chloride, carbon dioxide, and pH. These parameters will be measured prior to the initiation of the study. Thereafter, beginning with the acclimation period and continuing until the end of the study, water quality and temperature will be maintained at optimum levels according to standard aquaculture practices. Dissolved oxygen and water temperature will be measured daily with an YSI Model 55/12 FT (Yellow Springs Instruments, Inc., Yellow Springs, Ohio, USA; SOP No. 31.0). The pH of experimental tank water will be measured daily with a Accumet AB15 pH meter (Fisher Scientific, St. Louis, Missouri, USA; SOP No. 34.0). Alkalinity and hardness will be measured titrimetrically (APHA 1995; SOP No. 23.0) once a week. Photoperiod will be maintained at 14 h light and 10 h dark using an Intermatic timer (SOP No. 33.0) that is directly wired into the electrical system of the room. Photoperiod will begin at 700 h. Water temperature will be maintained at 28 + 1 0C for the duration of the study.

12.6 All fish will be fully acclimated to the facilities before treatments are administered. The

acclimation period will begin on the day the fish hatch and continue through 5 days post hatch. An additional tank of fish containing tilapia fry not selected in the initial random assignment to treatments will also be fed and observed during the acclimation period. During the acclimation period, fish will be observed four times daily. During the acclimation period and after the fish have absorbed their yolk sac, they will be fed to satiation four times daily. Feeding to satiation will enable all of the fish in the tank the opportunity to consume feed that is offered. Fish will be observed for signs of feeding during the acclimation period. Actively feeding fish will swim toward the surface and actively intake feed, resulting in less than or equal to half of the food remaining on the bottom of the tank. Any fish that has absorbed their yolk sac and is not observed swimming toward the food and actively taking in feed will be removed from the tank and replaced with a fish from the extra tank. If during the acclimation period, mortalities occur in any of the treatment tanks, fish will be replaced by fish from the tank of extra fish. Any fish that shows signs of disease or deformity will also be removed and replaced with fish from the extra tank. Replacement fish will be counted, weighed and measured and the information placed on the Fish Weight and Measurement Log form (Appendix B) for that tank, noting that the fish is a replacement.

Feed offered during the acclimation period will not be treated with MT. Each tank will

have its own container of either #0 or #1 tilapia starter feed. The container is to be weighed before the first feeding of the day and after each of the three feedings. These data will be recorded on the Daily Feed Log – Acclimation Period (Appendix B) for each tank.

Dead or moribund fish will be removed daily from the tanks during the acclimation

period, counted, recorded on the Mortality Data Sheet (Appendix B) and subjected to gross examination. Fish will then be placed in 10% buffered formalin and archived.

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12.7 Once the fish are 6 days of age, the MT and placebo feeds will be fed to fish as per the randomized treatment scheme. Once the treatment period begins, fish will not be replaced if mortalities occur. Prior to the initial feeding of the study diets, each tank of fish will be weighed as a group and 10 fish will be randomly selected and individually measured. Feed calculations will be determined from the initial weights. Feed will be offered 4 times daily in approximately equal portions. The amount of feed offered will initially be based on the biomass in the experimental tanks at allocation and then 10 fish from each tank will be weighed on a Mettler 8000SB1 balance (SOP No. 15.0) and measured every week to adjust the feeding amount. Weights and measurements will be recorded on the Fish Weight and Measurement Log form. Initial weights and lengths of fish starting at the initiation of feeding the experimental diets will be transferred to the Weekly Weight and Feed Adjustments form (Appendix B). Daily feed amounts will be adjusted based on the chart developed by Phelps and Pompa (2000).

Size range of fish (mm) Expected growth (mm) 8-12 0.25 9 0.31 10 0.38 11 0.44 12 0.5 13 0.56 14 0.62 15 0.69 16 0.75 17 0.81 18 0.87 19 0.94 20 1.0 21 1.06 22 1.12 23 1.19 24 1.21 25 1.27

For example, if a subsample of 10 fry in one experimental tank have an average length of 8 mm at the beginning of the treatment period and weigh 0.10 grams total, the feeding rate on day one will be (0.01g per fish x 0.15 feeding rate x 50 fish in tank) = 0.075 grams of feed. For day 2 the fish will be assumed to have grown 0.25 mm so the average size is 8.25 mm. The total weight of all fish in one tank will be determined using the equation:

Weight of fry = [0.02(Length)3/1000]*N Where: weight is in grams, length is the mean length in mm, and N is the number of fish in the tank. For the above example, on day 1 the fish averaged 8 mm and weighed 0.010 g and would receive 0.077 g of feed. On day 2 the fish would be 8.25 mm average length and would weigh 0.011 g each and receive 0.084 g of feed. The growth increment in length per day will be based on the length of the fish the last time they were measured. That is if the 10 fish average 8 mm then the growth rate per day in length will be 0.25 mm until the fish are weighed and measured the following week.

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One unmasked investigator, who will not conduct any data collection, will weigh feed on a Mettler 8000SB1 balance (SOP No. 15.0) into separate plastic bags, seal them, label them with study number, tank number, amount of feed, the date feed was first weighed, initials of the person who weighed the feed, date the feed is to be offered, and the name of the Study Director. The investigator will place the feed in the refrigerator where it will be stored until used. If mortalities occur, the daily ration for each tank will be reduced accordingly to maintain the 15% body weight feeding rate. The investigator will remove the feed to be fed on that day from the refrigerator. Empty plastic bags used to contain the daily rations will be retained until after the study is complete; the Study Director will inventory then dispose of the empty plastic bags by incineration.

