Approval Date: October 4, 1996
Freedom of Information Summary
NADA 140-828
I. GENERAL INFORMATION:
NADA 140-828 Sponsor: BAYER Corporation
Agriculture Division
Animal Health
P. O. Box 390
Shawnee Mission, Kansas 66201Generic Name: enrofloxacin Trade Name: Baytril® (enrofloxacin) 3.23% Concentrate Antimicrobial Solution Marketing Status: II. INDICATIONS FOR USE
Chickens - Baytril® (enrofloxacin) 3.23% Concentrate Antimicrobial Solution is indicated for the control of mortality associated with E. coli susceptible to enrofloxacin. Turkeys - Baytril® (enrofloxacin) 3.23% Concentrate Antimicrobial Solution is indicated for the control of mortality associated with E. coli and P. multocida (fowl cholera) susceptible to enrofloxacin.III. DOSAGE
A. DOSAGE FORM Baytril® (enrofloxacin) 3.23% Concentrate Antimicrobial Solution is available in 946 mL (one quart) and 3.8 L (one gallon) plastic containers. Each mL contains 32.3 mg enrofloxacin. B. ROUTE OF ADMINISTRATION Baytril® (enrofloxacin) 3.23% Concentrate Antimicrobial Solution should be added to clean drinking water and provided ad libitum. C. RECOMMENDED DOSAGES: Chickens and turkeys should be provided clean drinking water containing 25 to 50 ppm enrofloxacin. The medicated water should be administered continuously as the only source of drinking water for 3 to 7 days.
IV. EFFECTIVENESS
Data in support of this application were obtained from dose titration and clinical field studies. All were well controlled and conducted in compliance with the protocols covering these studies. The low end dose of enrofloxacin for controlling mortality due to Escherichia coli in chickens was established in a severe artificial infection dose titration study conducted under simulated use conditions. The efficacy of the 25 ppm dose given for three days duration was confirmed in clinical field studies conducted at three different geographic locations in chickens naturally infected with E. coli.
For turkeys, the low end dose of 25 ppm was established against fowl cholera in a severe infection model dose titration study with birds artificially infected with Pasteurella multocida. The efficacy of the 25 ppm dose was confirmed in clinical field studies where a three day treatment regimen was evaluated at three different geographic locations.
To establish the efficacy of enrofloxacin for the control of mortality associated with Escherichia coli in turkeys, a dose of 25 ppm given continuously for three days was evaluated in clinical field studies at three different geographic locations. This treatment regimen was selected based on the efficacy established against Pasteurella multocida (fowl cholera) and the similar susceptibility of this pathogen to E. coli.
1. Dose Range-Finding Study - Chickens
a. Type of Study: A dose titration study was conducted using enrofloxacin in broiler chickens artificially infected with Escherichia coli.
b. Investigator(s): Jeffrey N. Davidson, D.V.M., M.P.V.M.
Health Management Services
P.O. Box 2046
Tulare, California 93275c. General Design:
1) Purpose: The study was conducted to determine the efficacy of four graded doses of enrofloxacin and to select a low end dose for controlling mortality in chickens artificially infected with Escherichia coli.
2) Animals: Commercial Peterson x Arbor Acre chickens, 21 days of age, 360 males, 360 females (4 pens per treatment, 15 males and 15 females per pen) were used.
3) Control: Non-medicated water was available ad libitum from waterers for the duration of the study.
4) Challenge: The E. coli challenge was by intramuscular injection with an inoculum of the pathogenic organism.
5) Diagnosis: Colibacillosis was diagnosed by necropsy. A positive case was a dead bird, dying any time following the initiation of treatment, having a positive culture for E. coli and a lesion score >= 2 using the following scale: 1 = no lesions, 2 = minimal air sac lesions, 3 = moderate air sac and pericardial lesions, 4 = extensive air sac and pericardial lesions.
6) Dosage Form: The dosage form used was a concentrated aqueous solution with each mL containing 32.3 mg enrofloxacin. This formulation is the same as the marketed product.
7) Route of Administration: Orally, in the drinking water ad libitum
8) Dose: The doses tested in the study were 6.25, 12.5, 25 and 50 ppm, administered continuously in the drinking water for five consecutive days. Birds (except those in control groups) were started on the medicated water one hour after injection of the pathogenic organism.
9) Test Duration: The study continued for 10 days following challenge.
10) Pertinent Variables Measured: The primary parameter for determining efficacy was based upon a comparison of mortality due to E. coli between the unmedicated control group and each of the medicated groups.
d. Results: Results are shown in Table 4.1 below.
Table 4.1. Mean percent E. coli mortality from the initiation of medication to study end Group* Mortality: Mortality: Mortality: Males Females Combined UUC 0 (0/59) 3.3 (2/60) 1.7 (2/119) IUC 51.7 (31/60) 46.7 (28/60) 49.2 (59/120) 6.25 ppm 15.0 (9/60) 20.0 (12/60) 17.5 (21/120) 12.5 ppm 5.0 (3/60) 10.0 (6/60) 7.5 (9/120) 25 ppm 5.0 (3/60) 1.7 (1/60) 3.3 (4/120) 50 ppm 3.3 (2/60) 1.7 (1/60) 2.5 (3/120) *UUC = uninfected, unmedicated control; IUC = infected, unmedicated controle. Statistical Analysis: An analysis of variance showed that the five infected groups had significantly different mortality rates.A model fitting procedure using Anderson and Nelson's Linear-Plateau models was done on the mortality rates to help determine the recommended dose level. Although several models showed large R^2s and had good model fits, the Linear-Plateau model VI (1,3) was the best model. R^2 was equal to 0.992; the model p-value was highly significant, p = 3.25E-5; and the beta coefficients were highly significant (7.93E-8, 8.47E-4 and 3.33E-3). The residuals p-value was not significant, p = 0.804. The model is two linearly decreasing lines with significantly different slopes followed by a plateau (line parallel to the x-axis). The plateau occurs for dose levels of 25 and 50 ppm. Thus, the model indicates that the doses of 25 and 50 ppm give the best results but that there is not a significant difference between a dose of 25 ppm and a dose of 50 ppm.
f. Conclusions: Enrofloxacin was effective in controlling mortality associated with an artificially-induced E. coli infection. Based on the data from this study, 25 ppm was chosen as the lower end of the dose range.
g. Adverse Reactions: No adverse reactions were observed.
2. Dose Range-Finding Study - Turkeys
a. Type of Study: A dose titration study was conducted using enrofloxacin in growing turkeys artificially infected with Pasteurella multocida.
b. Investigator(s): Jeffrey N. Davidson, D.V.M., M.P.V.M.
Health Management Services
P.O. Box 2046
Tulare, California 93275c. General Design:
1) Purpose: The study was conducted to determine the efficacy of graded doses of enrofloxacin and to select an effective low end dose for controlling mortality in turkeys artificially infected with Pasteurella multocida.
2) Animals: Broad Breasted White turkeys, 12 weeks of age, 120 males, 120 females (4 pens per treatment, with 6 males and 6 females per pen) were used.
3) Control: Non-medicated water was available ad libitum from waterers for the duration of the study.
4) Challenge: The P. multocida challenge was initiated by swabbing the palatine-cleft with an inoculum of the pathogenic organism.
5) Diagnosis: Fowl cholera was diagnosed at necropsy by a positive culture for P. multocida.
6) Dosage Form: The dosage form used was a concentrated aqueous solution with each mL containing 32.3 mg enrofloxacin. This formulation is the same as the marketed product.
7) Route of Administration: Orally, in the drinking water ad libitum
8) Dose: The doses tested in the study were 12.5, 25, 50 and 100 ppm, administered continuously in the drinking water for five consecutive days. Birds (except unmedicated controls) were started on the medicated water one hour post-challenge.
9) Test Duration: The study continued for 8 days following challenge.
10) Pertinent Variables Measured: The primary parameter for determining efficacy was based upon a comparison of mortality due to P. multocida between the unmedicated control group and each of the medicated groups.
d. Results: Mortality results are shown in Table 4.2 below.
Table 4.2. Mean percent P. multocida mortality from initiation of medication to study end Dose Mortality: Mortality: Mortality: Enrofloxacin Males Females Combined 0 ppm 100 (23/23*) 95.8 (23/24) 97.8 (46/47) 12.5 ppm 8.3 (2/24) 8.3 (2/24) 8.3 (4/48) 25 ppm 0 (0/24) 0 (0/24) 0 (0/48) 50 ppm 0 (0/24) 0 (0/24) 0 (0/48) 100 ppm 0 (0/24) 0 (0/24) 0 (0/48) *One bird died which did not culture positive for Pasteurella multocida.e. Statistical Analysis: Statistical comparisons, with respect to mortality rates, were made with Fisher's exact test and supported by a Kruskal-Wallis test. A preliminary analysis indicated that the mortality rates of replicates within each group did not differ significantly and therefore all data were combined for group comparisons. All four active treatment groups had significantly lower (p<0.01) mortality rates than the control group. The mortality rate of the 12.5-ppm group (8.3%) was marginally significantly greater than the other three active treatment groups (p=0.06). f. Conclusions: Since mortality was reduced to zero at all dose levels except 12.5 ppm, it was concluded the 25 ppm should represent the low end of the dose range.g. Adverse Reactions: No adverse reactions were observed.
3. Field Study - Chickens: E. coli
a. Type of Study: A field study was conducted using enrofloxacin in broiler chickens with spontaneously occurring colibacillosis.
b. Investigator(s): Terry TerHune, D.V.M., Ph.D.
Health Management Services
P.O. Box 2046
Tulare, California 93275c. General Design:
1) Purpose: The study was conducted to confirm the efficacy of enrofloxacin for controlling mortality in chickens naturally infected with Escherichia coli.
2) Animals: Commercial Peterson x Arbor Acre chickens, 1-day-old, 500 males, 500 females (10 pens per treatment with 25 males and 25 females per pen) were used.
3) Control: Non-medicated water was available ad libitum from waterers for the duration of the study.
4) Challenge: The E. coli challenge was by natural infection. This was accomplished by applying stress to the birds at 18 days of age.
5) Diagnosis: The primary parameter for determining efficacy was based upon a comparison of mortality due to E. coli between the unmedicated control group and the medicated group. A positive case was a dead bird, dying any time following the initiation of treatment, having a positive culture for E. coli and a lesion score of > = 2 using the following scale: 0 = no lesions, 1 = minimal air sacculitis (slight cloudiness), 2 = moderate air sacculitis (non-coalesced fibrinous material) and/or mild pericarditis and/or perihepatitis, 3 = severe air sacculitis (coalesced fibrinous material) and/or moderate to severe pericarditis and/or perihepatitis.
6) Dosage Form: The dosage form used was a concentrated aqueous solution with each mL containing 32.3 mg enrofloxacin. This formulation is the same as the marketed product.
7) Route of Administration: Orally, in the drinking water ad libitum
8) Dose: The dose tested in the study was 25 ppm, administered continuously in the drinking water for three consecutive days. Medication was initiated when house mortality exceeded a level of 0.5% in a 24-hour period. This occurred at 21 days of age.
9) Test Duration: The study continued for 10 days following the end of the three day medication period.
10) Pertinent Variables Measured: The primary parameter for determining efficacy was based upon a comparison of mortality due to E. coli between the unmedicated control group and the medicated group.
d. Results: The mortality results are shown in Table 4.3 below. Treatment effect averaged across sex was significant (p = 0.004).
Table 4.3. Mean percent E. coli mortality from the initiation of medication to study end (Days 21 to 33) Dose Mortality: Mortality: Mortality: Enrofloxacin Males Females Combined 0 ppm 11 (27/246) 12.1 (29/240) 11.5 (56/486) 25 ppm 2.5 (6/242) 4.1 (10/245) 3.3 (16/487)e. Statistical Analysis: Mortality was transformed using an arc sine transformation and analyzed using analysis of variance techniques. The analysis model included terms for block and treatment. A significance level of 0.05 was used to test results. f. Conclusions: It was concluded that under the natural field conditions of the study, enrofloxacin was effective in controlling mortality associated with E. coli susceptible to enrofloxacin when administered at the rate of 25 ppm in drinking water for three consecutive days.g. Adverse Reactions: No adverse reactions were observed.
4. Field Study - Chickens: E. coli
a. Type of Study: A field study was conducted using enrofloxacin in broiler chickens with spontaneously occurring colibacillosis.
b. Investigator(s): Carey L. Quarles, Ph.D.
Colorado Quality Research
1401 Duff Drive, Suite 700
Fort Collins, CO 80524c. General Design:
1) Purpose: The study was conducted to confirm the efficacy of enrofloxacin for controlling mortality in chickens naturally infected with Escherichia coli.
