Date of Approval: [May 21, 2002]{.underline}

FREEDOM OF INFORMATION SUMMARY

####### Public Master File

####### 5667

####### Oxytetracycline hydrochloride

"\...for skeletal marking of fish fry (larvae) and fingerlings."

Sponsored by:

####### NRSP-7

**I. GENERAL INFORMATION**

> PMF Number: 5667
>
> Sponsor: NRSP-7\
> North Eastern Region\
> College of Veterinary Medicine\
> Cornell University\
> Ithaca, New York
>
> Generic Name: Oxytetracycline hydrochloride
>
> Marketing Status Over-the-counter
>
> Supplemental Effect: The approval of this supplement will allow for
> the use of oxytetracycline for the marking of otoliths and other
> skeletal structures of fish to assist in their identification and
> aging.
>
> *Minor Species Classification* All fish are classified as minor
> species. Therefore, this Public Master File addresses minor species
> requirements with respect to effectiveness and target animal safety
> data collection.

**II. INDICATION FOR USE**

> Oxytetracycline is indicated for the marking of bony tissues in fish
> as an aid in identification.

**III. [DOSAGE FORM, ROUTE OF ADMINISTRATION, AND DOSAGE]{.smallcaps}**

A. *Dosage Form*: Water-soluble Oxytetracycline hydrochloride.

B. *Route of Administration*: Immersion (bath)

> C. *Recommended Dosage*: Immerse larval or juvenile fish in 200 to 700
> mg/L oxytetracycline hydrochloride (buffered) for two to six hours.

**IV. EFFECTIVENESS**

> A combination of data from many different fish species reared in
> different temperatures and management systems were used to support the
> determination of effectiveness in all teleost (bony) fish, consistent
> with the *Guidance for Industry: FDA Approval of Animal Drugs for
> Minor Uses and for Minor Species* (FDA/CVM January 1999).
>
> Note also that Oxytetracycline in feed has been approved for skeletal
> marking of Pacific salmon under NADA 038-439.

Summaries of studies demonstrating the effectiveness of oxytetracycline
are provided.

A.  Field Study Conducted under INAD 8054

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1.  Type of Study: Clinical Field Trial

2.  Name and Address of Investigator:

> Wallace Jenkins\
> South Carolina Department of Natural Resources\
> P.O. Box 12559\
> Charleston, SC 29422-2559

3.  General Design of the Investigation:

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a.  Purpose of the Study: To evaluate the efficacy of using
    oxytetracycline (OTC) to mark red drum fingerlings for later
    identification as stocked fish in wild populations.

b.  Test Animals: Red drum fingerlings *(Sciaenops ocellatus)*, 13-39 mm
    total length

c.  Treatment Groups: Fish were harvested from ponds, treated in holding
    tanks, and restocked in a pond on the culture facility. A group of
    untreated control fish from each pond was held for comparison of
    survival.

d.  Dosage Form: Water-soluble oxytetracycline hydrochloride

e.  Route of Administration: Immersion (bath)

f.  Dosages Used: 0 and 500 mg oxytetracycline/L of water for 4 hours

g.  Test Duration: 7 days (from treatment to stocking)

h.  Parameter: examination of otoliths to identify marks and mortality
    associated with OTC immersion marking

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4.  Methods:

> Red drum were harvested from 5 ponds in the fall of 1996 and 4 ponds
> in the spring of 1997 for immersion treatment. Fish were placed in a
> holding tank and acclimated to 15 ppt salinity water. OTC was added to
> the tank to provide a 500 mg/L OTC concentration. Fish were held for 4
> hours. Fish were then transferred to a hauling trailer filled with 15
> ppt salinity water. Fish were restocked in a pond on the culture
> facility and held for 7 days. The salinity in the pond was raised to
> the concentration of the area to be stocked during the 7 day holding
> period. Fish were harvested and transported to stocking sites for
> release. A subsample of each group of marked fish was retained for 30
> days to confirm mark efficacy.

5.  Results:

> Visible marks could be detected on otoliths in all batches of treated
> fish. Samples of fish from the wild population indicated that up to 40
> % of stocked age 1 fish have OTC otolith marks.

6.  Conclusion:

> Based on this study, the recommended dose of oxytetracycline to
> achieve a fluorescent mark on the otolith is through four-hour
> immersion at 500 mg/L oxytetracycline hydrochloride.