12.8 After weighing, each tank of fish will be fed their assigned feeds and fish will be

observed 4 times daily for feed consumption, presence or absence of fecal material and for changes in behavior and response during the acclimation and treatment periods (SOP No. 21.1) .Fish distribution within the tanks will be recorded after lights are on but prior to tank cleaning as follows:

D= Dispersed: generally dispersed throughout the tank (normal) C= Crowding: generally crowded at the water flow inlet (abnormal)

P= Podding: generally congregated or podding in a shaded area of the tank (abnormal)

O= Other (abnormal): behavior characteristics other than those described above will be documented and described

Study personnel will assess fish distribution by slowly and cautiously moving to a

position near the tanks from which the observer can see the fish within the tank. The observer will note the general distribution of the fish within the tank and whether fish distribution was recorded before or after the initial flight/fright response was elicited. It is preferred to obtain fish distribution observation before the fish express a flight/fright response. Fish distribution should be assessed in all tanks with fish before attempting to assess flight/fright response.

Flight/fright response of fish within the tanks will be recorded following or concurrently

with fish distribution as follows: L = Lethargic: little to no flight/fright response, fish seem unresponsive (abnormal) N= Normal: fish dispersed throughout the water column react to movement by

podding or rapid movement away from the movement, appear to calm relatively quickly

F= Frantic: fish make erratic bursts to evade the movement source, seem to “bounce off the walls” (abnormal) O= Other (abnormal): behavior characteristics other than those described above will

be documented and described

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The preferred method for study personnel to assess fish flight/fright response is to move from their fish distribution observation position to a point near the tank at which they will then wave an arm once across the group of tanks. Because the fish may sense the presence of the observer during the observation of fish distribution, the observer will note whether or not the fish in the tank exhibited a response during the distribution observation, and the movement stimulus (arm wave) will still be administered before tank cleaning.

Study personnel will record the presence or absence of fecal material in the tank

concurrent with assessing fish distribution, flight/fright response, or immediately before tank cleaning.

Feeding activity will be recorded as follows:

0 = Poor: fish do not swim toward food or actively intake feed resulting in most or all of the food on bottom of the tank (abnormal)

1 =Average: majority of the fish swimming toward and actively taking in feed resulting in approximately half of the food remaining on the bottom of the tank (normal)

2 =Vigorous; all fish swimming toward feed and actively taking in feed resulting in little to no food on the bottom of the tank (normal)

Fish will also be observed for signs of flashing and loss of equilibrium concurrently with

fish distribution and response observations.

Study personnel offering the feed and making the observations will be unaware of the treatment assigned to each tank. Dead or moribund fish will be removed daily from the tanks during the treatment period, counted, weighed as a group (to the nearest 0.01 g) using a Mettler 8000SB1 balance (SOP No. 15.0 and SOP 22.0), individual total lengths measured (to the nearest mm; SOP 22.0) and recorded on the Mortality Data Sheet. Fish that die during the treatment period will be subjected to complete gross and histopathological examination (see below). The attending veterinarian will not be aware of the fish’s treatment group. The whole fish will be fixed in 10% formalin and sent to SIUC’s Histology Laboratory so that slides can be made. The slides will then be sent to the Fish Pathologist for analyses. If fish exhibit significant post-mortem changes (i.e. significant rotting or decay because of fungus or environmental bacteria) then individual fish gross necropsy will note this, and fish will not be subjected to histological analysis. However, the whole fish will be preserved. Daily, study personnel doing the feeding and observations will clean the tanks. Tanks are to be cleaned between 700 and 800 hours each morning. Each tank will have a mark on the side indicating the point at which 10% of the water volume has been removed. A siphon is started and feces and other debris (i.e. feed or algal particles) are siphoned into a bucket. No more than 10% of the water will be removed per day. If necessary, the sides of the aquaria will be scraped of algae using a coarse pad. The algae will be

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allowed to settle prior to siphoning of the tanks. Once tanks have been siphoned, dechlorinated water is added each aquarium such that the total volume of water is 18.9 L.

12.9 On the day after termination of dosing, all fish in the experimental tanks will be immobilized by immersion in an ice bath (one tank at a time) then euthanized by cervical severance (Close et al. 1996; Close et al. 1997; SOP 24.1). After fish from a tank are immobilized, the fish will be indiscriminately removed from the ice bath and assigned a number from 1 to 50, weighed, and their total length recorded. A random numbers table, including only the numbers 1-50, will be generated for each tank. Fish selected for histopatholgy will be those fish with the number corresponding to the first 10 numbers on the random generated table for that tank. In the event that a fish is not available (i.e. died prior to completion of the treatment period) for one of the first 10 numbers on the random table, the next number on the table will be used. If that fish is also not available, then the following number on the random numbers table will be used. This process will be repeated until 10 fish are available for histopathology. All fish in each tank will undergo a gross external exam. Any fish not chosen for histopathology as described above but with external abnormalities will also be sent for histopathological examination of any abnormal tissues as well (SOP 25.1). If the Study Director or the Veterinarian determines that a fish’s tail is sufficiently eroded so that determination of total length is not possible, then standard length to the nearest millimeter will be recorded for that fish and a comment added to the data sheet describing why standard length was recorded. Gross necropsies will be performed by the study veterinarian and histological slides will be read by the study Pathologist.