2) Animals: Commercial Peterson x Arbor Acre chickens, 1 day of age, 580 males, 580 females (10 pens per treatment with 29 males and 29 females per pen) were used.
3) Control: Non-medicated water was available ad libitum from waterers for the duration of the study.
4) Challenge: The E. coli challenge was by natural infection. This was accomplished by sourcing eggs from a breeder flock known to carry E. coli.
5) Diagnosis: The primary parameter for determining efficacy was based upon a comparison of mortality due to E. coli between the unmedicated control group and the medicated group. A positive case was a dead bird, dying any time following the initiation of treatment, having a positive culture for E. coli and a lesion score >= 2 using the following scale: 0 = no lesions, 1 = minimal air sacculitis (slight cloudiness), 2 = moderate air sacculitis (non-coalesced fibrinous material) and/or mild pericarditis and/or perihepatitis, 3 = severe air sacculitis (coalesced fibrinous material) and/or moderate to severe pericarditis and/or perihepatitis.
6) Dosage Form: The dosage form used was a concentrated aqueous solution with each mL containing 32.3 mg enrofloxacin. This formulation is the same as the marketed product.
7) Route of Administration: Orally, in the drinking water ad libitum
8) Dose: The dose tested in the study was 25 ppm, administered continuously in the drinking water for three consecutive days. Medication was started on Day 1 when mortality associated with E. coli had reached 0.5% in a 24-hour period.
9) Test Duration: The study continued for 10 days following the end of the 3-day medication period.
10) Pertinent Variables Measured: The primary parameter for determining efficacy was based upon a comparison of mortality due to E. coli between the unmedicated control group and the medicated group.
d. Results: The mortality results are shown in Table 4.4 below. Treatment effect averaged across sex was significant (p < 0.001).
Table 4.4. Mean percent E. coli mortality from the initiation of medication to study end (Days 1 to 13) Dose Mortality: Mortality: Mortality: Enrofloxacin Males Females Combined 0 ppm 8.7 (25/289) 9.4 (26/277) 9.0 (51/566) 25 ppm 0.7 (2/287) 0 (0/284) 0.4 (2/571)e. Statistical Analysis: Mortality was transformed using an arc sine transformation and analyzed using analysis of variance techniques. The analysis model included terms for block and treatment. A 0.05 level of significance was used to determine significant results. f. Conclusions: It was concluded that under the natural field conditions of the study, enrofloxacin was effective in controlling mortality associated with E. coli susceptible to enrofloxacin when administered at the rate of 25 ppm in drinking water for three consecutive days.g. Adverse Reactions: No adverse reactions were observed.
5. Field Study - Chickens: E. coli
a. Type of Study: A field study was conducted using enrofloxacin in broiler chickens with spontaneously occurring colibacillosis.
b. Investigator(s): John R. Glisson, D.V.M., Ph.D.
1111 Crestwood Court
Watkinsville, GA 30677c. General Design:
1) Purpose: The study was conducted to confirm the efficacy of enrofloxacin for controlling mortality in chickens naturally infected with E. coli.
2) Animals: Commercial Peterson x Arbor Acre chickens, one-day-old, 500 males, 500 females (10 pens per treatment with 25 males and 25 females per pen) were used.
3) Control: Non-medicated water was available ad libitum from waterers for the duration of the study.
4) Challenge: The E. coli challenge was by natural infection. This was accomplished by applying stress to the birds at 18 days of age.
5) Diagnosis: The primary parameter for determining efficacy was based upon a comparison of mortality due to E. coli between the unmedicated control group and the medicated group. A positive case was a dead bird, dying any time following the initiation of treatment, having a positive culture for E. coli and a lesion score> = 2 using the following scale: 0 = no lesions, 1 = minimal air sacculitis (slight cloudiness), 2 = moderate air sacculitis (non-coalesced fibrinous material) and/or mild pericarditis and/or perihepatitis, 3 = severe air sacculitis (coalesced fibrinous material) and/or moderate to severe pericarditis and/or perihepatitis.
6) Dosage Form: The dosage form used was a concentrated aqueous solution with each mL containing 32.3 mg enrofloxacin. This formulation is the same as the marketed product.
7) Route of Administration: Orally, in the drinking water ad libitum
8) Dose: The dose tested in the study was 25 ppm, administered continuously in the drinking water for three consecutive days. Medication was initiated when house mortality exceeded 0.5% in a 24 hour period. This occurred at 23 days of age.
9) Test Duration: The study continued for 10 days following the end of the three-day medication period.
10) Pertinent Variables Measured: The primary parameter for determining efficacy was based upon a comparison of mortality due to E. coli between the unmedicated control group and the medicated group.
d. Results: The mortality results are shown in Table 4.5 below. Treatment effect averaged across sex was significant (p < 0.001).
Table 4.5. Mean percent E. coli mortality from the initiation of medication to study end (Days 23 to 35) Dose Mortality: Mortality: Mortality: Enrofloxacin Males Females Combined 0 ppm 18.9 (43/228) 18.4 (45/244) 18.6 (88/472) 25 ppm 3.0 (7/237) 3.3 (8/242) 3.1 (15/479)e. Statistical Analysis: Mortality was transformed using an arc sine transformation and analyzed using analysis of variance techniques. The analysis model included terms for block and treatment. A 0.05 level of significance was used to determine significant results. f. Conclusions: It was concluded that under the natural field conditions of the study, enrofloxacin was effective in controlling mortality associated with E. coli susceptible to enrofloxacin when administered at the rate of 25 ppm in drinking water for three consecutive days.g. Adverse Reactions: No adverse reactions were observed.
6. Field Studies - Chickens: E. coli - Combined
a. Results: The results from the three chicken E. coli field studies were combined and statistically analyzed. The combined mortality data for these trials is summarized in Table 4.6.
Table 4.6. Mean percent E. coli mortality from the initiation of medication to study end for all 3 trials combined Dose Mortality: Mortality: Mortality: Enrofloxacin Males Females Combined 0 ppm 12.5 (95/763) 13.1 (100/761) 12.8 (195/1524) 25 ppm 2.0 (15/766) 2.3 (18/771) 2.1 (33/1537)b. Statistical Analysis: Mortality was transformed using an arc sine transformation and analyzed using analysis of variance techniques. The analysis model included terms for study, block within study, treatment, and study by treatment. The study by treatment interaction mean square was used to test for treatment significance. A 0.05 level of significance was used to determine significant results. A significant treatment effect was detected (P = 0.027) demonstrating that treatment efficacy was consistent across study locations. c. Conclusions: It was concluded that under the natural field conditions of these studies, enrofloxacin was effective in controlling mortality associated with E. coli susceptible to enrofloxacin when administered at the rate of 25 ppm in drinking water for three consecutive days.7. Field Study - Turkeys: E. coli
a. Type of Study: A field study was conducted using enrofloxacin in turkeys with spontaneously occurring colibacillosis.
b. Investigator(s): Terry TerHune, D.V.M., Ph.D.
Health Management Services
P.O. Box 2046
Tulare, California 93275c. General Design:
1) Purpose: The study was conducted to confirm the efficacy of enrofloxacin drinking water medication at a concentration of 25 ppm for 3 consecutive days for controlling mortality in turkeys naturally infected with E. coli.
2) Animals: Nicholas Turkeys, one-day-old, 800 males, 800 females (10 pens with 40 males and 10 pens with 40 females per treatment) were used.
3) Control: Non-medicated water was available ad libitum from waterers for the duration of the study.
4) Challenge: The E. coli challenge was by natural infection. This was accomplished by natural stress on the birds at 16 days of age.
5) Diagnosis: Colibacillosis was diagnosed by necropsy. A positive case was a dead bird, dying any time following the initiation of treatment, having a positive culture for E. coli and a lesion score of > = 2 using the following scale: 0 = no lesions, 1 = minimal air sacculitis (slight cloudiness), 2 = moderate air sacculitis (non-coalesced fibrinous material) and/or mild pericarditis and/or perihepatitis, 3 = severe air sacculitis (coalesced fibrinous material) and/or moderate to severe pericarditis and/or perihepatitis.
6) Dosage Form: The dosage form used was a concentrated aqueous solution with each mL containing 32.3 mg enrofloxacin. This formulation is the same as the marketed product.
7) Route of Administration: Orally, in the drinking water ad libitum
8) Dose: The dose tested in the study was 25 ppm, administered continuously in the drinking water for three consecutive days. Medication was initiated at 18 days of age (when house mortality exceeded a level of 0.5% in a 24-hour period).
9) Test Duration: The study continued for 10 days following the last day of medication.
10) Pertinent Variables Measured: The primary parameter for determining efficacy was based upon a comparison of mortality due to E. coli between the unmedicated control group and the medicated group.
d. Results: Mortality results are shown in Table 4.7.
Table 4.7. Mean percent E. coli mortality from the initiation of medication to ten days post medication (Days 19 to 31) Dose Mortality: Mortality: Mortality: Enrofloxacin Males Females Combined 0 ppm 5.0 (19/379) 5.1 (20/395) 5.0 (39/774) 25 ppm 0.3 (1/390) 0.3 (1/392) 0.3 (2/782)e. Statistical Analysis: Overall mortality was analyzed using mixed model methodology. Mortality was transformed using an arc sine transformation. Models included the random effects of blocks and their interactions. Fixed effects included treatments, sex, and their interactions. A significance level of 0.05 was used to test results. Treatment effect averaged across sex was significant (P = 0.0002). f. Conclusions: It was concluded that under the natural field conditions of the study, enrofloxacin was effective in controlling mortality associated with E. coli susceptible to enrofloxacin when administered at the rate of 25 ppm in drinking water for three consecutive days to turkeys.g. Adverse Reactions: No adverse reactions were observed.
8. Field Study - Turkeys: E. coli
a. Type of Study: A field study was conducted using enrofloxacin in turkeys with spontaneously occurring colibacillosis.
b. Investigator(s): Carey Quarles, Ph.D.
Colorado Quality Research
1401 Duff Drive, Suite 700
Fort Collins, CO 80524c. General Design:
1) Purpose: The study was conducted to confirm the efficacy of enrofloxacin drinking water medication at a concentration of 25 ppm for 3 consecutive days for controlling mortality in turkeys naturally infected with E. coli.
2) Animals: Nicholas Turkeys, 1- day-old, 800 males, 800 females (10 pens with 40 males and 10 pens with 40 females per treatment) were used.
3) Control: Non-medicated water was available ad libitum from waterers for the duration of the study.
4) Challenge: The E. coli challenge was by natural infection. This was accomplished by natural stress on the birds at 19 days of age.
5) Diagnosis: Colibacillosis was diagnosed by necropsy. A positive case was a dead bird, dying any time following the initiation of treatment, having a positive culture for E. coli and a lesion score of > = 2 using the following scale: 0 = no lesions, 1 = minimal air sacculitis (slight cloudiness), 2 = moderate air sacculitis (non-coalesced fibrinous material) and/or mild pericarditis and/or perihepatitis, 3 = severe air sacculitis (coalesced fibrinous material) and/or moderate to severe pericarditis and/or perihepatitis.
6) Dosage Form: The dosage form used was a concentrated aqueous solution with each mL containing 32.3 mg enrofloxacin. This formulation is the same as the marketed product.
7) Route of Administration: Orally, in the drinking water ad libitum
8) Dose: The dose tested in the study was 25 ppm, administered continuously in the drinking water for three consecutive days. Medication was initiated at 26 days of age (when house mortality exceeded a level of 0.5% in a 24-hour period).
9) Test Duration: The study continued for 10 days following the last day of medication.
10) Pertinent Variables Measured: The primary parameter for determining efficacy was based upon a comparison of mortality due to E. coli between the unmedicated control group and the medicated group.
d. Results: Mortality results are shown in Table 4.8.
Table 4.8. Mean percent E. coli mortality from the initiation of medication to ten days post medication (Days 26 to 38) Dose Mortality: Mortality: Mortality: Enrofloxacin Males Females Combined 0 ppm 4.6 (18/388) 5.2 (20/387) 4.9 (38/775) 25 ppm 0.3 (1/379) 0.5 (2/389) 0.4 (3/768)e. Statistical Analysis: Overall mortality was analyzed using mixed model methodology. Mortality was transformed using an arc sine transformation. Models included the random effects of blocks and their interactions. Fixed effects included treatments, sex, and their interactions. A 0.05 level of significance was used to determine significant results. Treatment effect averaged across sex was significant (P = 0.0001). f. Conclusions: It was concluded that under the natural field conditions of the study, enrofloxacin was effective in controlling mortality associated with E. coli susceptible to enrofloxacin when administered at the rate of 25 ppm in drinking water for three consecutive days to turkeys.g. Adverse Reactions: No adverse reactions were observed.