B.  Field Study Conducted Under INAD 8512

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1.  Type of Study: Clinical Field Trial

2.  Name and Address of Investigator:

> Michael L. Hendricks\
> Pennsylvania Fish & Boat Commission\
> Benner Spring Fish Research Station\
> 1225 Shiloh Road\
> State College, PA 16801-8495

3.  General Design of the Investigation:

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a.  Purpose of the Study: To apply unique multiple marks needed to
    discriminate between groups of fish from different egg source
    rivers, fish released as fry or fingerlings, and fish released at
    different sites.

b.  Test Animals: American shad fry *(Alosa sapidissima)*, 3-15 days old

c.  Treatment Groups: All fry produced received marks. Six different
    single and multiple marks were applied. A total of 8,500,000 fry
    were produced with all except 13,500 stocked in the Susquehanna
    River and its tributaries, or the Lehigh River. Fry were marked in
    1200 L rearing tanks (40 total), each containing up to 500,000 fry.

d.  Dosage Form: Water-soluble oxytetracycline hydrochloride

e.  Route of Administration: Immersion (bath)

f.  Dosages Used: 256 mg oxytetracycline/L of water for 4 hours

g.  Parameter: Fry from 6 tanks and raceways were sampled for otolith
    mark retention.

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4.  Results:

> Retention of immersion marks for American shad fry was 100 % for all
> production groups in 1996. Refer to the following table.
>
> **Table B.1.** Oxytetracycline mark retention for American shad reared
> in 1996.

  ----------- ----------- -------------- ---------- ------------ ----------- -----------
  **Tank/\    **Mark      **Marks        **Number   **Number     **Percent   **Number
  Raceway**   Applied**   Visualized**   Marked**   Examined**   Marked**    stocked**

  Race F1     9,12,15     9,12,15        19         19           100         171,700

  Race F3     3,9,12,15   3,9,12,15      19         19           100         277,100

  Race E1     3,6,9       3,6,9          18         18           100         42,900

  Race F2     3,9,12      3,9,12         19         19           100         561,100

  Race F4     3,6,9,12    3,6,9,12       19         19           100         682,500

  Not Sampled 3           3              \-         \-           \-          5,730,200

  Tank J4\*   3           3              17         17           100         Not stocked
  ----------- ----------- -------------- ---------- ------------ ----------- -----------

> \*Sampled at 28 days of age.

5.  Conclusion:

> Based on this study, the recommended dose of oxytetracycline to
> achieve a fluorescent mark on the otolith is through four-hour
> immersion at 256 mg/L oxytetracycline hydrochloride.

C.  Field Studies Conducted Under INAD 8427

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1.  Type of Study: Clinical Field Trial

2.  Name and Address of Investigator:

> Kirby D. Cottrell,\
> Hatcheries Section Head\
> Illinois Department of Conservation\
> Lincoln Tower Plaza\
> 524 South Second Street\
> Springfield, Illinois 62701-1787

3.  General Design of the Investigation:

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a.  Purpose of the study: To determine the effectiveness of
    oxytetracycline hydrochloride in the marking of otoliths of fry
    (larval) and fingerling fish of different species.

b.  Test animals and Treatment Groups: Largemouth bass (*Micropterus
    salmoides*) fingerlings, Walleye (*Stizostedion vitreum*) fry and
    fingerlings, and Sauger (*Stizostedion canadense*) fry and
    fingerlings.

c.  Treatment Groups: The fish were assigned to either treatment (500
    mg/L for 6 hours) or smaller control groups. The following table
    includes the number of each fish species treated in 1997.

  ------------------------ ---------------------- -----------------------
  **Species**              **Treated Fish**       **Control Fish**

  Largemouth bass          25,000                 300
  fingerlings                                     

  Walleye fry              5,837,4000             100,000

  Walleye fingerlings      143,306                1,000

  Sauger fry               2,400,000              100,000

  Sauger fingerlings       166,934                1,000
  ------------------------ ---------------------- -----------------------

d.  Dosage Form: Water-soluble oxytetracycline hydrochloride

e.  Route of Administration: Immersion (bath)

f.  Dosages Used: 0 and 500 mg oxytetracycline/L of water for 6 hours.

g.  Test Duration: 30 to 45 days (from treatment to sampling).

h.  Parameters: Clinical observations and examination of otoliths to
    identify marks (50 fish from each treated group, and 50 from each
    control group).

###### Methods:

> Marking of fry -- Groups of 100,000 fry were placed in a 500 mg/L
> concentration of oxytetracycline in 3 gallons of water within a
> plastic fish hauling bag for six hours. To ascertain mark retention,
> 100,000 of the marked and control fry were placed into separate
> rearing ponds. At the end of the rearing period (45 days for walleye
> and 60 days for sauger), samples of 50 fish per group were collected
> at harvest and checked for mark efficacy.
>
> Marking of fingerlings -- Fish were immersed in a 500 mg/L
> concentration of oxytetracycline for six hours. Marked and control
> fish were retained for 30 days post-treatment and then harvested for
> examination. Samples of at least 50 fish from each group were checked
> for mark efficacy.