If necropsy data are recorded by someone other than the veterinarian, the data recorder must be masked. In the event that someone other than the veterinarian records the necropsy data, then all observations recorded to describe gross pathology observed during gross necropsy will be reviewed and confirmed by the Study Veterinarian. The external surface of each necropsied fish will be inspected, noting coloration as normal, dark or pale on the Necropsy Form. Throughout the necropsy, any grossly visible external or internal abnormalities will be recorded on the Necropsy Form, and noted for histopathological evaluation. The pathologist will be made aware so that the abnormality can be viewed histologically. Each fish will be opened and examined grossly by a veterinarian. Due to the relatively small size of the fish, (estimated to be < 25 mm at the end of the study) individual organs and organ systems will not be weighed. The veterinarian conducting the gross pathological exam will be unaware of the treatment assignment when gross necropsies are performed. Following gross necropsy, the 10 selected fish will be placed in 10% neutral buffered formalin for a period of no less than 48 hours. Whole fish will be processed onto slides according to SIUC Histology Center’s SOPs entitled Labeling Specimens, Tracking Specimens for Paraffin Processing, V.I.P.- Processing Tissue, V.I.P. –Unloading Tissue and Paraffin Embedding, Paraffin Microtomy and Routine Stains: H&E. Processed slides will be sent to the pathologist. The remaining fish in the tank will be placed into formalin and archived.

12.10 Pathology The study pathologist will examine all histological samples. The pathologist will be

provided copies of the necropsy reports and will be aware of the identity of the different

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treatment groups and any clinical signs observed for each fish. Tissues to be examined during histopathology include liver, anterior (head) kidney, posterior (trunk) kidney, spleen, heart, gill, pituitary, and gonads.

Tissues from the 5X treatment group and the untreated control group will be examined

first. If no histopathological changes are observed, then no further examination of tissues will be required. If histopathological changes are observed in the 5X treatment group then only tissues corresponding to the abnormal tissues in the 5X group will be examined in the 3X treatment group. If no histopathological changes are observed in the 3X treatment group then no further examination of tissues will be required. Similarly, if histopathological changes are observed in the 3X treatment group then only tissues corresponding to the abnormal tissues in the 3X group will be examined in the 1X group. If a histopathological change is observed in any tissue, then that tissue must be examined within the next lower groups until the pathology is not observed.

12.11 Adverse Events

The Study Director will be advised immediately of any mortality or other abnormality associated with or suspected to result from treatment. The Study Director will be advised immediately if any adverse events, such as power outages, pump failures, excess mortality due to disease outbreak occurs. The Study Director will then notify Study Management and discuss whether or not the mortality or abnormality has the potential to compromise study conclusions and if the study should be continued or terminated. The adverse event should be documented with an explanation as to what the event was, when it occurred, the duration of the event, and steps taken to mitigate the event and the final decision of whether the study should be continued or not. If any event occurs such as those described above occur, the event will be documented on the Adverse Events Form. A full explanation of why fish were removed from the study will be described in the Final Study Report.

12.12 Disposal of Wastes

Any remaining medicated feed, excluding archived samples, can be used in other studies or disposed of by incineration.

13 Data Analysis 13.1 Definition of the experimental unit and replicates

The experimental unit is herein defined as the tank of fish receiving feed containing a specific dose of MT (n = 50 test fish per experimental unit per replicate). There will be three replicates per MT dosage fed.

13.1 Response variables:

The primary response variables for this study will be necropsy findings, histopathological findings and mortality. These variables for statistical purposes will be defined as binary variables as follows:

14


Necropsy: presence or absence of lesions Histopathology: For each organ, presence or absence of lesions Mortality: fish died or did not die during treatment.

Secondary response variables will include:

Changes in weight of fish in tanks Changes in body lengths of fish in tanks Feed consumption Behavior data (i.e. fish distribution, flight or fright response, and feed response)

13.2 Statistical methodology

Secondary variable data generated will be summarized by exposure concentration. For example, for each concentration tested, we will: A. Calculate and graph “mean changes in weight of fish” and “mean changes in length of fish” for each concentration of MT fed (0, 60, 180, and 300mg/kg) to demonstrate differences, should they exist. Each mean will be based on the number of replicate groups of fish tested (n = 3 replicate groups), and a 95% CI for each mean will be calculated and graphed if one or more of the replicate groups has < 100% survival; B. Tabulate or graph behavior results to show the relative frequency of occurrence of normal and abnormal behaviors observed during exposure and recovery periods; C. Tabulate or graph fish feed consumption results for each concentration of MT fed (0, 60, 180, and 300mg/kg) to demonstrate differences, should they exist.

All binary variables will be analyzed using a generalized linear mixed model with the effects treatment (fixed) and block (random). A binomial error and logit link and the Kenward-Rogers degrees of freedom will be used. The analysis will be done in SAS Proc GLIMMIX using the following code:

proc glimmix; model y/n = treatment /error=binomial link=logit ddfm=kr; random block; lsmeans treatment /diff; run;

Here, y represents the number of successes in a tank and n represents the total number of fish sampled.

If the data do not converge in Proc GLIMMIX, the proportion of successes will be analyzed using a linear mixed model with the effects treatment (fixed) and block (random). Prior to analysis, the proportions will be transformed using the arcsine square root function. The Kenward-Rogers degrees of freedom will be employed. The analysis will be done in SAS Proc Mixed using the following code:

proc mixed; model response = treatment /ddfm=kr;

15


random block; lsmeans treatment /diff; run;

Here, response represents the arcsine-square-root-transformed proportion of success.

Treatment will be tested at significance level α = 0.10. If treatment is significant, pairwise comparisons will be tested between the control group and each treated group (lsmeans statement in SAS code above) at unadjusted significance level α = 0.10.