9. Field Study - Turkeys: E. coli
a. Type of Study: A field study was conducted using enrofloxacin in turkeys with spontaneously occurring colibacillosis.
b. Investigator(s): John R. Glisson, D.V.M., Ph.D.
1111 Crestwood Court
Watkinsville, GA 30677c. General Design:
1) Purpose: The study was conducted to confirm the efficacy of enrofloxacin drinking water medication at a concentration of 25 ppm for 3 consecutive days for controlling mortality in turkeys naturally infected with E. coli.
2) Animals: Nicholas Turkeys, one day-old, 800 males, 800 females (10 pens with 40 males and 10 pens with 40 females per treatment) were used.
3) Control: Non-medicated water was available ad libitum from waterers for the duration of the study.
4) Challenge: The E. coli challenge was by natural infection. This was accomplished by natural stress on the birds at 18 days of age.
5) Diagnosis: Colibacillosis was diagnosed by necropsy. A positive case was a dead bird, dying any time following the initiation of treatment, having a positive culture for E. coli and a lesion score of > = 2 using the following scale: 0 = no lesions, 1 = minimal air sacculitis (slight cloudiness), 2 = moderate air sacculitis (non-coalesced fibrinous material) and/or mild pericarditis and/or perihepatitis, 3 = severe air sacculitis (coalesced fibrinous material) and/or moderate to severe pericarditis and/or perihepatitis.
6) Dosage Form: The dosage form used was a concentrated aqueous solution with each mL containing 32.3 mg enrofloxacin. This formulation is the same as the marketed product.
7) Route of Administration: Orally, in the drinking water ad libitum
8) Dose: The dose tested in the study was 25 ppm, administered continuously in the drinking water for three consecutive days. Medication was initiated at 25 days of age (when house mortality exceeded a level of 0.5% in a 24-hour period).
9) Test Duration: The study continued for 10 days following the last day of medication.
10) Pertinent Variables Measured: The primary parameter for determining efficacy was based upon a comparison of mortality due to E. coli between the unmedicated control group and the medicated group.
d. Results: Mortality results are shown in Table 4.9.
Table 4.9. Mean percent E. coli mortality from the initiation of medication to ten days post medication (Days 25 to 37) Dose Mortality: Mortality: Mortality: Enrofloxacin Males Females Combined 0 ppm 12.9 (45/350) 10.7 (40/375) 11.7 (85/725) 25 ppm 1.9 (7/361) 1.1 (4/381) 1.5 (11/742)e. Statistical Analysis: Overall mortality was analyzed using mixed model methodology. Mortality was transformed using an arc sine transformation. Models included the random effects of blocks and their interactions. Fixed effects included treatments, sex, and their interactions. A 0.05 level of significance was used to determine significant results. Treatment effect averaged across sex was significant (P = 0.0001). f. Conclusions: It was concluded that under the natural field conditions of the study, enrofloxacin was effective in controlling mortality associated with E. coli susceptible to enrofloxacin when administered at the rate of 25 ppm in drinking water for three consecutive days to turkeys.g. Adverse Reactions: No adverse reactions were observed.
10. Field Studies - Turkeys: E. coli - Combined
a. Results: The results from the three turkey E. coli field studies were combined and statistically analyzed. The combined mortality data for these trials is summarized in Table 4.10.
Table 4.10. Mean percent E. coli mortality from the initiation of medication to ten days post medication for all 3 trials combined Dose Mortality: Mortality: Mortality: Enrofloxacin Males Females Combined 0 ppm 7.3 (82/1117) 6.9 (80/1157) 7.1 (162/2274) 25 ppm 0.9 (10/1140) 0.5 (6/1152) 0.7 (16/2292)e. Statistical Analysis: Overall mortality was analyzed using mixed model methodology. Mortality was transformed using an arc sine transformation. The random effects included locations, blocks nested within locations and their interactions. Fixed effects included treatments, sex, and their interactions. A 0.05 level of significance was used to determine significant results. A significant treatment effect was detected (p = 0.0421) demonstrating that treatment efficacy was consistent across study locations. f. Conclusions: It was concluded that under the natural field conditions of these studies, enrofloxacin was effective in controlling mortality associated with E. coli susceptible to enrofloxacin when administered at the rate of 25 ppm in drinking water for three consecutive days to turkeys.11. Field Study - Turkeys: P. multocida
a. Type of Study: A field study was conducted using enrofloxacin in turkeys naturally infected with Pasteurella multocida (fowl cholera).
b. Investigator(s): Terry TerHune, D.V.M., Ph.D.
Health Management Services
P.O. Box 2046
Tulare, CA 93275c. General Design:
1) Purpose: The study was conducted to confirm the efficacy of enrofloxacin drinking water medication at a concentration of 25 ppm for 3 consecutive days for controlling mortality in turkeys naturally infected with Pasteurella multocida (fowl cholera).
2) Animals: Nicholas Turkeys, 7 weeks of age, 240 males, 300 females (10 pens with 12 males and 10 pens with 15 females per treatment) were used.
3) Control: Non-medicated water was available ad libitum from waterers for the duration of the study.
4) Challenge: Fowl cholera exposure was accomplished when the birds were eight weeks of age by randomly selecting two birds from each pen (seeder birds) and challenging them with virulent P. multocida using the palatine cleft swab method.
5) Diagnosis: Fowl cholera was diagnosed by necropsy. A positive case was a dead bird, dying any time following the initiation of treatment, having a positive culture for P. multocida and lesions compatible with fowl cholera. Lesions in the lungs were scored according to the following scale: 0 = no lesions; 1 = mild lung involvement (approximately 1/4 of the lung); 2 = moderate lung involvement (approximately 1/2 of the lung); 3 = severe lung involvement (> 1/2 of the lung).
6) Dosage Form: The dosage form used was a concentrated aqueous solution with each mL containing 32.3 mg enrofloxacin. This formulation is the same as the marketed product.
7) Route of Administration: Orally, in the drinking water ad libitum
8) Dose: The dose tested in the study was 25 ppm, administered continuously in the drinking water for three consecutive days. Medication was initiated when house mortality due to fowl cholera exceeded a level of 1.0% in a 24-hour period in the non-seeder birds. This occurred on the third day following challenge of the seeder birds.
9) Test Duration: The study continued for 10 days post medication.
10) Pertinent Variables Measured: The primary parameter for determining efficacy was based upon a comparison of mortality due to P. multocida between the unmedicated control group and the medicated group.
d. Results: Mortality results (excluding seeder birds) are shown in Table 4.11.
Table 4.11. Mean percent P. multocida mortality (excluding seeder birds) from the initiation of medication to ten days post medication Dose Mortality: Mortality: Mortality: Enrofloxacin Males Females Combined 0 ppm 62.7 (52/83) 59.2 (59/98) 60.8 (110/181) 25 ppm 2.2 (2/91) 4.6 (5/108) 3.5 (7/199)e. Statistical Analysis: Overall mortality was analyzed by sex using general linear models methodology. Mortality was transformed using an arc sine transformation prior to analysis. Models included the effects of blocks and treatments. A 0.05 level of significance was used to determine significant results. Treatment effect was significant for both males and females (p = 0.0017 and p = 0.0022, respectively). f. Conclusions: It was concluded that under the natural field conditions of the study, enrofloxacin was effective in controlling mortality associated with P. multocida susceptible to enrofloxacin when administered at the rate of 25 ppm in drinking water for three consecutive days to turkeys.g. Adverse Reactions: No adverse reactions were observed.
12. Field Study - Turkeys: P. multocida
a. Type of Study: A field study was conducted using enrofloxacin in turkeys naturally infected with Pasteurella multocida (fowl cholera).
b. Investigator(s): Carey Quarles, Ph.D.
Colorado Quality Research
1401 Duff Drive, Suite 700
Fort Collins, CO 80524c. General Design:
1) Purpose: The study was conducted to confirm the efficacy of enrofloxacin drinking water medication at a concentration of 25 ppm for 3 consecutive days for controlling mortality in turkeys naturally infected with Pasteurella multocida (fowl cholera).
2) Animals: Nicholas Turkeys, 9 weeks of age, 240 males, 300 females (10 pens with 12 males and 10 pens with 15 females per treatment) were used.
3) Control: Non-medicated water was available ad libitum from waterers for the duration of the study.
4) Challenge: Fowl cholera exposure was accomplished when the birds were ten weeks of age by randomly selecting two birds from each pen (seeder birds) and challenging them with virulent P. multocida using the palatine cleft swab method.
5) Diagnosis: Fowl cholera was diagnosed by necropsy. A positive case was a dead bird, dying any time following the initiation of treatment, having a positive culture for P. multocida and lesions compatible with fowl cholera. Lesions in the lungs were scored according to the following scale: 0 = no lesions; 1 = mild lung involvement (approximately 1/4 of the lung); 2 = moderate lung involvement (approximately 1/2 of the lung); 3 = severe lung involvement (> 1/2 of the lung).
6) Dosage Form: The dosage form used was a concentrated aqueous solution with each mL containing 32.3 mg enrofloxacin. This formulation is the same as the marketed product.
7) Route of Administration: Orally, in the drinking water ad libitum
8) Dose: The dose tested in the study was 25 ppm, administered continuously in the drinking water for three consecutive days. Medication was initiated when house mortality due to fowl cholera exceeded a level of 1.0% in a 24- hour period in the non-seeder birds. This occurred on the third day following challenge of the seeder birds.
9) Test Duration: The study continued for 10 days post medication.
10) Pertinent Variables Measured: The primary parameter for determining efficacy was based upon a comparison of mortality due to P. multocida between the unmedicated control group and the medicated group.
d. Results: Mortality results (excluding seeder birds) are shown in Table 4.12.
Table 4.12. Mean percent P. multocida mortality (excluding seeder birds) from the initiation of medication to ten days post medication Dose Mortality: Mortality: Mortality: Enrofloxacin Males Females Combined 0 ppm 63.9 (62/97) 45.1 (55/122) 53.4 (117/219) 25 ppm 11.3 (11/97) 5.6 (7/124) 8.1 (18/221)e. Statistical Analysis: Overall mortality was analyzed by sex using general linear models methodology. Mortality was transformed using an arc sine transformation prior to analysis. Models included the effects of blocks and treatments. A 0.05 level of significance was used to determine significant results. Treatment effect was significant for both males and females (p = 0.0001 for both).f. Conclusions: It was concluded that under the natural field conditions of the study, enrofloxacin was effective in controlling mortality associated with P. multocida susceptible to enrofloxacin when administered at the rate of 25 ppm in drinking water for three consecutive days to turkeys.
g. Adverse Reactions: No adverse reactions were observed.
13. Field Study - Turkeys: P. multocida
a. Type of Study: A field study was conducted using enrofloxacin in turkeys naturally infected with Pasteurella multocida (fowl cholera).
b. Investigator(s): John R. Glisson, D.V.M., Ph.D.
1111 Crestwood Court
Watkinsville, GA 30677c. General Design:
1) Purpose: The study was conducted to confirm the efficacy of enrofloxacin drinking water medication at a concentration of 25 ppm for 3 consecutive days for controlling mortality in turkeys naturally infected with Pasteurella multocida (fowl cholera).
2) Animals: Nicholas Turkeys, 7 weeks of age, 240 males, 300 females (10 pens with 12 males and 10 pens with 15 females per treatment) were used.
3) Control: Non-medicated water was available ad libitum from waterers for the duration of the study.
4) Challenge: Fowl cholera exposure was accomplished when the birds were eight weeks of age by randomly selecting two birds from each pen (seeder birds) and challenging them with virulent P. multocida using the palatine cleft swab method.
5) Diagnosis: Fowl cholera was diagnosed by necropsy. A positive case was a dead bird, dying any time following the initiation of treatment, having a positive culture for P. multocida and lesions compatible with fowl cholera. Lesions in the lungs were scored according to the following scale: 0 = no lesions; 1 = mild lung involvement (approximately 1/4 of the lung); 2 = moderate lung involvement (approximately 1/2 of the lung); 3 = severe lung involvement (> 1/2 of the lung).
6) Dosage Form: The dosage form used was a concentrated aqueous solution with each mL containing 32.3 mg enrofloxacin. This formulation is the same as the marketed product.
7) Route of Administration: Orally, in the drinking water ad libitum
8) Dose: The dose tested in the study was 25 ppm, administered continuously in the drinking water for three consecutive days. Medication was initiated when house mortality due to fowl cholera exceeded a level of 1.0% in a 24- hour period in the non-seeder birds. This occurred on the third day following challenge of the seeder birds.
9) Test Duration: The study continued for 10 days post medication.
10) Pertinent Variables Measured: The primary parameter for determining efficacy was based upon a comparison of mortality due to P. multocida between the unmedicated control group and the medicated group.
d. Results: Mortality results (excluding seeder birds) are shown in Table 4.13.