4.  Results:

> Results of otolith marking field efficacy trials in 1997

  -------------- ----------------- ------------- ------------- ------------
  **Species**    **Treatment**     **Number      **Number      **Percent
                                   Examined**    Marked**      Marked**

  Walleye fry    OTC Immersion     50            50            100

                 Control           50            7             14

  Walleye        OTC Immersion     50            100           100
  fingerling                                                   

                 Control           50            0             0

  Sauger         OTC Immersion     50            50            100
  fingerling                                                   

                 Control           50            0             0

  Sauger fry     OTC Immersion     50            50            100

                 Control           50            0             0

  Largemouth     OTC Immersion     50            50            100
  Bass                                                         
  fingerling                                                   

                 Control           50            0             0
  -------------- ----------------- ------------- ------------- ------------

5.  Conclusion:

> This study was also conducted in 1995 and 1996 with similar numbers of
> fish and with similar results. Based on these studies, the recommended
> dose of oxytetracycline to achieve a fluorescent mark on the otolith
> of walleye fry and fingerlings, sauger fry and fingerlings, and
> largemouth bass fingerlings is through six-hour immersion in 500 mg/L
> oxytetracycline hydrochloride.

D.  Field Studies Conducted Under INAD 8096

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1.  Type of Study: Clinical Field Trial

2.  Name and Address of Investigator:

> David O. Lucchesi\
> Division of Wildlife\
> South Dakota Department of Game, Fish, and Parks\
> Foss Building\
> Pierre, South Dakota 57501

3.  General Design of the Investigation:

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a.  Purpose of the Study: To determine the effectiveness of
    oxytetracycline hydrochloride in the marking of otoliths of walleye
    fry (larval) and fingerlings.

b.  Test Animals: Walleye (*Stizostedion vitreum*) fry and fingerlings.

  ---------------------------------- ------------------------------------
  **Species**                        **Number of Fish**

  Walleye fry                        12,000,000

  Walleye fingerlings                150,000
  ---------------------------------- ------------------------------------

c.  Treatment Groups: Natural rearing ponds were stocked with either 500
    mg/L or 700 mg/L marked fry, or 500 mg/L marked fingerlings. Equal
    numbers of marked and unmarked fry were stocked into 0.8 ha hatchery
    ponds. Two ponds were stocked with 500 mg/L marked fry and two ponds
    were stocked with 700 mg/L marked fry.

d.  Dosage Form: Water-soluble oxytetracycline hydrochloride.

e.  Route of Administration: Immersion (bath).

f.  Dosages Used: Walleye fry were marked at either 500 mg/L or 700
    mg/L. The fingerlings were marked at 500 mg/L. Immersion was for 6
    hours.

g.  Test Duration: 30 days to 3 months (from treatment to sampling).

h.  Parameters: examination of otoliths to identify marks and mortality
    associated with OTC immersion marking

###### Methods:

> To produce the marking bath, an OTC slurry was mixed in a 20 L plastic
> bucket and was then buffered to a neutral pH using sodium phosphate
> (dibasic, Na~2~HPO~4~). Walleye fry were immersed for 6 hours in 683-L
> fiberglass raceways containing either 500 or 700 mg/L OTC. Density of
> fry did not exceed 2,000 fry per liter. Pond-reared fingerlings were
> marked by immersion for 6 hours in a 500 mg/L OTC solution in 632-L
> fiberglass transfer tanks. Fingerling densities did not exceed 50 fish
> per liter.
>
> To evaluate marking efficacy and mark retention, OTC-marked fry and
> fingerlings were stocked into natural rearing ponds that had complete
> fish kills the previous winter. These test ponds contained marked
> individuals from one of three test groups: fry immersed in 500 mg/L or
> 700 mg/L OTC, or fingerlings immersed in 500 mg/L. Test ponds were
> electrofished in early fall (approximately 3 months after marking) to
> recover walleyes for evaluation of the mark quality.
>
> To evaluate mortality associated with OTC immersion marking, the 0.8
> ha ponds were seined approximately 30-40 days after stocking. A sample
> of 100 fingerlings per pond was examined for OTC otolith marks.

4.  Results:

> Visibility of walleye otolith marks 3 months after marking as fry.