13.2.1 Other data: Tabulated in final report 13.3 Conclusions about the study’s objectives

Data used in comparisons will be submitted as a text file or in an Excel spreadsheet with the FSR. All statistical output from the statistical package used to do any analysis for this study will be submitted with the FSR.

13.4 Statistical software

The statistical graphics/software package used will be Statistical Analysis System, version 9.1 (SAS Institute, Inc., Cary, North Carolina, USA) or an equivalent. Commonly used, commercially available statistical software packages, such as those used in these studies, will not be validated.

14 Study Records and Archiving

All data generated in the study will be recorded in loose-leaf laboratory notebooks. All data sheets and electronic files will be encoded with the study number when the data are generated and stored in secure files. Raw data, prepared slides, tissue blocks, preserved residual carcasses, laboratory notebooks, electronic files and the final report will be filed in the archives of Fisheries and Illinois Aquaculture Center, Southern Illinois University, Carbondale, IL as per 21 CFR Part 58.195 (SOP 30.0).

14.1 Deviations All deviations from this approved protocol will be documented in the laboratory

notebook at the time they occur and discussed with the Study Director, who will determine the impact of the deviations. The Study Director will notify management and the Quality Assurance Unit of any deviations to the protocol including their impact on the study. Deviations from the established Study Protocol that could potentially negatively impact the outcome of the study will be documented in the FSR. If deviations occur, the Study Director will be contacted immediately for advice. If possible, protocol deviations will be documented fully when they occur, accompanied by a written explanation of what

16


happened, why, and what steps were taken to mitigate the deviation. If deviation statements are entered in log books as raw data, they will be signed and dated.

14.2 Amendments

Protocol addendums will be attached to the revised version of study protocol “The Safety of 17α-Methyltestosterone Administered in the Feed for Induction of Sex Inversion in Tilapia”. If a protocol addendum is sent to FDA’s CVM, a cover letter will accompany the addendum referencing the submission date of the above protocol, and describing the purpose of the addendums. Addendums and copies of the cover letter sent to FDA will be kept with the study protocol.

A signed copy of the Study Protocol will be retained by the Study Director during the acclimation and treatment periods. At any time before a study begins, desired changes to the Study Protocol should be brought to the attention of the Study Director. The desired changes will be documented. Documentation will include date of amendment, and the amended text. The amendment will be kept with the original study protocol. The amendment will be signed by the Study Director. All study personnel will be advised of protocol amendments prior to starting a study.

15. Attachments and Appendices Attachment 1. Layout of the aquaria room. Attachment 2. Schedule of study procedures Appendix A. MSDS for Methyltestosterone Appendix B. Data forms referenced Appendix C. Standard Operating Procedures Appendix D. Personnel and Curriculum Vitae

a. Study Director Anita M. Kelly, Ph.D. b. Quality Assurance Officer William Muhlach, Ph.D. c. Other Study Personnel Christopher C. Green, M.S. Rick Echols, B.S.

Joe Simeca, DVM, Ph.D. Maureen L. Doran, B.A., HTL (ASCP) Andrew E. Goodwin, Ph.D. LITERATURE CITED

APHA (American Public Health Association). 1995. Standard methods for the examination of

water and wastewater. 19th edition. APHA, Washington, D.C. Berger, A. and S. Rothbard. 1987. Androgen induced sex-reversal of red tilapia fry stocked in

cages within ponds. Bamidgeh 39: 49-57. Close, B. (Chair), K. Banister, V. Baumans, E. Bernoth, N. Bromage, J. Bunyan, W. Erhardt, P.

Flecknell, N. Gregory, H. Hackbarth, D. Morton, and C. Warwick. 1996.

17


Recommendations for euthanasia of experimental animals: Part 1. Laboratory Animals. 30:293-316.

Close, B. (Chair), K. Banister, V. Baumans, E. Bernoth, N. Bromage, J. Bunyan, W. Erhardt, P. Flecknell, N. Gregory, H. Hackbarth, D. Morton, and C. Warwick. 1997. Recommendations for euthanasia of experimental animals: Part 2. Laboratory Animals. 31:1-32.

Guerrero, R.D. 1975. Use of androgens for the production of all male Tilapia aurea (Steindachner). Transactions of the American Fisheries Society 104:342-348.

Guerrero, R.D. and L.A. Guerrero. 1988. Feasibility of commercial production of sex reversed Nile tilapia fingerlings in the Philippines. Pages 183-186 in: R.S.V. Pullin, T. Bhukaswan, K. Tonguthai, and J.L. Maclean, editors. Proceedings of the Second International Symposium on Tilapia in Aquaculture, 16-20 March 1987, Bangkok, Thailand ICLARM Conference Proceedings, vol. 15.

Hanson, T.R., R.O. Smitherman, W.L. Shelton, and R.A. Dunham. 1983. Growth comparisons of monosex tilapia produced by separation of sexes, hybridization, and sex reversal. In: W.L. Shelton, and Z. Yaron, editors. Proceedings of the First International Symposium on Tilapia in Aquaculture. Tel Aviv University, Israel, pp. 570-579.

Kirk, R.G. 1972. A review of recent developments in Tilapia culture, with special reference to fish farming in the heated effluents of power stations. Aquaculture 1:45-60.

MacIntosh, D.J. and D.C. Little. 1995. Nile tilapia Oreochromis niloticus. Pages 277-320 in: N.R. Bromage, and R.J. Roberts, editors. Broodstock Management and Egg and Larval Quality. Blackwell Publishing Co., Cambridge, MA, USA.

Marwah, A., P. Marwah, and H. Lardy. 2005. Development and validation of a high performance liquid chromatography assay for 17α-methyltestosterone in fish feed. Journal of Chromatography B 824:107-115.