Table 4.13. Mean percent P. multocida mortality (excluding seeder birds) from the initiation of medication to ten days post medication Dose Mortality: Mortality: Mortality: Enrofloxacin Males Females Combined 0 ppm 66.3 (57/86) 76.3 (71/93) 71.5 (128/179) 25 ppm 14.4 (13/90) 15.7 (16/102) 15.1 (29/192)e. Statistical Analysis: Overall mortality was analyzed by sex using general linear models methodology. Mortality was transformed using an arc sine transformation prior to analysis. Models included the effects of blocks and treatments. A 0.05 level of significance was used to determine significant results. Treatment effect was significant for both males and females (p = 0.0001 for both). f. Conclusions: It was concluded that under the natural field conditions of the study, enrofloxacin was effective in controlling mortality associated with P. multocida susceptible to enrofloxacin when administered at the rate of 25 ppm in drinking water for three consecutive days to turkeys.g. Adverse Reactions: No adverse reactions were observed.
14. Field Studies - Turkeys: P. multocida - Combined
a. Results: The results from the three turkey P. multocida field studies were combined and statistically analyzed. The combined mortality data (excluding seeder birds) for these trials is summarized in Table 4.14.
Table 4.14. Mean percent P. multocida mortality (excluding seeder birds) from the initiation of medication to ten days post medication for three field trials combined Dose Mortality: Mortality: Mortality: Enrofloxacin Males Females Combined 0 ppm 64.3 (171/266) 58.8 (184/313) 61.3 (355/579) 25 ppm 9.4 (26/278) 8.4 (28/334) 8.8 (54/612)e. Statistical Analysis: Overall mortality was analyzed by sex using mixed model methodology. Mortality was transformed using an arc sine transformation prior to analysis. The random effects included locations, blocks nested within locations and locations by treatment interactions. Fixed effects included treatments. A 0.05 level of significance was used to determine significant results. A significant treatment effect was detected for both males and females (p = 0.0071 and p = 0.0134, respectively) demonstrating that treatment efficacy was consistent across study locations. f. Conclusions: It was concluded that under the natural field conditions of these studies, enrofloxacin was effective in controlling mortality associated with P. multocida susceptible to enrofloxacin when administered at the rate of 25 ppm in drinking water for three consecutive days to turkeys.B. Studies Supporting Labeling
1. In vitro Susceptibility Study - Chickens and Turkeys
a. Type of Study: This was a study to determine the In vitro susceptibility of selected poultry pathogens to enrofloxacin.
b. Testing Laboratory: Colorado Animal Research Enterprises, Inc. (CARE)
6200 East County Road 56
Fort Collins, Colorado 80524c. General Design:
1) Purpose: The purpose of this study was to determine the minimum inhibitory concentrations (MICs) of selected pathogens to enrofloxacin. Isolates representative of pathogens known to cause disease in poultry were tested. This study was intended to provide baseline In vitro susceptibility data for the target pathogens (E. coli and P. multocida) and also to provide data for labeling purposes.
2) Samples: Isolates obtained from natural infections in chickens and turkeys were sent to Colorado Animal Research Enterprises, Inc. from nine laboratories representing five different geographical areas. The identity of each isolate was confirmed via observation of colony morphology and gram stain and each was biochemically confirmed using the Biolog Microstation 3( identification system of bacteria identification.
3) Procedure: Each bacterial isolate with a confirmed identity was subjected to broth dilution susceptibility testing using the microdilution technique. All tests were performed according to National Committee for Clinical Laboratory Standards (NCCLS) document M31-P, Vol. 14, No. 20, December, 1994. Documented modifications of the technique were performed to accommodate the unique veterinary pathogens.
4) Test Duration: Isolates tested were collected from clinical cases occurring from 1994 through 1996.
5) Pertinent Parameters Measured: The MIC50 (minimum inhibitory concentration for 50% of the isolates) and the MIC90 (minimum inhibitory concentration for 90% of the isolates) were determined and the ranges of MIC values were also reported.
d. Results: MIC50, MIC90 and MIC ranges are reported for each isolate by animal source (chicken or turkey) in Table 4.15.
Table 4.15. MIC values of enrofloxacin against bacterial isolates from natural infections (µg/mL) Pathogen Source No. Isolates MIC range MIC50 MIC90 E. coli chicken 82 0.015-1 0.03 0.06 E. coli turkey 59 0.015-0.06 0.03 0.06 P. multocida chicken 59 <= 0.008-0.125 0.03 0.06 P. multocida turkey 45 <= 0.008-0.125 0.03 0.03 S. aureus chicken 81 0.06-0.5 0.125 0.25 Salmonella spp. chicken 54 0.03-0.125 0.06 0.125 Salmonella spp. turkey 55 0.03-2 0.06 0.125 M. gallisepticum turkey 30 0.06-0.25 0.06 0.125e. Conclusions: The information from this study, when combined with the results of chicken and turkey pharmacokinetic studies, suggest that most isolates tested would be susceptible to enrofloxacin when administered within the proposed dose range. These data, therefore, support the results of the clinical field trials and the proposed dose range. 2. Pharmacokinetics of Enrofloxacin in Chickensa. Type of Study: This was a study to determine the plasma profile for enrofloxacin.
b. Investigator(s): Terry TerHune, D.V.M., Ph.D.
Health Management Services
P.O. Box 2046
Tulare, California 93275c. General Design:
1) Purpose: The purpose of this study was to determine the concentrations of enrofloxacin in plasma during ad libitum access to water containing 25 or 50 ppm enrofloxacin.
2) Animals: Healthy growing chickens from a commercial grower were used in this study. A total of 440 chickens, (220 males and 220 females), were assigned to 20 pens (10 pens of each sex), with 22 birds per pen.
3) Route of Administration: Orally, in the drinking water ad libitum
4) Doses: Doses tested were 25 and 50 ppm administered in the drinking water for seven consecutive days. Blood samples were taken at 0 (prior to medication), 6, 12, 18, 24, 36, 48, 60, 72, 84, 96, 108, 120, 132, 144, 156, and 168 hours after initiation of medication.
5) Test Duration: The study spanned seven days.
6) Pertinent Variables Measured: Enrofloxacin levels in plasma were measured using an HPLC assay for parent drug.
d. Results: The plasma concentrations determined are presented in Table 4.16.
Table 4.16. Mean plasma concentrations (µg/mL) of enrofloxacin in plasma of chickens administered enrofloxacin in drinking water ad libitum at 25 and 50 ppm for seven consecutive days Time of sampling (hr) after initiation of medication Dose level 6 12 24 to 168 25 ppm 0.241 0.317 0.381 50 ppm 0.464 0.653 0.712e. Conclusions: Following initiation of medication at either the 25 or 50 ppm treatment rate, the first plasma levels measured (at 6 hours) were more than 8X and 15X the MIC50 values, respectively, for E. coli isolates from chickens. These values support the efficacy observed in clinical trials conducted at the low end of the dose range (25 ppm). 3. Pharmacokinetics of Enrofloxacin in Turkeysa. Type of Study: This was a study to determine the plasma profile for enrofloxacin.
b. Investigator(s): Terry TerHune, D.V.M., Ph.D.
Health Management Services
P.O. Box 2046
Tulare, California 93275c. General Design:
1) Purpose: The purpose of this study was to determine the concentrations of enrofloxacin in plasma during ad libitum access to water containing 25 or 50 ppm enrofloxacin.
2) Animals: Healthy growing turkeys from a commercial grower were used in this study. A total of 440 turkeys (220 males and 220 females) were assigned to 20 pens, (10 pens of each sex), with 22 birds per pen.
3) Route of Administration: Orally, in the drinking water ad libitum
4) Doses: Doses tested were 25 and 50 ppm administered in the drinking water for seven consecutive days. Blood samples were taken at 0 (prior to medication), 6, 12, 18, 24, 36, 48, 60, 72, 84, 96, 108, 120, 132, 144, 156 and 168 hours after initiation of medication.
5) Test Duration: The study spanned seven days.
6) Pertinent Variables Measured: Enrofloxacin levels in plasma were measured using an HPLC assay for parent drug.
d. Results: The plasma concentrations determined are presented in Table 4.17.
Table 4.17. Mean plasma concentrations (µg/mL) of enrofloxacin in plasma of turkeys administered enrofloxacin in drinking water ad libitum at 25 and 50 ppm for seven consecutive days Time of sampling (hr) after initiation of medication Dose level 6 24 to 168 25 ppm 0.204 0.240 50 ppm 0.352 0.458e. Conclusions: Following initiation of medication at either the 25 or 50 ppm treatment rate, the first plasma levels measured (at 6 hours) were more than 6X and 11X the MIC50 values, respectively, for E. coli and P. multocida isolates from turkeys. These values support the efficacy observed in clinical trials conducted at the low end of the dose range (25 ppm).
V. ANIMAL SAFETY
A. Pivotal Studies
1. Target Animal Safety Study - Chickens
a. Type of Study: This was a 28-day study in broilers in which enrofloxacin was administered in the drinking water at 0, 25, 125, and 625 ppm for 21 consecutive days followed by a 7-day observation period.
b. Study Director: Jeffrey N. Davidson, D.V.M., M.P.V.M.
Health Management Services
P. O. Box 2046
Tulare, CA 93275c. General Design:
1) Purpose: This study was designed to determine the toxicological effects of enrofloxacin when administered in the drinking water to chickens. Potential target organs and tissues were to be identified through clinical observations, gross necropsy and histological examination. Total blood cell counts, packed cell volume, clotting time, estimate of buffy coat, body weight, feed and water consumption were other variables of interest.
2) Animals: One-day-old Hubbard broiler chicks (n = 160) were used in the study. For each of 4 treatments, there were 2 pens of 10 chicks for each sex (80 males, 80 females). The average initial weight was 33.0 grams.
3) Control: Non-medicated water was available ad libitum.
4) Dosage Form: An aqueous formulation containing 32.3 mg enrofloxacin per milliliter was used. The dosage form is the marketed formulation of Baytril (enrofloxacin) 3.23% Concentrate Antimicrobial Solution.
5) Dose: The doses were 0, 25, 125 and 625 ppm enrofloxacin.
6) Route of Administration: Orally, in the drinking water ad libitum
7) Study Duration: The study included 21 consecutive days of treatment followed by 7 days of observation.
8) Pertinent Measurements/Observations: Daily clinical observations, mortality, hematological measurements (Days 22 and 28), body weight (pen basis on days 7, 14, 21, and 28), feed consumption (pen basis on days 7, 14, 21, and 28), water consumption (pen basis daily during medication, then at weekly intervals), gross necropsy and histopathology (Day 28) were evaluated to assess the potential toxicity in broilers.
d. Results:
1) Clinical Observations: The only clinical sign observed was a transient droopiness of the eyelid in the 625-ppm group.
2) Mortality: No deaths occurred during the study.
3) Body Weight: On Days 21 and 28, the 625-ppm group weighed less than the other three groups.
4) Feed Conversion: Feed conversion was determined by taking total feed consumption at each time period and dividing by total body weight. There were no differences between groups in feed conversion.
5) Water Consumption: Water consumption measured on Days 21 and 28 revealed reduced water consumption in the 625-ppm group when compared to the other groups. At Day 28, the consumption was also reduced in the 125-ppm group.
6) Hematology: There were no statistically significant differences between groups for total cell counts, clotting times or packed cell volumes on Days 22 and 28. All birds had adequate buffy coats as measured on the microhematocrit.
7) Gross and Histological Observations: No drug related gross lesions were observed at necropsy in any of the groups. There were some minimal histological changes described as atrophy of the testicles in the medicated birds; this finding was not dose related.
e. Statistical Analysis: A randomized complete block design was used with four treatments in four replicate blocks. An analysis of variance was used to measure treatment differences.
f. Conclusions: Results from this study, in conjunction with the lack of adverse effects in the clinical field trials indicate that Baytril( (enrofloxacin) 3.23% Concentrate Antimicrobial Solution is safe when used in chickens in accordance with label directions.
2. Target Animal Safety Study - Turkeys
a. Type of Study: This was a 28-day study in growing turkeys, in which enrofloxacin was administered in the drinking water at 0, 25, 125, and 625 ppm for 21 consecutive days followed by a 7-day observation period.
b. Study Director: Jeffrey N. Davidson, D.V.M., M.P.V.M.
Health Management Services
P.O. Box 2046
Tulare, CA 93275c. General Design:
1) Purpose: This study was designed to determine the toxicological effects of enrofloxacin when administered in the drinking water to turkeys. Potential target organs and tissues were to be identified through clinical observations, gross necropsy and histological examination. Total blood cell counts, packed cell volume, clotting time, white blood cell count, body weight, feed and water consumption were other variables of interest.