  ------------------------- ------------- ----------- ----------- -----------
  **OTC Concentration       **Mark                                
  (mg/L)**                  Intensity**                           

                            **0**         **1**       **2**       **3**

  500 (1996)                50%           40%         10%         0

  700 (1996)                0             0           18%         82%

  700 (1997)                0             0           2%          98%
  ------------------------- ------------- ----------- ----------- -----------

> These findings suggest that better quality marks are obtained when
> fish are immersed in a bath containing 700 mg/L OTC. Problems with OTC
> precipitating out of solution typically occurred at a pH of higher
> than 8.0. Therefore, the pH of the bath was maintained at 7.0 to 7.6,
> even when hatchery water pH exceeded that range.

5.  Conclusion:

> Based on this study, the recommended dose of oxytetracycline to
> achieve a fluorescent mark on the otolith of walleye fry is through
> six-hour immersion at 700 mg/L OTC.
>
> [Supporting Articles]{.smallcaps}
>
> The following published articles were submitted to support the
> effectiveness of oxytetracycline for the marking of otoliths in bony
> fish:

1.  Brooks, Ronald C., Roy C. Heidinger, and Christopher C. Kohler.
    Mass-Marking Otoliths of Larval and Juvenile Walleyes by Immersion
    in Oxytetracycline, Calcein, or Calcein Blue. North American Journal
    of Fisheries Management 14: 43-150, 1994.

2.  Hendricks, M.L., Bender, T.R., and Mudrak, V.A. Multiple Marking of
    American Shad Otoliths with Tetracycline Antibiotics. North American
    Journal of Fisheries Management, 11:212-219, 1991.

3.  Secor, D.H., Miller, White, M.G., and Dean, J.M. Immersion Marking
    of Larval and Juvenile Hatchery-Produced Striped Bass with
    Oxytetracycline. Transactions of the American Fisheries Society,
    120:261-266, 1991.

4.  Thomas, Leston M. Chemical Mark Application in Red Drum (*Sciaenops
    ocellatus*). Thesis for Master of Science at Corpus Christi State
    University, Corpus Christi, Texas. 44 pages, 1993.

5.  Younk, J.A., Cook, M.F. Fluorescent Chemical Marking of Walleye
    Larvae with a Selected Literature Review of Similar Investigations.
    Minnesota Department of Natural Resources Investigational
    Report 408. 18 pages, 1991.

**V. TARGET ANIMAL SAFETY**

A.  "Toxicity of Oxytetracycline and Calcein to Juvenile Striped Bass."

> (DRAFT manuscript submitted to INAD 8054 and Transactions of the
> American Fisheries Society)

1.  Type of Study: Target animal safety

2.  Name and Address of Investigators:

> Bumguardner, BW and King, TL\
> Texas Parks and Wildlife Department\
> HC 2 Box 385\
> Palacios, Texas 77465

3.  General Design of the Investigations:

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a.  Purpose of the Study: The study was designed to identify adverse
    effects of exposing fish to a geometric sequence of oxytetracycline
    by immersion.

b.  Test Animals: juvenile striped bass (*Morone saxatilis*),
    approximately 48 mm total length and 2.2 g

c.  Treatment Groups: 3 replicates per concentration tested. 10 fish per
    replicate. The first set of tests included 6 different OTC
    concentrations. The second set of tests included 3 higher
    concentrations.

d.  Dosage Form: Water-soluble oxytetracycline hydrochloride

e.  Route of Administration: Immersion (bath).

f.  Dosages Used: concentrations of 0, 55.8, 111.6, 223.3, 446.5, and
    893; second set of tests: 893, 1786, and 3572 mg/L. 6 hour exposure.

g.  Test Duration: 26 days (from set up to final observations).

h.  Parameters: survival and behavior at 1 hour intervals during the 6
    hour treatment and for 6 hours after treatment. Then, mortality
    daily for next 4 days. Water conditions were also monitored for
    temperature, salinity, pH, and total hardness.

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4.  Methods:

> After a 22-day acclimation period in a recirculating raceway, the fish
> were transferred to the test chambers. The test chambers were 4.5-L
> aquaria containing salt water (enough to make the total volume after
> addition of OTC solution 2 liters). OTC HCl unbuffered solution was
> added to provide concentrations of 0, 55.8, 111.6, 223.3, 446.5, and
> 893 mg/L. Since no deaths were seen, the 893 mg/L concentration was
> repeated and 1786 and 3572 mg/L concentrations were added. Fish were
> immersed for 6 hours. Then fish were placed in beaker while aquaria
> were rinsed. Fish then were placed back into aquaria. Water was
> exchanged every 24 hrs for 4 days.