Mc Geachin, R., E.H. Robinson, and W.H. Neill. 1987. Effect of feeding high levels of androgens on the sex-ratio of Oreochromis aureus. Aquaculture 61:317-321.

Melard, C. 1995. Production of a high percentage of male offspring with 17α-ethynylestradiol sex-reversed Oreochromis aureus. I. Estrogen sex-reversal and production of F2 pseudofemales. Aquaculture 130:25-34.

Phelps, R.P. and T.J. Pompa. 2000. Sex reversal of tilapia. Pages 34-59 in: B.A. Costa-Pierce and J.E. Rokacy, editors. Tilapia Aquaculture in the Americas, Volume II. World Aquaculture Society, Baton Rouge, LA.

Ruwet, J.C., J. Voss, L. Hanon, and J.C. Micha. 1976. Biologie et élevage du tilapia. Pages 332-364 in FAO, editors, Symposium on Aquaculture in Africa, 30 September-2 October 1975, Accra, Ghana. CIFA Technical Paper 4.

Sanico, A.F. 1975. Effects of 17α-ethynltestosterone and esterone on sex ratio and growth of Tilapia aurea (Steindachner). Master’s thesis, Auburn University, Auburn, Alabama. 36 pp.

Tayamen, M.M. 1977. The inducement of sex reversal in Sarotherodon niloticus (Linnaeus). Master’s thesis, Auburn University, Auburn, Alabama. 50 pp.

Vera-Cruz, E.M.V., and G.C. Mair. 1994. Conditions for effective androgen sex-reversal in Oreochromis niloticus (L.). Aquaculture 122:237-248.

Yamamoto, T. 1969. Sex differentiation. Pages 117-175 in: W.S. Hoar, and D.J. Randall, editors. Fish physiology. Volume III. Academic Press, New York.

18


Attachment 1. Figure 1. Layout of the aquaria room in the vivarium in Life Science II on the campus of Southern Illinois University Carbondale. Numbers within the aquaria are tank numbers. Numbers to the right of the bracket are block numbers.

3

2

1

12

11

10

9

8

7

6

5

4

3

2

1

Aquaria stand with aquaria

Door

19

Attachment 2.

Table 1. Schedule of Study Procedures. The following list is the anticipated study day on which

the listed activity will occur. The exact date on which an activity occurs will be recorded.

Anticipated Date of Activity

Activity TA Day 1

Tank Acclimation (TA) – Weigh, measure and allocate newly

atched yolk-sac fish to experimental tanks h TA Day 1-5

Observe fish for absorption of yolk sac. Once yolk sac has been absorbed, offer un-medicated diet. Observe fish during yolk-ac and post yolk-sac. s

D osing Day 1, 7, 14, and 21 W eigh and measure fish in experimental tanks D osing Day 1-28 O ffer experimental diets, observe fish. Dosing Day 29 Euthanize all surviving fish and weigh and measure. Necropsy

and begin preparing histological samples for selected fish.


21

Appendix A. MSDS for Methyltestosterone

Appendix B. Data Forms Referenced

Test Article Inventory From Random Assignment Form for Feed Random Assignment Form for Fish to Tanks Random Assignment Form for Feed to Tanks Water Quality Log Fish Weight and Measurement Log Weekly Weight and Feed Adjustments Daily Feed Log – Acclimation Period Daily Feed Log- Treatment Period Acclimation Period Observation Record Treatment Period Observation Record Necropsy Record Histopathology Record Adverse Events Form


27

TEST ARTICLE INVENTORY

THESE PAGES ARE NOT TO BE SHOWN TO MASKED INVESTIGATORS INSTRUCTIONS: Complete one form for each batch of test article including control feed - attach extra pages if necessary. Fill in all blanks - use N/A if not applicable. For feed code, state the type of feed, i.e. #0 or #1 tilapia starter diet, and state the numerical code (from the Numerical Assignment Form for Feed) corresponding to the concentration of MT in the feed. Batch number should include the batch number of the feed obtained from the manufacturer. MT Concentration __________ Feed code ____________ Batch No. ___________Expiration Date ___________ Date No. Grams

Received From No. Grams Used By No. Grams

Remaining Remainder (check one): Destroyed Returned Date ____________ Initials _______________


28

Numerical Assignment Form for Feed

THESE PAGES ARE NOT TO BE SHOWN TO MASKED INVESTIGATORS INSTRUCTIONS: The diets will have a four digit number assigned to each treatment group by the Study Director. The Study Director will pick the numbers from a randomized table. The Study Director will keep this information in a secure place. This information will not be revealed to the masked study investigators or the veterinarian. MT Concentration in Feed Four Digit Code Study Director ____________________________________ Date____________________


29

Random Assignment Form for Fish to Tanks INSTRUCTIONS: Assignment of fish randomly to tanks will be done by placing 12 pieces of paper numbered 1-12 in a box. A masked investigator will pull a number from the box, write in the first blank space below, weigh and measure 50 fish and record the data on the Procurement and Treatment data form and place those 50 fish in that tank.