2) Animals: One-day-old Broad Breasted White turkey poults (n = 160) were used in the study. For each of the 4 treatments, there were 2 pens of 10 poults each for each sex (80 males, 80 females). The average initial weight was 56.6 g.
3) Control: Non-medicated water was available ad libitum.
4) Dosage Form: An aqueous formulation containing 32.3 mg enrofloxacin per milliliter was used. The dosage form is the marketed formulation of Baytril (enrofloxacin) 3.23% Concentrate Antimicrobial Solution.
5) Dose: The doses were 0, 25, 125 and 625 ppm enrofloxacin.
6) Route of Administration: Orally, in the drinking water ad libitum
7) Study Duration: The study included 21 consecutive days of treatment followed by 7 days of observation.
8) Pertinent Measurements/Observations: Daily clinical observations, mortality, hematological measurements (Days 22 and 28), body weight (pen basis on days 7, 14, 21 and 28), feed consumption (pen basis on Days 7, 14, 21, and 28), water consumption (pen basis daily during medication, then at weekly intervals), gross necropsy and histopathology (Day 28) were evaluated to assess the potential toxicity in turkeys.
d. Results:
1) Clinical Observations: There were no adverse clinical signs noted in the 0-, 25-, and 125-ppm groups. In the 625-ppm dose group, starting on about the fifth day, birds demonstrated a marked decrease in activity and listlessness. They appeared dehydrated and spent more time under the brooding heaters. Mortality started occurring around the seventh day. By the eleventh day the activity of the birds appeared normal. The adverse signs could not be attributed to any one system, i.e., locomotor, respiratory, etc.
2) Mortality: There was no mortality in the 0-, 25-, and 125-ppm groups. Eleven of the 40 birds in the 625-ppm group died between Days 7 to 10. Post-mortem findings revealed dehydration and emaciation with no feed or ingesta in the digestive tract. There were no other abnormal findings.
3) Body Weight: At Day 14, the 125-ppm group was heavier than the control birds. The birds treated with 625 ppm weighed less than the controls at Day 7 and Day 21. The weights were not different between groups at the end of the study.
4) Feed Consumption: There was no difference in feed consumption except on Day 14 when the 625-ppm group showed a decrease in cumulative feed consumption compared to the controls.
5) Water Consumption: During the first week, the 625-ppm group consumed less water than the controls. After the first week, there was no difference in water consumption among the groups.
6) Hematology: There were no statistically significant differences between groups for total cell counts, clotting times, packed cell volumes or white blood cell counts. All values were within normal ranges for birds of this age.
7) Gross and Histological Observations: No drug related gross lesions or abnormal histopathological changes were observed in any of the groups.
e. Statistical Analysis: A randomized complete block design was used with four treatments in four replicate blocks. An analysis of variance was used to measure treatment differences.
f. Conclusions: There were no mortalities or adverse clinical signs noted in birds receiving either the 25 or 125 ppm dose. Birds in the 625-ppm dose group appeared to be listless beginning on about the fifth day and some mortality occurred between the seventh and tenth days in this group. Activity was normal for the duration of the study. Necropsy results from the birds that died indicated water and feed were not being consumed. There were no other abnormal findings. Results from this study, in conjunction with the lack of adverse effects in the clinical field trials indicate that Baytril® (enrofloxacin) 3.23% Concentrate Antimicrobial Solution is safe when used in turkeys in accordance with labeling.
3. Target Animal Safety Study - Turkeys
a. Type of Study: This was a 35-day study in growing male turkeys, in which enrofloxacin was administered in the drinking water at 0, 25, and 125 ppm for 21 consecutive days followed by a 14-day observation period.
b. Study Director: Jeffrey N. Davidson, D.V.M., M.P.V.M.
Health Management Services
P.O. Box 2046
Tulare, CA 93275c. General Design:
1) Purpose: This study was designed to determine the toxicological effects of enrofloxacin when administered in the drinking water to male turkeys. Clinical observations and gross necropsy were to be performed along with histological examination of the testes and proximal end of the tibia. Body weight, feed and water consumption were other variables of interest.
2) Animals: Male, 21 days of age, Broad Breasted White turkey poults (n = 60) were used in the study. For each of the 3 groups, there were 2 pens of 10 poults per pen. The average initial weight was 438 grams.
3) Control: Non-medicated water was available ad libitum.
4) Dosage Form: An aqueous formulation containing 32.3 mg enrofloxacin per milliliter was used. The dosage form is the marketed formulation of Baytril( (enrofloxacin) 3.23% Concentrate Antimicrobial Solution.
5) Dose: The doses were 0, 25 and 125 ppm enrofloxacin.
6) Route of Administration: Orally, in the drinking water ad libitum
7) Study Duration: The study included 21 days of treatment beginning at 21 days of age followed by a 14-day observation period.
8) Pertinent Measurements/Observations: Daily clinical observations, mortality, body weight and feed consumption (pen basis at 28, 35, 42, and 56 days of age), water consumption (pen basis daily during medication, then at weekly intervals), gross necropsy and histopathology (five birds from each replicate at 43 days of age and five birds from each replicate at 56 days of age) were evaluated to assess the potential toxicity in turkeys.
d. Results:
1) Clinical Observations: No adverse clinical signs were noted in any group.
2) Mortality: No mortality occurred in any of the groups.
3) Body Weight: There was no negative effect on body weights at any of the intervals measured. The treated groups weighed more than the controls (28, 35, 42, and 56 days of age).
4) Feed Consumption: Enrofloxacin did not depress feed intake. The treated groups consumed more feed than the controls at all intervals measured (28, 35, 42, and 56 days of age).
5) Water Consumption: Enrofloxacin did not depress water consumption. Consumption was similar for all groups.
6) Gross and Histological Observations: No drug related gross lesions were observed at necropsy in any of the groups. No treatment related histopathology changes were observed in the tissues examined.
e. Statistical Analysis: A randomized complete block design was used with three treatments in two replicate blocks. Statistical analyses of the data were not performed since the primary purpose of the study was to evaluate gross lesions and histological changes of the testes and proximal end of the tibia. Weight gain and feed consumption were not analyzed because both treated groups ate more feed and gained more weight than the controls.
f. Conclusions: The results of this study confirm the results of an earlier study in younger birds which demonstrated Baytril® (enrofloxacin) 3.23% Concentrate Antimicrobial Solution is safe for use in turkeys at the labeled dose and duration.
4. Reproductive Safety Study - Chickens
a. Type of Study: This was a 235-day study in which enrofloxacin was administered to chickens via the drinking water at the rate of 150 ppm. One group received treatment for 7 consecutive days beginning at 1, 50, 100, 150, and 200 days of age. Another group received the same treatment for seven consecutive days beginning at 100, 150, and 200 days of age, while a third group served as a non-medicated control. On Day 157, females and males in each group were co-mingled for the remainder of the study. Egg production was monitored and chicks hatched from eggs were also monitored.
b. Study Director: Terry TerHune, D.V.M., Ph.D.
Health Management Services
P.O. Box 2046
Tulare, CA 93275c. General Design:
1) Purpose: This study was designed to determine the effects of enrofloxacin on reproduction when administered in the drinking water to chickens. Egg production and egg weights were recorded along with hatchability and chick viability. Reproductive organs from treated birds were examined histologically and the gonads of chicks from hatched eggs were also examined histologically. Body weights of the treated birds were also monitored.
2) Animals: One-day old Arbor Acres chicks (n = 900) were used in the study. Each of three groups consisted of 10 pens containing 20 females and 10 pens containing 10 males. On Day 157, preselected females and males were co-mingled for the remainder of the study. The average initial weight for females was 44.0 g and for males was 43.0 g.
3) Control: Non-medicated water was available ad libitum.
4) Dosage Form: An aqueous formulation containing 32.3 mg enrofloxacin per milliliter was used. The dosage form is the marketed formulation of Baytril (enrofloxacin) 3.23% Concentrate Antimicrobial Solution.
5) Dose: The dose used was 150 ppm for seven consecutive days repeated three times in one group and five times in the other treated group.
6) Route of Administration: Orally, in the drinking water
7) Study Duration: The study spanned 235 days beginning at one day of age.
8) Pertinent Measurements/Observations: Body weights (pen basis on Days 1, 100, 157, and 206), egg production, egg weight, hatchability and chick viability along with histopathology of male and female reproductive organs of treated birds and testicles and ovaries of seven day old chicks hatched from eggs of treated birds were evaluated to assess effect on reproduction.
d. Results:
1) Body Weights: Body weights were similar among the three groups throughout the study. The data were not statistically analyzed because under the commercial poultry practices utilized in the study the amount of feed offered to pens was limited to control the weight of the chickens. This is necessary to optimize reproductive function.
2) Egg Production: The mean eggs produced increased with time during the observation period for all three groups. There was no significant difference in egg production among the three groups (p > 0.1).
3) Egg Weight: Mean egg weights increased with time during the observation period for all three groups. There was no significant difference in egg weights among the three groups (p > 0.1).
4) Hatchability: Overall, the median percent of chicks hatched normal was 73.3, 75.3, and 77.5 for the control, 5-treatment and 3-treatment groups, respectively. There was no significant difference among the groups with respect to the percent of chicks hatched normal (p > 0.1).
5) Viability: Chick viability was evaluated based on six measurements: weight less than 30 g; deformed; navel not healed; dehydrated; abnormal down color; and abnormal stance. There was no significant difference among the three groups with respect to any of the individual measured viability parameters (p > 0.1). The median percentage of chicks evaluated as abnormal with respect to at least one parameter was greater in the control group (median = 7.4%) when compared with both the 5-treatment group (median = 6.9%) and the 3- treatment group (median = 4.5%). There was no significant difference among the three groups with all viability parameters combined (p > 0.1).
6) Histological Observations: No significant histological changes were observed in the male or female reproductive organs from the treated birds necropsied on Day 157. Further, no lesions were observed in the testicles or ovaries of the seven day old chicks hatched from eggs produced by treated birds.
e. Statistical Analysis: A randomized complete block design was used with each block containing one pen from each group. The pen was the experimental unit. All data were analyzed on a per pen basis so that the sample size/group was 10 pens. A repeated measures analysis of variance was used to measure treatment differences.
f. Conclusions: Results of this study indicate that at the proposed label dose and duration, Baytril® (enrofloxacin) 3.23% Concentrate Antimicrobial Solution is safe for use in broiler breeder chickens of both sexes.
VI. HUMAN SAFETY
A. Toxicity Studies
1. Acute Oral Single Dose Studies in Rats, Mice, Rabbits, and Dogs
a. Study Report Number: 73075
b. Study Completion: August, 1984 (start: June, 1984)
c. Study Director: Dr. M. Schmidt
Bayer Institute for Toxicology
Wuppertal, Germanyd. Substance and Dosage Form: Technical drug substance; oral suspension
e. Species and Strain of Animal: Rats (Wistar), Mice (BOR:CFW1),
Rabbits (Chinchilla), Dogs (Beagle)f. Number of Animals per Sex per Treatment Group: Rats - 5, Mice - 5, Rabbits -2, Dogs - 2
g. Levels and Duration of Dosing:
Rats- 630, 1000, 5000 mg/kg - single day;
Mice - 1000, 2000, 4000, 5000 mg/kg - single day;
Rabbits - 320, 500, 800, 1200, 2000 mg/kg - single day;
Dogs - 1000, 5000 mg/kg - single dayh. Route of Administration: Oral
i. Parameters Tested and Discussion of Results:
Clinical Signs: Observations for 14 days. Symptoms observed were reduced mobility, trembling, tonic convulsions, labored breathing and staggering gait. Signs appeared as early as 15 minutes after exposure with some persisting for 10 days.
Gross Necropsy: Pulmonary congestion and hemorrhage.
Conclusions: Oral LD50's: Male and Female Rats = >5000 mg/kg. Male Mice = >5000 mg/kg and Female Mice = 4336 mg/kg. Male and Female Rabbits = ~ 500 - 800 mg/kg. Male and Female Dogs = Not established due to vomition.
2. 90-Day Oral Toxicity Study in Rats
a. Study Report Number: 73194
b. Study Completion: December, 1984 (start: September 1984)
c. Study Director: R. L. Kowalski
Miles Laboratories
Elkhart, Indianad. Substance and Dosage Form: Technical drug substance; incorporated into feed
e. Species and Strain of Animal: Rats (Sprague-Dawley)
f. Number of Animals per Sex per Treatment Group: 15
g. Levels and Duration of Dosing: 0, 500, 2000, 7500 ppm for a minimum of 91 days.
h. Route of Administration: Oral
i. Parameters Tested and Discussion of Results:
Clinical Signs and Survival: No test article related signs of toxicosis and no deaths occurred.
Physical Examination: Weekly examinations including direct ophthalmoscopy at termination were not suggestive of any test article-related effects.