### Results:

a.  Water conditions:

+-------------------+-----------------------+--------------------------+
| Condition         | Pre Treatment         | During Treatment         |
+-------------------+-----------------------+--------------------------+
| Temperature       | 26 C                  | ### 26 C                 |
+-------------------+-----------------------+--------------------------+
| Salinity          | 5 [+]{.underline} 1.9 | 7 o/oo                   |
|                   | o/oo                  |                          |
+-------------------+-----------------------+--------------------------+
| pH                | 8.3                   | 3.25-8.56\*              |
+-------------------+-----------------------+--------------------------+
| Total hardness    |                       | 33.4 mg/L (as CaCO3)     |
+-------------------+-----------------------+--------------------------+

> \*pH varied depending on OTC concentration
>
> A white precipitate was observed in the higher OTC concentration test
> chambers.

b.  Mean Mortality (%):

+--------------+-----------------+-----------------+-----------------+
| C            | ### D           | After Treatment | Total (and      |
| oncentration | uring Treatment |                 | range)          |
+--------------+-----------------+-----------------+-----------------+
| 0 (control)  | 0               | 6.7             | 6.7 (0-10)      |
+--------------+-----------------+-----------------+-----------------+
| 55.8 mg/L    | 0               | 0               | 0               |
+--------------+-----------------+-----------------+-----------------+
| 111.6 mg/L   | 0               | 0               | 0               |
+--------------+-----------------+-----------------+-----------------+
| 223.3 mg/L   | 0               | 0               | 0               |
+--------------+-----------------+-----------------+-----------------+
| 446.5 mg/L   | 0               | 30              | 30 (10-60)      |
+--------------+-----------------+-----------------+-----------------+
| 893 mg/L     | 0               | 81.7            | 81.7 (60-100)   |
+--------------+-----------------+-----------------+-----------------+
| 1786 mg/L    | 23.3            | 73.3            | 96.7 (90-100)   |
+--------------+-----------------+-----------------+-----------------+
| 3572 mg/L    | 100             | \-              | 100             |
+--------------+-----------------+-----------------+-----------------+

> Stressed behavior (rapid swimming at the water's surface) was reported
> in concentrations of OTC 446.5 mg/L and higher.

6.  Conclusion:

> The pH of the immersion solution may have caused or contributed to the
> death of the fish in the highest dose groups. The mean pH of test
> aquaria water after addition of OTC solution began to decrease
> significantly at 893 mg OTC/L. At 1786 mg/L, the pH was more than 1.5
> units lower than that of the control group; at 3572 mg/L, the pH was
> more than 5 units lower than that of the control group.
>
> Water quality, especially pH, must be monitored and controlled when
> using higher concentrations of OTC for immersion of fish. Buffering of
> the water can be done to maintain a healthy pH when OTC is used.

B.  Field Study Conducted Under INAD 8054

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1.  Type of Study: Clinical Field Trial

2.  Name and Address of Investigator:

> Wallace Jenkins\
> South Carolina Department of Natural Resources\
> P.O. Box 12559\
> Charleston, SC 29422-2559

3.  General Design of the Investigation:

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a.  Purpose of the Study: This study was primarily designed to
    demonstrate effectiveness, but during the fall of 1996 and spring
    1997 fish were stocked in hatchery ponds following treatment to
    evaluate post-treatment mortality.

b.  Test Animals: Red drum *(Scianenops ocellatus)* fingerlings. The
    mean weights and lengths are shown in the table in the results
    section

c.  Treatment Groups: Ponds were stocked with fingerlings following
    marking. The number of fingerlings in each group is shown in the
    table in the result section.

d.  Dosage Form: Water soluble oxytetracycline hydrochloride

e.  Route of Administration: immersion (bath)

f.  Dosages Used: 500 mg oxytetracycline/L for 4 hours

g.  Test Duration: 7 days (from treatment to release)

h.  Parameters: mortality occurring one week following treatment

######## Methods:

> Red drum fingerlings were place in a holding tank on trailer and
> acclimated to approximately 15 ppt salinity. Adequate OTC was added to
> provide a 500 mg/L concentration. Fish were held for 4 hours and then
> transferred to a hauling trailer. Fish were stocked in a pond on the
> culture facility and held for one week. During that week pond salinity
> was slowly raised to the same concentration as the area to be stocked
> (29-30 ppt). Then fish were harvested and transported to stocking
> sites for release.

4.  Results:

> Mean size and survival of red drum fingerlings treated from 9/96-5/97
> with OTC at a concentration of 500 mg/L for 4 hours.