Order in which tanks are stocked with Tilapia

Investigator ____________________________________ Date____________________


30

Random Assignment Form for Feed and Tanks

THESE PAGES ARE NOT TO BE SHOWN TO MASKED INVESTIGATORS INSTRUCTIONS: The tanks will be assigned to blocks for this study (Figure 1). The first four tanks (1-4) closest to the door will assign to block 1. The next four tanks (5-8) will be block 2 and the last four tanks (9-12) will be block 3. Each block of tanks will have one of each treatment group represented. The Study Director will then assign the tanks within each block randomly to treatment groups. Four pieces of paper, each containing one of the diet numbers is placed into a box. In a separate box, four pieces of paper are placed containing the tank numbers within one block of tanks. The Study Director will pick one piece of paper from the random numbers box and one piece of paper from the tank number box. The tank within that block is then assigned that treatment number. The assignment of treatment to tank will be recorded on the Tank Treatment Assignment form. Once one block of tanks have been assigned one of each of the treatment groups, the Study Director will replace the treatment numbers in the box and shake it. The next group of tank numbers is then placed into the second box and the process for random assignment is repeated. The Study Director will keep this information in a secure place. This information will not be revealed to the masked study investigators or the veterinarian. Block Number Tank Number 1 1 1 1 2 2 2 2 3 3 3 3 4 4 4 4 Study Director ____________________________________ Date____________________

Date Time Temp. °C

Water hardness

Water Alkalinity

Dissolved oxygen

Nitrate Nitrite Ammonia Chloride Water Carbon dioxide

Water pH

Recorded by

Safety of 17a-methyltestosterone For Induction of Sex-Inversion in Tilapia:

WATER QUALITY RECORD

INSTRUCTIONS: Complete one form for each tank used in the study - attach extra pages if necessary. Fill in all blanks - use "N/A" if not applicable. Prior to stocking yolk sac fish hardness, alkalinity, dissolved oxygen, nitrate, nitrite, ammonia, chloride, carbon dioxide, temperature and pH are to be measured prior to the initiation of the study. Dissolved oxygen, water temperature, pH and ammonia are to be measured daily. Alkalinity and hardness are to be measured once a week. Tank No. ________

Fish Weight and Measurement Log INSTRUCTIONS: Complete one form for each tank of fish in the study and for each day fish are weighed and measured- attach extra pages if necessary. During the initial stocking of yolksac fish, count and weigh 50 fish and randomly measure 10 fish. Fifty pieces of paper numbered 1-50 are placed into a box. Ten pieces of paper are removed. Fish are counted and those fish matching the numbers selected from the box will be weighed. If during the acclimation period, i.e. hatch until 5 days post hatch, mortalities or deformities or disease occur remove the fish from the tank and replace with fish from the extra tank. Count, weigh and measure the fish placing the data in one of the blank rows below. Place the combined weight of the replacement fish in the space marked combined weight of fish and a comment noting that this/these is/are the weight(s) of the replacement fish. In the comment column state that it is a replacement and the reason for that replacement. Fill in all blanks - use "N/A" if not applicable. Date ________________ Tank No. ________________ Combined weight of fish ____________________

Fish No. Length (mm)

Comments

1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Weighed and Measured By (signature) __________________________________Date ______________ Data Recorded By: ___________________________________________ Date ____________________


Page 1 of 2 Weekly Weight and Feed Adjustments

THESE PAGES ARE NOT TO BE SHOWN TO MASKED INVESTIGATORS INSTRUCTIONS: Complete one form for each tank of fish in the study - attach extra pages if necessary. Average length and weight of fish at the start of feeding the experimental diets will be transferred to column 1 below and average length to column 2. Daily feed amounts will be adjusted based on the chart developed by Phelps and Pompa (2000).

Size range of fish (mm) Expected growth (mm) 8-12 0.25 9 0.31 10 0.38 11 0.44 12 0.5 13 0.56 14 0.62 15 0.69 16 0.75 17 0.81 18 0.87 19 0.94 20 1.0 21 1.06 22 1.12 23 1.19 24 1.21 25 1.27

For example, if a subsample of 10 fry in one experimental tank have an average length of 8 mm at the beginning of the treatment period and weigh 0.10 grams total, the feeding rate on day one will be (0.01 x 0.15 x 50 fish per tank) = 0.075 grams of feed. For day 2 the fish will be assumed to have grown 0.25 mm so the average size is 8.25 mm. The total weight of all fish in one tank will be determined using the equation: Weight of fry = 0.02(Length)3/1000*N Where weight is in grams, length is the mean length in mm, and N is the number of fish in the tank. For the above example, on day 1 the fish being an average of 8 mm would weigh 0.01 g and would receive 0.077 g of feed. On day 2 the fish would be 8.25 mm average length and would weigh 0.011 g and receive 0.084 g of feed. The growth increment in length per day will be based on the length of the fish. That is if the 10 fish average 8 mm then the growth rate per day in length will be 0.25 mm until the fish are weighed and measured the following week. Once a week fish will be weighed and measured to verify and adjust feeding amounts. Days for weighing fish are marked as “weigh fish” on the data sheet below. Fill in all blanks - use "N/A" if not applicable. Transfer the amount of feed to the Daily Feed Log – Treatment Period for determination of feeding amounts per feeding.