Body Weights: A statistically significant reduction in mean body weight for males and females from the high-dose group.
Food Consumption: Feed intake was slightly increased for the high-dose animals.
Hematology: No treatment related trends were observed in these parameters.
Clinical Chemistries: Total protein levels were significantly decreased for both sexes in the high-dose group after Weeks 6 and 13. Aspartate aminotransferase was decreased for the high-dose males after 6 and 13 weeks. A decrease in total bilirubin occurred in the high dose males. A dose-dependent increase in inorganic phosphorous was reported in the males and females after 13 weeks.
Urinalysis: A dose-dependent trend toward decreasing urine sodium output for males in the mid- and high-dose groups at 6 weeks and for females in the mid- and high-dose groups after 13 weeks.
Organ Weights: Heart weights were decreased for mid-dose and high-dose animals. Mean prostate weights were significantly lower for mid- and high-dose males. Liver weights were decreased at the high-dose levels.
Gross Pathology: Swollen external ears and distention of the cecum were primarily in the high-dose animals.
Microscopic Pathology: Auricular chondropathy was observed in all groups including the controls, but appeared to be dose related (1 control, 1 low-dose, 6 mid-dose and 10 high-dose). Microscopic changes of questionable relationship to the test article were observed in the knee joints of 3 of 30 rats in the high-dose group. Microscopic change occurred in the epididymides and testes of male rats from the high-dose group. This change appeared to represent a degenerating spermatid.
Conclusion: The Agency concluded a NOEL of 500 ppm (40 mg/kg).
3. 90-Day Oral Toxicity Study in Dogs
a. Study Report Number: 73146
b. Study Completion: December, 1984 (start: September 1984)
c. Study Director: M. C. Porter
Miles Laboratories
Elkhart, Indianad. Substance and Dosage Form: Technical drug substance; incorporated into feed
e. Species and Strain of Animal: Canine (Beagle)
f. Number of Animals per Sex per Treatment Group: 4
g. Levels and Duration of Dosing: 0, 320, 800 and 2000 ppm; 91 days
h. Route of Administration: Oral
i. Parameters Tested and Discussion of Results:
Clinical Signs and Survival: Scattered incidences of emesis and an aversion to the test diet during the early phase in mid-dose and high-dose animals. No adverse effect upon physical condition or deaths occurred in test animals which were 12 to 13 months of age at initiation of study.
Physical Examination: Conducted prior to treatment and at termination including opthalmoscopy with no adverse effects.
Body Weights: Mean body weights which were recorded at weekly intervals during the course of the study were not affected.
Food Consumption: Diet consumption returned to normal after the initial aversion phase.
Hematology: No adverse effects at the 3 evaluations during course of study including termination.
Clinical Chemistries: Evaluated at 3 intervals including termination with a decreased serum globulin/total protein early, but returning to normal as the study progressed.
Urinalysis: Evaluated at 3 intervals including termination with no adverse effects.
Organ Weights: Organ weights were within the normal historical range for the laboratory.
Gross Pathology: No drug-related observations.
Microscopic Pathology: A high incidence of lipofuscin pigment was observed in the epithelial cells of the proximal convoluted tubules of the kidneys for high-dose animals, but was mild in intensity. The initial ages of the dogs used in the study were beyond the sensitive periods for the development of joint or testicular lesions associated with quinolone compounds in growing animals.
Conclusions: Agency concluded no toxic effect was observed in this study, but the test animals were older than stated in the guidelines.
4. Additional Subchronic Oral Toxicity Study in Dogs
a. Study Report Number: 73775
b. Study Completion: August, 1986 (start: May, 1986)
c. Study Director: M.C. Porter
Miles Laboratories
Elkhart, Indianad. Substance and Dosage Form: Technical drug substance; incorporated into feed.
e. Species and Strain of Animal: Canine (Beagle)
f. Number of Animals per Sex per Treatment Group: 4
g. Levels and Duration of Dosing: 0, 100, 320, 2500 ppm for 91 days
h. Route of Administration: Oral
i. Parameters Tested and Results:
Clinical Signs and Survival: No deaths occurred, but abnormal gait and/or posture was observed in the high-dose animals by the second week of the study. Daily observations were conducted.
Physical Examination: Radiographic examination revealed that neither bone growth nor density were significantly affected. Physical examinations including direct ophthalmoscopy were conducted pretreatment and at termination with the only finding described in the clinical signs section.
Body Weights: Body weights (recorded at weekly intervals) and body weight gains were not significantly different for treated and control animals.
Food Consumption: Diet consumption, which was recorded daily, was not affected.
Hematology: Samples collected at 2 and 6 weeks as well as at termination showed no abnormal findings.
Clinical Chemistries: Evaluated at 2 and 6 weeks and at termination with no abnormalities found.
Urinalysis: Collected after Weeks 2 and 6, and at termination with crystal formation in the urine of dogs receiving the high-dose treatment.
Organ Weights: Absolute and relative organ weights for treated animals did not differ significantly from those of controls, but testicular weights were higher for the treated animals.
Gross Pathology: Superficial erosions of articular cartilage surfaces were seen in all high-dose dogs and one mid-dose male with no joint lesions in the remaining mid-dose or low-dose animals.
Microscopic Pathology: Microscopically, the joint lesions were characterized by splitting of the articular cartilage surface and disorganization of chondrocytes. Necrosis and disintegration of hyaline cartilage was seen in some cases.
A marked variation occurred in the appearance of testes including stage of maturity, diameter of lumen of seminiferous tubules and vacuolar changes in the lining cells of the tubules. One dog from the control group and 3 dogs from the mid-dose group had signs of testicular maturity as evidenced by the production of tail-containing spermatozoa. None of the low-dose or high-dose animals had any evidence of tailed-spermatozoa. Three dogs from the control group and 1 each from the mid- and high-dose groups had immature testes that were characterized by small seminiferous tubules with little or no lumen and containing a single layer of spermatogonial cells. Testes from 4 dogs, one each from the low- and high-dose groups and 2 from the mid-dose group, contained seminiferous tubules with dilated lumen and often contained more than a single layer of lining cells. Similar, but less advanced, tubules were observed for one dog from the control group. Vacuolar change in the apical parts of the spermatogonial cells occurred in 2 dogs of the low-dose group, 3 dogs of the high-dose group, and 1 control. The tubular morphology observed for the 2 low-dose and 3 high-dose animals appeared to be beyond the normal limits.
Conclusions: The Agency concluded a NOEL of 100 ppm or equivalent of 3 mg/kg for this study following conduction of additional subchronic dog studies.
5. Additional Subchronic Oral Toxicity Study in Male Dogs
a. Study Report Number: 73788
b. Study Completion: September, 1987 (start: June, 1987)
c. Study Director: M. C. Porter
Miles Laboratories
Elkhart, Indianad. Substance and Dosage Form: Technical drug substance; incorporated into feed.
e. Species and Strain of Animal: Canine (Beagle)
f. Number of Animals per Sex per Treatment Group: 4 (Males only)
g. Levels and Duration of Dosing: 0, 10, 20, 40 and 3200 ppm
h. Route of Administration: Oral
i. Parameters Tested and Results:
Clinical Signs and Survival: No deaths occurred and overt indications of toxicity were limited to the musculoskeletal system in high-dose dogs.
Physical Examination: Observed daily for appearance and behavior with physical examination including opthalmoscopy prior to termination. Abnormalities were described in clinical signs section.
Body Weights: Recorded weekly with body weight gains not affected except for high-dose animals during first 5 weeks of study.
Food Consumption: Diet consumption was similar for all groups including controls.
Organ Weights: Absolute and relative testicular weights for treated animals did not differ meaningfully or in a dose-related manner from those of controls.
Gross Pathology: Testes and epididymides were examined for all dogs at termination. No gross lesions or abnormalities were noted.
Microscopic Pathology: The microscopic appearance of the testes was variable due to stages of sexual maturity. All dogs were considered within normal physiologic limits except for one dog in the high-dose group which had distinct bilateral degenerative changes.
Conclusions: The NOEL was 40 ppm (approximately 1.2 mg/kg/day).
6. Additional Subchronic Toxicity Study in Male Dogs Followed by a 13-Week Withdrawal
a. Study Report Number: 73789
b. Study Completion: December, 1987 (start: June, 1987)
c. Study Director: M. C. Porter
Miles Laboratories
Elkhart, Indianad. Substance and Dosage Form: Technical drug substance; incorporated into feed.
e. Species and Strain of Animal: Canine (Beagle)
f. Number of Animals per Sex per Treatment Group: 4 (Males only)
g. Levels and Duration of Dosing: 0, 10, and 40 ppm
h. Route of Administration: Oral
i. Parameters Tested and Results:
Clinical Signs and Survival: No deaths occurred and no overt indications of toxicity were observed.
Physical Examination: No abnormalities were observed in examination at pretreatment and at termination which included ophthalmoscopy.
Body Weights: Body weight gains were not adversely affected.
Food Consumption: Diet consumption was similar for all groups including controls.
Organ Weights: Absolute and relative testicular weights for treated animals did not differ meaningfully or in a dose-related manner from those of controls.
Gross Pathology: No gross lesions were noted in the testes and epididymides.
Microscopic Pathology: No test article-related changes occurred in either testes or epididymides with both organs appearing normal and containing normal, mature spermatozoa.
Conclusions: The NOEL was 40 ppm.
7. Teratology (Segment II) Study in the Rat
a. Study Report Number: 73159
b. Study Completion: October, 1984 (start: September, 1984)
c. Study Director: G.R. Clemens
Miles Laboratories
Elkhart, Indianad. Substance and Dosage Form: Technical drug substance
e. Species and Strain of Animal: Rat (Charles River)
f. Number of Animals per Sex per Treatment Group: 28 (Females only)
g. Levels and Duration of Dosing: 0, 50, 210, 875 mg/kg for 10 consecutive days from the sixth through the fifteenth day of gestation.
h. Route of Administration: Oral
i. Parameters Tested and Results:
Maternal Survival and Signs of Toxicity: All dams survived and there were no remarkable observations attributable to the test article.
Food Consumption and Body Weights: Food consumption was significantly higher in the high-dose group, but overall weight gain was significantly reduced in that group.
Reproductive Performance: There was no statistically significant differences in any reproductive parameter for any test article group when compared with the control.
Fetal Parameters: Fetal weights for both males and females were significantly reduced for the high-dose group. Male and combined fetal weights were significantly reduced also for the mid-dose group. No gross morphological external and visceral changes attributable to the test article were observed in the fetuses, but they were generally smaller in the high-dose group compared to the controls.
Skeletal Examination: There was no increase in incidence of common skeletal variations, but a statistically significant delay in ossification was observed in the mid- and high-dose groups which accompanied the overall reduction in body weights for these groups.
Conclusions: The Agency concluded a NOEL of 50 mg/kg/day for the study.
8. Embryotoxicity/Teratogenicity Study in the Rabbit
a. Study Report Number: 73705
b. Study Completion: Conducted in 2 phases: January 1986 and January 1989
c. Study Director: H. Becker
Research and Consulting Co.
Itingen, Switzerlandd. Substance and Dosage Form: Technical drug substance
e. Species and Strain of Animal: Rabbit (Chinchilla)
f. Number of Animals per Sex per Treatment Group: 16 (Females only)
g. Levels and Duration of Dosing: 0, 1, 5, 25, and 75 mg/kg from Day 6 through Day 18 post-breeding.
h. Route of Administration: Oral gavage
i. Parameters Tested and Results:
Maternal Survival and Signs of Toxicity: No treated dams expired with one in the high-dose group aborting on Day 19.
Food Consumption and Body Weights: Mean food consumption was similar for the 3 lower-dosed groups, but a statistically significant decrease was noted for the high-dose females. This group also had a slightly decreased body weight gain.
Reproductive Performance: No statistically significant differences were observed for the 3 lower-dosed groups. Treatment at the higher dose caused increased post-implantation loss.
Fetal Parameters: No differences occurred in mean body weights of fetuses in the treated groups. Malformations observed were considered to be incidental and within the normal spontaneous range.
Skeletal Examination: No test article or dose-related differences were evident and the stage of development in all groups was similar.
Conclusions: The Agency concluded a NOEL of 25 mg/kg.
9. Specialized Male Fertility Study in the Rat
a. Study Report Number: 73800
b. Study Completion: December, 1986 (start: May, 1986)
c. Study Director: G.R. Clemens
Miles Laboratories
Elkhart, Indianad. Substance and Dosage Form: Technical drug substance; incorporated into feed.
e. Species and Strain of Animal: Rat (Sprague-Dawley)
f. Number of Animals per Sex per Treatment Group: 60 (males only)
g. Levels and Duration of Dosing: 0 and 7500 ppm for 90 days
h. Route of Administration: Oral
i. Parameters Tested and Results:
The study design consisted of treating males for 90 days with interim sacrifices at 3, 6, 9, and 13 weeks. Fifteen males from each group were retained, placed on control diet and then mated during the late treatment phase and at intervals during the recovery phase to non-treated females. The females were terminated on day 20 of gestation and evaluated for reproductive indices.