  ------------- ------------ ----------- ------------ ------------ -------------
  **Treatment   **Mean       **Mean      **Number of  **Survival   **Number
  Date**        Length       Weight      Fish         (%)**        Harvested**
                (mm)**       (g)**       Treated**                 

  9/16          39           0.59        37,047       72.2         26,746

  10/15         37           0.67        63,898       43.1         28,061\*

  10/16         25           0.21        381,028      66.6         253,637

  10/22         37           0.68        43,332       71.1         30,801

  11/7          13           0.10        265,593      86.4         229,712\*\*

  5/15          33           0.36        65,810       82.8         54,474

  5/22          28           0.25        114,423      80.7         92,395

  5/27          29           0.24        470,611      86.8         408,653

  5/29          34           0.31        143,589      93.9         134,900
  ------------- ------------ ----------- ------------ ------------ -------------

\*This pond had a dense growth of macrophytic algae. Fish got stranded
in the algal mat during harvest. \*\*A salinity acclimation error during
post-treatment resulted in 20% mortality during harvest and release,
therefore only 183,770 fish were released alive.

5.  Conclusion:

> Based on these trials, the recommended dose of oxytetracycline to
> achieve a fluorescent mark on the otolith through four-hour immersion
> in 500 mg/L oxytetracycline is safe to red drum fingerlings.

C.  Field Studies Conducted Under INAD 8427

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1.  Type of Study: Clinical Field Trial

2.  Name and Address of Investigator:

> Kirby D. Cottrell\
> Hatcheries Section Head\
> Illinois Department of Conservation\
> Lincoln Tower Plaza\
> 524 South Second Street\
> Springfield, Illinois 62701-1787

3.  General Design of the Investigations:

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a.  Purpose of the Study: The studies were primarily designed to
    demonstrate effectiveness, but they involved the treatment of many
    species and large numbers of fish. The studies demonstrate several
    issues related to target animal safety.

b.  Test Animals: Largemouth bass (*Micropterus salmoides*) fingerlings,
    Walleye (*Stizostedion vitreum*) fry and fingerlings, and Sauger
    (*Stizostedion canadense*) fry and fingerlings.

c.  Treatment Groups: The fish were assigned to either treatment (500
    mg/L for\
    6 hours) or smaller control groups according to the following table.

  -------------------------- ---------------------- ---------------------
  SPECIES                    No. in OTC Group       No. in Control Group

  Largemouth Bass            25,000                 300
  fingerlings                                       

  Walleye fry                5,837,400              100,000

  Walleye fingerlings        143,306                1,000

  Sauger fry                 2,400,000              100,000

  Sauger fingerlings         166,934                1,000
  -------------------------- ---------------------- ---------------------

d.  Dosage Form: Water-soluble oxytetracycline hydrochloride

e.  Route of Administration: Immersion (bath).

f.  Dosages Used: 0 and 500 mg oxytetracycline/L of water for 6 hours

g.  Test Duration: 1 day (fish stocked the day of treatment)

h.  Parameters: mortality and adverse reactions

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4.  Methods:

> Marking of fry - Groups of 100,000 fry were placed in a 500 mg/L
> concentration of oxytetracycline in 3 gallons of water within a
> plastic fish hauling bag for six hours.
>
> Marking of fingerlings - Fish were immersed in a 500 mg/L
> concentration of oxytetracycline for six hours.

5.  Results:

> Mortality and adverse reactions observed in 1997 at Jake Wolf, Little
> Grassy and LaSalle Hatcheries in Illinois.

  ----------------------- ---------------- ----------------- ----------------
  **Species**             **Number         **Mortalities**   **Adverse
                          Treated**                          Reactions**

  Largemouth bass         25,000           0                 None
  fingerlings                                                

  Walleye fry             5,824,080        0                 None

  Walleye fingerlings     138,626          0                 None

  Sauger fry              22,000,000       0                 None

  Sauger fingerlings      166,934          0                 None
  ----------------------- ---------------- ----------------- ----------------

> No adverse reactions or mortality occurred in control groups in any of
> the species treated.

6.  Conclusion:

> This study was repeated with similar numbers of fish and with similar
> results in 1996. Based on this study, the recommended dose of
> oxytetracycline to achieve a fluorescent mark on the otolith through
> six-hour immersion at 500 mg/L is safe to fry and fingerlings.