33


Page 2 of 2 Weekly Weight and Feed Adjustments

Tank No. ________________ Date Average

length (mm)

Calculated weight [Weight of fry = 0.02(Length)3/1000*N]

Amount of feed [Total weight X 0.15]

By

(weigh fish)

(weigh fish)

(weigh fish)

(weigh fish)

34


Daily Feed Log – Acclimation Period INSTRUCTIONS: Fish will be distributed into tanks on the first day after hatching. To determine the amount of feed consumed by fish during the acclimation period, 20 grams of #0 tilapia starter diet will be weighed into a separate container for each tank of fish. The container will be weighed before the first feeding and the weight recorded. Fish will then be fed to satiation four times during the day. After each feeding period the container will be weighed again and the weight recorded. The amount of feed fed will be determined by subtracting the after feeding weights of the container from the before feeding rates. Date___________________ Tank Feed container

weight before first feeding

Feed container weight after first feeding

Feed container weight after second feeding

Feed container weight after third feeding

Total Fed Amount

Feed weighed By

1 2 3 4 5 6 7 8 9 10 11 12

35


Daily Feed Log – Treatment Period

THESE PAGES ARE NOT TO BE SHOWN TO MASKED INVESTIGATORS

INSTRUCTIONS: This task is to be completed by unmasked investigators. Total feed amounts fed on a daily basis should be obtained from the Weekly Weight and Feed Adjustments form. For each tank of fish, the total amount of feed is to be divided equally among four feedings. Record the amount of feed weighed for each feeding in the chart below. Place the feed into a bag, seal them, label them with study number, tank number, amount of feed, the date feed was first weighed, initials of the person who weighed the feed, date the feed is to be offered, and the name of the Study Director. The investigator will place the feed in the refrigerator where it will be stored until feed to fish by a masked investigator. Date___________________ Tank/ Treatment code

Amt of feed from Weekly Weight and Feed Adjustment Form

Amt Weighed for Feeding 1




Total Fed Amount

Feed weighed By

1/ 2/ 3/ 4/ 5/ 6/ 7/ 8/ 9/ 10/ 11/ 12/

36


ACCLIMATION PERIOD OBSERVATION RECORD INSTRUCTIONS: Complete one form for each tank of fish in the study (fish are observed 4 times daily)- attach extra pages if necessary. Fill in all blanks - use "N/A" if not applicable. In the observation column, the presence of yolk sac, fish distribution and the flight or fright response should be recorded along with any other observations. Yolk sac presence will be defined as (1) present; (2) absent. Fish distribution will be defined as: (1) Dispersed (fish generally dispersed throughout the tank; normal); (2) Crowded (fish generally crowded at the water flow inlet; abnormal); or (3) Podded (fish generally congregated in a shaded area of the tank; abnormal). The flight/fright response is to be assessed by slowly waving an arm over each tank. During the first observation of the day, the fright or flight response needs to be tested prior to tank cleaning. The flight/fright response will be categorized as follows: (1) Lethargic (fish seem unresponsive with little to no flight/fright response; abnormal); (2) Normal (fish are dispersed throughout the water column and react to movement by podding or rapid movement away from the movement, then appear to calm relatively quickly); or (3) Frantic (fish make erratic bursts to evade the movement source, seem to bounce off the walls; abnormal). Feeding observations will be categorized as follows: 0 = Poor: fish do not swim toward food or actively intake feed resulting in most or all of the food on bottom of the tank (abnormal); 1 =Average: majority of the fish swimming toward and actively taking in feed resulting in approximately half of the food remaining on the bottom of the tank (normal); 2 =Vigorous; all fish swimming toward feed and actively taking in feed resulting in little to no food on the bottom of the tank. (normal). Record the number of dead and moribund fish on the Mortality Record Sheet. If dead fish occur, contact veterinarian for necropsy. Tank No. ______________________________

DATE COMMENTS/OBSERVATIONS

Yolk sac:

Fish Distribution:

Flight or Fright Response:

Feed Response:

Initials

Additional Comments:


Yolk sac:

Fish Distribution:


Feed Response:

Initials



Yolk sac:

Fish Distribution:


Feed Response:

Initials



Yolk sac:

Fish Distribution:


Feed Response:

Initials


37


TREATMENT OBSERVATION RECORD INSTRUCTIONS: Complete one form for each tank of fish in the study (fish are observed 4 times daily)- attach extra pages if necessary. Fill in all blanks - use "N/A" if not applicable. In the observation column, the presence of yolk sac, fish distribution and the flight or fright response should be recorded along with any other observations. Fish distribution will be defined as: (1) Dispersed (fish generally dispersed throughout the tank; normal); (2) Crowded (fish generally crowded at the water flow inlet; abnormal); or (3) Podded (fish generally congregated in a shaded area of the tank; abnormal). The flight/fright response is to be assessed by slowly waving an arm over each tank. During the first observation of the day, the fright or flight response needs to be tested prior to tank cleaning. The flight/fright response will be categorized as follows: (1) Lethargic (fish seem unresponsive with little to no flight/fright response; abnormal); (2) Normal (fish are dispersed throughout the water column and react to movement by podding or rapid movement away from the movement, then appear to calm relatively quickly); or (3) Frantic (fish make erratic bursts to evade the movement source, seem to bounce off the walls; abnormal). Feeding observations will be categorized as follows: 0 = Poor: fish do not swim toward food or actively intake feed resulting in most or all of the food on bottom of the tank (abnormal); 1 =Average: majority of the fish swimming toward and actively taking in feed resulting in approximately half of the food remaining on the bottom of the tank (normal); 2 =Vigorous; all fish swimming toward feed and actively taking in feed resulting in little to no food on the bottom of the tank. (normal). Record the number of dead and moribund fish on the Mortality Record Sheet. If dead fish occur, contact veterinarian for necropsy. Tank No. ______________ DATE COMMENTS/OBSERVATIONS

Fish Distribution:


Feed Response:


Initials


Fish Distribution:


Feed Response:


Initials


Fish Distribution:


Feed Response:


Initials


Fish Distribution:


Feed Response:


Initials

38


39

Mortality Record Sheet

INSTRUCTIONS: Complete one form for each tank of fish in the study. Dead fish are to be netted out of the tank, counted, and weighed as a group (0.1 g) and individual total lengths measured (mm). The veterinarian will perform a gross necropsy on the fish. Fish are then to be placed into a vial of 10% buffered formalin, which is labeled with the date, tank number, number of dead fish, and the investigator’s initials. If mortalities occur during the acclimation period, a gross necropsy only is required. If mortalities occur during the treatment period fish will be subjected to gross necropsy and histopathology. Date _____________________ Tank Number of ID ___________________________ Number of mortalities ________________________________ Gross necropsy performed? yes no Date necropsy performed _______________________

Histopathology required? yes no Date histopathology performed _____________________

If no necropsy or histopathology was performed, please provide justification

__________________________________________________________________________________

__________________________________________________________________________________

__________________________________________________________________________________

_________________________________

Weight of all fish ______________________ Individual Fish Lengths (mm)

NECROPSY RECORD INSTRUCTIONS: Complete one form for each fish in each tank of the study - attach extra pages if necessary. Color of the fish should be noted as normal, dark or pale. If lesions are found they should be scored as follows: (0) no lesions; (1) mild lesions; (2) moderate lesions; (3) severe lesions. Location of lesion should be noted on the fish drawing. If more than one lesion is present, alphabetically label the lesions followed by the lesion score. For organ appearance note if the organ is normal or abnormal. If abnormal, state the reason in the comment column. Fill in all blanks - use "N/A" if not applicable. If data are recorded by someone other than the veterinarian, the data recorder must be masked. In the event that someone other than the veterinarian records the necropsy data, then all observations recorded to describe gross pathology observed during gross necropsy will be reviewed and confirmed by the Study Veterinarian. Date _____________ Tank No. ___________ Fish Number ______ Weight ____________ Length __________

External Color________________________________ Lesions______________________________________ Organ Gross Appearance Comments

Gills

Liver

Spleen

Kidney

Data Recorded by _____________________________________________ Date___________ Veterinarian’s signature ________________________________________ Date_________


41

HISTOLOGY RECORD Instructions: Fill in the date and the fish ID #. The following organs were collected for histological examination. The severities of inflammatory, degenerative and proliferative changes should all be noted and each graded on a scale of one to five (1 = minimal, 2 = mild, 3 = moderate, 4 = moderately-severe, and 5 = severe). Any additional tissue sent for analysis should be listed under other. Indicate the slide number, block number and tissue name in the spaces provided. If additional sheets are necessary, please attach. Date_________________ Fish ID#_____________________ Organ

Slide number

Block number

Tissue Appearance

Gonads

Pituitary

Liver

Spleen

Gills

Kidney (anterior and posterior)

Thyroid

Other (list):

Pathologist signature ________________________________________ Date_________


42

ADVERSE EVENTS FORM

Instructions: If any adverse events, such as power outages, pump failures, excess mortality due to disease outbreak occurs, this form needs to be completed for each event. The Study Director should be notified immediately. The Study Director will then notify Study Management and discuss whether or not the mortality or abnormality has the potential to compromise study conclusions and if the study should be continued or terminated. What was the adverse event?______________________________________________________ Date the event occurred__________________________________________________________ How long did the event last?______________________________________________________ What steps were taken to mitigate the event? _________________________________________ ______________________________________________________________________________

______________________________________________________________________________

______________________________________________________________________________

______________________________________________________________________________

______________________________________________________________________________

______________________________________________________________________________

______________________________________________________________________________

______________________________________________________________________________

______________________________________________________________________________

Was the study continued or terminated? _____________________________________________

Other comments________________________________________________________________

Signature _____________________________________________________________________


43

APPENDIX C. Standard Operating Procedures

SIUC Histology Laboratory SOPs Referenced in the Protocol

Labeling Specimens Tracking of Specimens for Paraffin Processing V.I.P.-Processing Tissue V.I.P.- Unloading Tissue and Paraffin Embedding Paraffin Microtomy Routine Stains: H&E

Fisheries and Illinois Aquaculture Center SOPs Referenced in the Protocol

SOP Number Title 15.0 Balance Calibration and Basic Use 21.1 Observation of Fish 22.0 Weighing and Measuring Fish 23.0 Water Quality Monitoring 24.1 Euthanasia of Test Fish 25.1 Test Organism Necropsy Methods 30.0 Archive 31.0 Dissolved Oxygen and Temperature Meter (YSI55) 32.0 AquaClear 20 Multistage Filtration System 33.0 Intermatic Timer 34.0 Accumet AB15 pH Meter Fisheries and Illinois Aquaculture Center SOPs Not Referenced in the Protocol but Critical to the Implementation of the Study SOP Number Title 1.0 Definition of Standard Operating Procedure (SOP) 2.0 Development, Revision, Deletion and Review of SOPs 3.0 Numbering and Format Systems for SOPs 4.0 Duties and Responsibilities Testing Facility Management 5.0 Study Director Criteria 6.0 Study Director Duties and Responsibilities 7.0 Preparation and Management of Resumes 8.0 Quality Assurance Unit (QAU) Responsibilities 9.1 Employee Training 10.1 Documentation of Personnel Training 11.0 Signature Sheet 12.0 Standard Procedure for Explaining Notebook Entry Errors 13.0 Receipt and Distribution of of Control Substances 14.0 Storage of Test and Control Substances 18.0 Test Organism Identification, Care, Transfer and Handling 26.0 Significant Figures and Number Rounding 27.0 Telephone, Conversation, and Meeting Documentation


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28.0 Computer Entry of Data 35.0 Study Conduct for Target Animal Safety Studies


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Appendix D. Personnel and Curriculum Vitae

17MT TAS Protocol Revision 4

Documents

Transcript of 17MT TAS Protocol Revision 4