Clinical Signs and Survival: There were no overt effects on appearance and/or behavior of the males during the study.
Food Consumption and Body Weights: Feed consumption and body weight gain were significantly decreased during the exposure period with both returning to normal by the end of the recovery period.
Male Breeding Performance: Libido was unaffected, but a significant decrease occurred in fertility.
Male Testes/Weights and Histopathology: There was an increase in mean testicular weight during the treatment period, but by the end of the recovery period, the weights were significantly less than the controls. Epididymal weights for the test article group were lower than the control group throughout the study. Abnormal sperm were observed in the treated animals after Week 3, but a nearly complete reversal of this change occurred during the recovery period.
Female Reproductive Performance: An adverse effect was seen on fertility of the untreated females at the initial mating (last 2 weeks of treatment phase), but during each successive breeding, fertility improved and was judged to return to normal by the 7th week of the recovery phase.
Conclusions: The treatment of 7500 ppm caused a marked reduction in male fertility due to the adverse effect on sperm development. Functional fertility returned to normal for 13 of the 15 males during the withdrawal period, but two continued to have complete bilateral testicular atrophy and subsequent infertility.
10. 90-Day Feeding Study Followed by a 13-Week Withdrawal in Male Rats
a. Study Report Number: 73812
b. Study Completion: December, 1987 (start: June, 1987)
c. Study Director: J. J. Bare
Miles Laboratories
Elkhart, Indianad. Substance and Dosage Form: Technical drug substance; incorporated into feed
e. Species and Strain of Animal: Rat (Sprague-Dawley)
f. Number of Animals per Sex per Treatment Group: 135 (males only)
g. Levels and Duration of Dosing: 0, 125, 500, and 7500 ppm for 91 days
h. Route of Administration: Oral
i. Parameters Tested and Results:
The study design consisted of treating males for a period of 91 days with a subgroup receiving 7500 ppm terminated after 14 days on the diet. Fifteen animals from each group were terminated after 91 days. The remaining animals were maintained on a control diet for another 90 days and then terminated. Testes and epididymides were evaluated.
Clinical Signs and Survival: One low-dose animal died, but no other deaths occurred. No signs of toxicosis were observed.
Body Weights: There was no effect on weight gain in the low or mid-dose groups. The high-dose animals had a statistically significant decrease for body weight gain during the treatment phase, but gained rapidly during the reversible phase.
Physical Examination: All animals appeared to be in good physical condition.
Histopathology: Abnormal spermatozoa were present in the testes of 10 of the 15 high-dose rats terminated at Day 14. At the end of the withdrawal period, no abnormal spermatozoa were detected in the testes, but bilateral testicular atrophy was seen in 2 high-dose rats.
Organ Weights: At both terminations, a dose-dependent decrease in mean epididymal weights occurred which were statistically significant in the mid- and high-dose groups. There was a significant increase in weight of testes in the high-dose groups on Day-91 sacrifice.
Conclusions: The NOEL was judged to be 125 ppm or approximately 9.9 mg/kg/day.
11. Two-Generation Reproduction Study in Rats
a. Study Report Number: 73314
b. Study Completion: August, 1985 (start: November, 1984)
c. Study Director: G.R. Clemens
Miles Laboratories
Elkhart, Indianad. Substance and Dosage Form: Technical drug substance; incorporated into feed
e. Species and Strain of Animal: Rat (Sprague-Dawley)
f. Number of Animals per Sex per Treatment Group: 120 in the F0 generation
g. Levels and Duration of Dosing: 0, 500, 2000 and 7500 ppm over a duration of two generations.
h. Route of Administration: Oral
i. Parameters Tested and Results:
Clinical Observations: No overt toxicity signs at any dietary concentration. General behavior and appearance was essentially normal for the F0 and F1 generations. Swollen pinnae were reported in both the F0 and F1 generations.
Body Weight and Food Consumption: Body weights for the high-dose males and females from the F0 and F1 generations were significantly reduced during most of the study. A significant reduction in food intake for the F0 and F1 generation was seen in high-dose males.
Reproductive Performance of F0 and F1 Males: Libido was unaffected and breeding performance was considered favorable for males from both generations.
Reproductive Performance of F0 and F1 Females: No meaningful alteration occurred in any reproductive parameter for dams exposed to 500 ppm or 2000 ppm of test article in the diet. There was a marked reduction in reproductive performance in the dams receiving the 7500-ppm level. The parameters affected included increased gestation length, the pregnancy rates, total pups born, litter size, number of implantations and birth index which were significantly reduced for the high-dose group.
Neonatal Growth and Development: There was no increase in the number of stillbirths for any dose group from either generation when compared to the controls. The high-dose level reduced neonatal survival and neonatal weight gain during lactation which were significantly reduced. Unilaterally, small testes were reported.
Microscopic Pathology: No changes occurred in the female reproductive tissues. Spermatic morphological alterations were seen in the high-dose males from both generations. Cecal dilatation, unilateral testicular atrophy and reduced prostatitis were observed in the various treatment groups. Histopathological evaluations of the pinnae were not conducted.
Conclusions: The Agency concluded a NOEL of 2000 ppm (165 mg/kg) for the study.
12. Additional Two-Generation Reproduction Study in Rats
a. Study Report Number: 73892
b. Study Completion: February, 1987 (start: May, 1986)
c. Study Director: R. L. Kowaski
Miles Laboratories
Elkhart, Indianad. Substance and Dosage Form: Technical drug substance; incorporated into feed
e. Species and Strain of Animal: Rat (Sprague-Dawley)
f. Number of Animals per Sex per Treatment Group: 120 in the F0 generation
g. Levels and Duration of Dosing: 0, 125, 300 and 2000 ppm over a duration of two generations
h. Route of Administration: Oral
i. Parameters Tested and Results:
Clinical Observations: No adverse behavioral effects or signs of toxicosis were attributed to the test article at any of the 3 concentrations.
Food Consumption: Food consumption was not affected by consumption of the test substance.
Reproductive Performance of Males: Male libido was unaffected by the dietary intake of the test compound.
Reproductive Performance of Females: No effect occurred upon performance. Estrus cycling and fertility in general (pregnancy rates, implantation, litter size) for animals in treated groups compared favorably with those of the controls.
Neonatal Growth and Development: No meaningful alterations occurred in neonatal development at any of the exposure levels. There was a slight, but significant decrease in body weight gain in the F2 generation.
Microscopic Pathology: The only definite test article-related change was seen in the testes and epididymides of mid- and high-dose F0 and F1 males. The change was characterized as abnormal spermatozoa.
Organ Weights: Epididymal weights of the high-dose group were decreased in both the F0 and F1 generation with the variation in the F1 being statistically significant.
Conclusions: No effects on fertility or reproductive performance were noted. The Agency concluded a NOEL of 125 ppm.
13. Rat Primary Hepatocyte Unscheduled DNA Synthesis Assay
a. Study Report Number: 73055
b. Study Completion: October, 1984 (start: July, 1984)
c. Study Director: M. A. Cifone
Litton Bionetics
Kensington, Marylandd. Substance and Dosage Form: Technical drug substance
e. Species and Strain of Animal: Rat (Fischer)
f. Number of Animals per Sex per Treatment Group: Not Applicable
g. Levels and Duration of Dosing: 1 µg/mL to 500 µg/mL
h. Route of Administration: Cell culture perfusion
i. Parameters Tested and Results:
The test material did not induce significant changes in the nuclear labeling of primary rat hepatocytes for an applied concentration range of 1 µg/mL to 200 µg/mL. Treatment with 500 µg/mL was completely lethal. No criteria used to indicate UDS were approached by the treatments and no dose-related response was observed.
Conclusions: The test material was concluded to be tentatively negative in the Primary Rat Hepatocyte UDS Assay.
14. Micronucleus Test on the Mouse to Evaluate for Mutagenic Effect a. Study Report Number: 74164
b. Study Completion: June, 1985 (start: December, 1984)
c. Study Director: B. Herbold
Institute for Toxicology
Wuppertal, Germanyd. Substance and Dosage Form: Technical drug substance; oral suspension
e. Species and Strain of Animal: Mouse (NMRI)
f. Number of Animals per Sex per Treatment Group: 5
g. Levels and Duration of Dosing: 0, 1000, 1500, and 2000 mg/kg; single dose. The test was conducted as 2 experiments as follows:
Experimental Group Dose (mg/kg) Sacrifice Time (hours) 1) Control 0 24 Bay Vp 2674 2000 24 Bay Vp 2674 2000 48 Bay Vp 2674 2000 72 Cyclophosphamide 20 24 2) Control 0 24 Bay Vp 2674 1000 24 Bay Vp 2674 1500 24 Bay Vp 2674 2000 24 Cyclophosphamide 20 24h. Route of Administration: Oral i. Parameters Tested:The micronucleus test detects possible mutagenic effect utilizing femoral marrow and specifically detects polychromatic erythrocytes containing micronuclei.
j. Results: Erythrocyte formation as measured by the ratio of polychromatic to normochromatic erythrocytes, was adversely affected in the 72-hour group of the first experiment. No indications of a relevant mutagenic effect with Bay Vp 2674 were found following a single dose of up to 2000 mg/kg.
k. Conclusions: The Agency reserved comment and final conclusions. 15. Mammalian Cell Chromosomal Aberration Mutagenicity Assay
a. Study Report Number: 74165
b. Study Completion: June, 1988 (start: December, 1987)
c. Study Director: R. Taalman
Hazleton Biotechnologies
Veenendaal, Netherlandsd. Substance and Dosage Form: Technical drug substance; in vitro
e. Species and Strain of Animal: Chinese Hamster Ovary Cells (CHO-WBI)
f. Number of Animals per Sex per Treatment Group: Not applicable
g. Levels and Duration of Dosing: Non-activated 25.0, 50.0, 100.0, and 250.0 µg/mL; activated 1.0, 100.0, 250.0, and 500.0 µg/mL.
h. Route of Administration: Infusion into cell culture.
i. Parameters Tested and Results:
The assay evaluated for clastogenic activity in the Chinese Hamster Ovary Cells measuring chromosomal aberrations using duplicate cultures in the presence and absence of a metabolic activation system.
The test article was considered positive for chromosomal aberration induction under the non-activation conditions. In the activation part of the assay, the test article did not exhibit clastogenic activity.
j. Conclusions: The Agency concluded the test substance to be positive for mutagenicity activity in this assay.
16. Rat Bone Marrow Chromosomal Aberration Mutagenicity Assay a. Study Report Number: 74166
b. Study Completion: November, 1986 (start: July, 1986)
c. Study Director: J. Boatman
Life Science Research Limited
Suffolk, Englandd. Substance and Dosage Form: Technical drug substance; suspension
e. Species and Strain of Animal: Rat (CD)
f. Number of Animals per Sex per Treatment Group: 5 and/or 15
g. Levels and Duration of Dosing: 0, 40, 200, and 1000 mg/kg; single dose
h. Route of Administration: Oral gavage
i. Parameters Tested and Results:
The effect of Bay Vp 2674 on chromosomal structure was investigated in bone marrow cells of the rat following acute oral administration. Two hours before the surviving animals were killed, bone marrow cell division was arrested at metaphase by the administration of a spindle poison. At termination, bone marrow cells were harvested and metaphase chromosome spreads were prepared on microscope slides. The mitotic index was calculated for each animal, giving an indication of the gross toxicity to dividing bone marrow cells. Fifty metaphases were examined from each animal and all observed chromosomal aberrations were recorded.
j. Conclusions: This assay was sensitive to the positive control chlorambucil, but no significant damage to chromosomal structure in rat bone marrow was seen following administration of enrofloxacin.
17. Sister Chromatid Exchange Mutagenicity Assay a. Study Report Number: 74178
b. Study Completion: April to August, 1987 and October, 1987 to February, 1988
c. Study Director: B. Herbold
Institute for Toxicology
Wuppertal, Germanyd. Substance and Dosage Form: Technical drug substance; suspension
e. Species and Strain of Animal: Hamster (Chinese)
f. Number of Animals per Sex per Treatment Group: 5
g. Levels and Duration of Dosing: 2000, 4000, and 8000 mg/kg; single treatment
h. Route of Administration: Oral, stomach tube
i. Parameters Tested and Results:
This in vivo test system evaluates the induction of sister chromatid exchange (SCE) in the bone marrow of Chinese hamsters. Cyclophosphamate was used as a positive control. The evaluation was conducted in two independent phases due to an indication of a dose-dependent trend in the initial evaluation. The values of the second phase did not suggest a dose-dependent trend, but there was a significant difference (P <= 0.05) between the negative control and the 4000-mg/kg group.