D.  Field Study Conducted Under INAD 6990

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1.  Type of Study: Clinical Field Trial

2.  Name and Address of Investigator:

> R. T. Colesante\
> Senior Aquatic Biologist\
> New York State Department of Environmental Conservation\
> Oneida Fish Culture Station\
> P.O. Box 303 Hatchery Road\
> Constantia, NY 13044

3.  General Design of the Investigation:

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a.  Purpose of the Study: Evaluate the survival of walleye fry after
    oxytetracycline (OTC) marking and stocking in earthen ponds.

b.  Test Animals: Walleye (*Stizostedion vitreum*) fry

c.  Treatment Groups. Treated and untreated groups of 20,000 fry stocked
    in 12 ponds.

d.  Dosage Form: Water-soluble oxytetracycline hydrochloride

e.  Route of Administration: Immersion (bath)

f.  Dosages Used: 0 and 500 mg oxytetracycline/L of water for 6 hours

g.  Test Duration: 45-55 days (treatment to harvest)

h.  Parameter: Survival

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4.  Methods:

> Fry were immersed in a 500 mg/L oxytetracycline static bath for 6
> hours. Six ponds were stocked with marked fry and an additional six
> ponds were stocked with unmarked fry. Ponds were harvested after 45-55
> days.

5.  Results:

> Analysis of mean survival using a "t" test indicated no significant
> difference between treated and untreated fry. Survival results from
> each pond are shown in the following table.

  --------------- ---------------- ------------------- -------------------
  **Pond No.**    **Fry Stocked**  **Fingerling        
                                   Return**            

                                   **Unmarked**        **Marked**

  1               20,000           \-                  22,728

  2               20,000           \-                  15,557

  3               20,000           \-                  11,347

  4               20,000           5,360               \-

  5               20,000           12,964              \-

  6               20,000           \-                  14,118

  7               20,000           18,184              \-

  8               20,000           \-                  13,432

  9               20,000           12,367              \-

  10              20,000           12,727              \-

  11              20,000           13,457              \-

  12              20,000           \-                  11,697

  **Mean                           **62.5 %**          **74.1 %**
  Survival**                                           
  --------------- ---------------- ------------------- -------------------

6.  Conclusion:

> This study was conducted in 1993 with similar results. Based on this
> study, the recommended dose of oxytetracycline to achieve a
> fluorescent mark on the otolith through six-hour immersion at 500 mg/L
> is safe to walleye fry.

VI. **HUMAN FOOD SAFETY**

The human food safety of the use of oxytetracycline to mark larval, fry,
or fingerling finfish by immersion has been met by data in a public
master file (PMF), data in the public literature, and by the long
inherent withdrawal period which occurs between treatment of the fish
and possible consumption by humans. A tolerance and regulatory method
for oxytetracycline have been established previously. An additional
withdrawal time beyond the grow-out period is not needed for larval,
fry, or fingerling finfish marked with oxytetracycline by immersion.

A. Tolerance

A tolerance of 2 ppm as the sum of tetracycline residues has been
previously codified for the edible tissue of salmonids and catfish (21
CFR 556.500). This tolerance is now being extended to all finfish.

B. Residue Depletion Data in a Public Master File

Residue data from fingerling fish marked with oxytetracycline via
medicated feed are described in PMF 3265. The data in this PMF were used
to support the approval for marking the skeletal tissue of Pacific
salmon (not over 30 grams body weight) with 250 mg OTC/kg of fish/day in
medicated feed for 4 consecutive days (21 CFR 558.450). The data
demonstrate that in salmon fingerlings fed 1.0 g oxytetracycline/kg body
weight/day for 4 days, the oxytetracycline residues after 7 days
withdrawal ranged from \<0.15 to 0.95 ppm in muscle and from \<0.25 to
3.0 ppm in skin. After 22 days withdrawal, the oxytetracycline residues
ranged from \<0.1 to 0.2 ppm in muscle and from 0.28 to 1.15 ppm in
skin. It was concluded that this use of oxytetracycline for marking is
safe to humans because the treated fish have a prolonged grow-out period
of 6 months to several years during which the drug residues have time to
deplete to the tolerance (which was 0.1 ppm at that time and has since
been revised, 61 FR 67453).

C. Residue Depletion Data in the Public Literature

1\. Unkenholz, E. et al. 1997. Oxytetracycline marking efficacy for
yellow perch fingerlings and temporal assays of tissue residues. Prog.
Fish-Culturist. 59:280-284.

> Yellow perch fingerlings (90 to 120 days old) were held in 1,250 L
> fiberglass raceways at 12°C. Solutions of OTC HCl were added to each
> raceway to give calculated final concentrations of 300, 500, or 700 mg
> OTC/L. The exposure time was 12 hours. The treated fish were sampled
> at 3, 6, 9, and 12 hours after the test period started to determine
> uptake. Fish were held in unmedicated water during the withdrawal
> period. The fish were sampled at 7, 14, 42, and 110 days withdrawal.
> OTC levels were analyzed by HPLC in composite samples (containing
> muscle, skin, scales, and bones) from all timepoints and in muscle
> samples from 110 days withdrawal. The following results were reported:

  ----------------- ----------------- ----------------- -----------------
  OTC Concentration Withdrawal Time   OTC Residues      OTC Residues
  (mg OTC/L)        (Days)            (ppm) in          (ppm) in Muscle
                                      Composite Samples 

  309               7                 0.59              \-\--

                    14                0.66              \-\--

                    42                0.42              \-\--

                    110               0.57              \<0.05

  534               7                 0.82              

                    14                1.06              

                    42                0.41              

                    110               0.56              \<0.05

  748               7                 0.63              

                    14                0.89              

                    42                0.43              

                    110               0.44              \<0.05
  ----------------- ----------------- ----------------- -----------------

# 2. Brown, M. L. 2002. In-Transit Oxytetracycline Marking, Non-Lethal Mark Detection, and Tissue Residue Depletion in Yellow Perch. North American Journal of Fisheries Management. 22:236-242.

> Adult yellow perch (73.9 grams [+]{.underline} 2.0) were held in a
> 1,250 L fiberglass raceway with a water temperature of 19°C. The fish
> were immersed in 600 mg OTC/L for 6 hours. After treatment, the
> medicated waters were flushed away. The fish were held in unmedicated
> water throughout the withdrawal period. Groups of 3 fish were sampled
> from 1 to 1,080 hours withdrawal and sacrificed for tissue analyses.
> The samples were stored at -20°C prior to analysis. The skin was
> removed from the fillet. The muscle tissue was pooled (n=3) and
> homogenized to form a composite sample for each time period. The
> samples were analyzed with an HPLC method (method not provided). The
> HPLC analyses were performed by the Pharmacy/Biochemistry Department
> at South Dakota State University. The results were as follows:

  ----------------------------------- -----------------------------------
  OTC Residues in Pooled Muscle       
  Samples (n=3) of Adult Yellow Perch 
  Immersed in 600 mg OTC/L for 6      
  Hours.                              

  Withdrawal Time in Hours            OTC Residues (ppm)

  1                                   2.56

  14                                  0.217

  24                                  0.246

  48                                  0.321

  72                                  0.131

  144                                 0.320

  216                                 0.171

  288                                 0.226

  360                                 0.120

  504                                 0.186

  792                                 0.048

  1080                                0.017
  ----------------------------------- -----------------------------------

D. Inherent Withdrawal Period

Fish will be marked by immersion in oxytetracycline at the early life
stages of larvae, fry, or fingerling. This early life stage treatment
allows for an inherent withdrawal time of six months to several years
between treatment and growth to market/legal size. Based on the residue
data described above in sections B and C, the inherent withdrawal time
will provide an adequate amount of time for oxytetracycline residues to
deplete to below the tolerance of 2 ppm.

E. Regulatory Method

The analytical method for the detection of residues of oxytetracycline
is a microbiological assay using *Bacillus cereus* var. *mycoides*. This
method may be found in "Antibiotic Residues in Milk, Dairy Products, and
Animal Tissues: Methods, Reports, and Protocols" (revised October 1968,
reprinted December 1974. National Center for Antibiotic and Insulin
Analysis, FDA, Washington, DC 20204). The method is on file at the
Center for Veterinary Medicine, 7500 Standish Pl., Rockville, MD 20855.

**VII. AGENCY CONCLUSIONS**

> The data submitted in this public master file (PMF) satisfy in part
> the requirements of Section 512 of the Food, Drug, and Cosmetic Act
> with regard to the proposed use of water-soluble oxytetracycline
> hydrochloride to mark skeletal tissues of fry and fingerling finfish.
> Fish are a minor species of animals defined under 21 CFR 514.1(d)(ii).
> The data submitted meet the requirements of that regulation, and FDA's
> "Guidance for Industry: FDA Approval of New Animal Drugs for Minor
> Uses and for Minor Species" (January 1999). FDA will consider this
> information along with other required data as support for new animal
> drug applications (NADAs) that may be filed for this use of
> water-soluble oxytetracycline hydrochloride in finfish.
>
> FDA is publishing a notice of availability of this PMF to encourage
> sponsors to file NADAs for water-soluble oxytetracycline for the use
> covered by this PMF. Sponsors of NADAs or supplemental NADAs may,
> without further authorization, reference the PMF to support approval
> of an application filed under 21 CFR 514.1(d), i.e., minor use animal
> drugs. An NADA or supplemental NADA must include, in addition to
> reference to the PMF, animal drug labeling and other information
> needed for approval, such as data concerning manufacturing methods,
> facilities, and controls.
>
> Oxytetracycline is already approved as an over the counter drug for
> use in feed under CFR 558.450 in chickens, turkeys, beef cattle,
> salmonids, catfish, and lobsters.