Conclusions: The Agency chose to reserve comment regarding the adequacy of the test as performed and the results obtained.
18. Salmonella/Microsome Assay to Evaluate for Point-Mutagenic Effects (Ames Test)
a. Study Report Number: 72213
b. Study Completion: December, 1984 (start: November, 1984)
c. Study Director: B. Herbold
Institute for Toxicology
Wuppertal, Germanyd. Substance and Dosage Form: Technical drug substance; agar/bacterial cell culture
e. Species and Strain of Animal: Salmonella typhimurium (LT-2); TA 1535, TA 100, TA 1537, TA 98
f. Number of Animals per Sex per Treatment Group: Not applicable
g. Levels and Duration of Dosing: 8, 40, 200, and 1000 µg per plate
h. Route of Administration: Agar/plate inoculation
i. Parameters Tested and Results:
Bay Vp 2674 was tested in the Ames Assay for the induction of bacterial mutation using four histadine-auxotrophs of Salmonella typhimurium. The stated drug concentrations were evaluated with and without the S-9 metabolic activator. Positive controls were used.
Conclusions: The Agency concluded that this microbiological assay did not satisfy the criteria for a valid test to assess the mutagenicity of an antimicrobial compound.
19. Study of Chronic Toxicity and Carcinogenicity in Mice a. Study Report Number: 74229
b. Study Completion: October, 1988 (start: October, 1986)
c. Study Director: E. Bomhard
Institute for Toxicology
Wuppertal, Germanyd. Substance and Dosage Form: Technical drug substance; incorporated into feed
e. Species and Strain of Animal: Mouse (B6C3F1)
f. Number of Animals per Sex per Treatment Group: 50
g. Levels and Duration of Dosing: 0, 1000, 3300, and 10,000 ppm for 24 months
h. Route of Administration: Oral
i. Parameters Tested and Results:
Ten additional mice of both sexes were assigned to the above stated treatments, plus 10 of each sex at 20,000 ppm for interim sampling at 12 months.
Inspection of Animals: The body surfaces, orifices, eyes, general behavior, posture, breathing and excretion products of the treated animals did not show any unusual features compared to the controls when inspected twice daily.
Ophthalmological Investigations: No evidence of treatment induced damage to the eyes in the groups receiving up to and including 3300 ppm at the end of the study. The females in the 10,000-ppm group showed a clearly elevated incidence of focal lenticular opacity. No histological correlation could be assigned to the effect.
Mortality: A slightly higher increase in mortality of the males in the 3300-ppm group and females in the 10,000 ppm- group was observed.
Body Weights: Body weights were determined once a week with animals in the 2 lower dosed groups being significantly higher than the controls with the 10,000-ppm group occasionally higher and the 20,000-ppm group comparable to the controls.
Food Consumption: Intake was monitored once a week, with no difference in groups receiving up to and including 10,000 ppm and the control.
Hematology: The total white blood cell counts were decreased in the 3300- and 10,000-ppm groups for the males and in the 3300-ppm group for the females. Significantly lower hemoglobin, hematocrit, MCV and MCH values were seen at the 10,000 ppm level.
Clinical Chemistries: Samples obtained after 12 and 24 months showed lower alkaline phosphatase levels at the 3300 and 10,000 ppm levels. A dose dependent trend was observed in total protein values with the reduction in total plasma protein attributed to a decrease in the globulin fraction.
Gross Pathology: No specific findings were observed at the 12-month sacrifice. After 24 months, an incidence of cecal dilation was observed which increased with increasing dose. Males treated at 1000 ppm had an incidence of 4.0%, 42.0% at 3300 ppm and 82.0% at 10,000 ppm. Females had an incidence of 26.0% at 3300 ppm and 64.0% at 10,000 ppm.
Organ Weights: No changes occurred in absolute or relative organ weights except for increases in kidney weights of females receiving 20,000 ppm.
Microscopic Pathology: On histopathological examination, there was neither an increase in the incidence nor a decrease in the time of appearance of tumors in the dosed groups compared to the controls. Intrahepatic bile-duct changes were detected in the 3300- and 10,000-ppm dose groups. Focal papillary mucosal hyperplasia in the gall bladder was observed in the 3300-ppm males and the 10,000- ppm females.
Conclusions: The Agency concluded a NOEL of 1000 ppm (323 mg/kg) for all effects. There was no evidence of carcinogenic effect in mice dosed with 1000 to 10,000 ppm enrofloxacin for two years.
20. Study of Chronic Toxicity and Carcinogenicity in Rats a. Study Report Number: 74230
b. Study Completion: September, 1988 (start: August, 1986)
c. Study Director: E. Bomhard
Institute for Toxicology
Wuppertal, Germanyd. Substance and Dosage Form: Technical drug substance; incorporated into feed
e. Species and Strain of Animal: Rat (Wistar)
f. Number of Animals per Sex per Treatment Group: 60
g. Levels and Duration of Dosing: 0, 770, 2000, and 6000 ppm for 105 wk
h. Route of Administration: Oral
i. Parameters Tested and Results:
Additionally, 10 rats of both sexes were assigned to the above stated treatments, plus 10 of each sex at 10,000 ppm for interim sampling at 12 mo.
Inspection of Animals: The body surfaces, orifices, general behavior, posture, breathing and excretion products of the treated animals did not show any unusual features compared to the controls when inspected twice daily, with a detailed inspection once a week.
Mortality: Mortality increased for both males and females at the 6000-ppm level.
Body Weights: The body weight development of males was highly retarded at the 10,000-ppm level with a slight retardation at 6000 ppm. Slight growth reduction was recorded for females at 10,000 ppm. Body weights were recorded weekly until week 13 and then every 2 weeks.
Food Consumption: Intake was monitored once a week, with a significant increase for both males and females at the 10,000-ppm level. Levels up to and including 6000 ppm did not differ appreciably from the controls.
Hematology: Investigations were carried out after 6, 12 and 18 months and at the end of the study. Leukopenia was seen in the 770- to 10,000-ppm groups for both the males and females. Temporarily decreased erythrocyte, hemoglobin, hematocrit, and MCV values were observed at the higher doses.
Clinical Chemistries: Samples were obtained at 6, 12, and 18 months and at the end of the study. Total protein was significantly decreased in males at all doses and at all sampling intervals. Total protein decreases were less for females.
Urinalysis: Performed at 6 month intervals and overall, the results indicated the test substance did not lead to toxicologically relevant kidney damage.
Ophthalmological Investigations: No evidence of treatment-induced changes in the refractile media, the ocular fundus or the pupillary reflex after one year and at the end of the study.
Gross Pathology: A statistically significant increase in the incidence of hepatic cysts was found in both sexes after 2000- and 6000-ppm treatments and a biologically significant increase in males at 770 ppm. Both sexes showed a significant increase in the incidence of dilated cecum after 6000 ppm. Size of testes was reduced and consistency altered in male treatments at 2000 to 10,000 ppm.
Organ Weights: Absolute and relative liver weights were decreased in males at 2000 ppm and above. The same decreases were seen in the females at the interim, but not at the study termination. In the 6000-ppm males, absolute weights of the brain, testes and adrenals were significantly decreased, but the relative weights were no different than the controls.
Microscopic Pathology: The incidence of bile-duct hyperplasia in the liver increased in a dose-response in all treatment groups and both sexes. Sclerotic changes were statistically significant from 770 ppm in the males and from 2000 ppm in females. Cystic bile-duct hyperplasia increased with drug exposure from 770 ppm in the males and 2000 ppm in females. A marked increase in the incidence of cardiomyopathy in males at the 6000-ppm dose and in females dosed at 770 ppm and above. There was also a significant increase in the number of subendocardial proliferative lesions in males and females at the highest dose, as well as an elevated number of subendocardial mesenchymal tumors. There was a marked increase in the number of sarcolemmal nuclei in skeletal muscles of animals in the 6000-ppm treatments. This lesion is generally associated with degenerative changes in the muscle fibers. Marked degenerative changes were also noted in the sciatic nerve. Fewer females in the treated groups showed mammary alveolar development and milk secretions than the control animals.
Conclusions: The Agency concluded a NOEL was not established for the study due to incidence of bile duct hyperplasia and cardiomyopathy. There was no evidence of carcinogenic effect in rats dosed with 770 to 6000 ppm enrofloxacin for two years.
21. Chronic Toxicity Study in Rats After Administration in Feed Over a Period of Two Years
a. Study Report Number: 74387
b. Study Completion: July, 1991 (start: July, 1989)
c. Study Director: K. Leser
Institute for Toxicology
Wuppertal, Germanyd. Substance and Dosage Form: Technical drug substance; incorporated into feed
e. Species and Strain of Animal: Rat (Wistar)
f. Number of Animals per Sex per Treatment Group: 50
g. Levels and Duration of Dosing: 0, 100, and 500 ppm for 24 months
h. Route of Administration: Oral
i. Parameters Tested and Results:
Histological evaluation was limited only to the heart and liver. Additionally, 10 rats of each sex per group were used for an intermediate necropsy after 12 mo.
Inspection of Animals: Twice daily observations with a detailed inspection once a week did not detect abnormalities in body surfaces, orifices, general behavior, posture, respiration or excreta.
Mortality: No statistically significant differences were observed compared to the controls.
Body Weights: Weights were recorded weekly with treated rats having comparable gains to the corresponding controls.
Food Consumption: Feed intake was determined on individual animals weekly until Week 13 and then every 4 weeks. There were no significant differences compared to the controls.
Hematology: Investigations were conducted after 6, 12, 18, and 24 months on 10 rats per group. There was no evidence of treatment related damage.
Clinical Chemistries: Again, the samples were evaluated at 6, 12, 18, and 24 months. No correlation was observed between parameter values and dose or time in the study period.
Urinalysis: Performed in 10 rats per dose and per sex after 27, 52, 79, and 104 weeks were of no toxicological significance.
Ophthalmological Examinations: No evidence of any treatment related changes after one year, or at the end of the study.
Gross Pathology: No abnormalities were noted at the interim sacrifices. In the males at final necropsy, there was a trend toward an increased number of swollen livers for the males.
Organ Weights: No toxicological significance was attached to differences.
Microscopic Pathology: Sclerotic bile-duct hyperplasia was more common in males and females treated with enrofloxacin than the control animals. The incidence increased in males after 52 and 104 weeks of treatment at 100- and 500-ppm dose levels. By 104 weeks, there were as many affected males in the 100-ppm dose group and the lesions were of equal severity. As the incidence at 52 weeks is approximately the same as at 104 weeks, the administration of enrofloxacin accelerates the development of bile-duct sclerosis in male rats. In females, there was an increased incidence of bile-duct hyperplasia at 104 weeks in the 500-ppm dose group. The average severity increased in both the 100- and 500-ppm doses when compared to the control females. The total number of females affected was markedly less than the number of males. No evidence of carcinogenicity was seen in the hearts or livers in this study.
j. Conclusions: A NOEL for enrofloxacin had not been demonstrated by this study based on significant increases in the bile-duct hyperplasia in all of the exposure groups of male rats. The Agency set the NOEL at 100 ppm after considering the spectrum of data from all of the chronic rat studies and opinions submitted to CVM. The 100 ppm NOEL corresponded with doses of 5.3 and 7.2 mg enrofloxacin/kg bw for the male and female rats, respectively.
22. Evaluation of Bile Duct Hyperplasia in Male Rat Livers
a. Study Report Number: 74388
b. Study Completion: May, 1992
c. Study Director: R. Squire
Johns Hopkins School of Medicine
Baltimore, Marylandd. Substance and Dosage Form: Technical drug substance; incorporated into feed
e. Species and Strain of Animal: Rat (Wistar)
f. Number of Animals per Sex per Treatment Group: 50
g. Levels and Duration of Dosing: 0, 100, and 500 ppm for 24 months
h. Route of Administration: Oral
i. Parameters Tested and Results:
Dr. Squire conducted a second and independent histological evaluation of the liver slides for the male rats from both the interim and terminal kills for the previously described Study No. 74387. This assessment was arranged and conducted at the request of Bayer. Conclusions were: "There was an increase over controls in the incidence of bile duct hyperplasia in both dose groups at the interim kill, although this was probably a spurious finding. At the terminal kill, an increased incidence occurred only at the 500-ppm level. No clear effect on lesion severity was noted in any group. The 100-ppm dose was a no-effect level in this study."
23. Chronic Toxicity Study in Rats with Administration in Feed Over a Period of Two Years
a. Study Report Number: 74472
b. Study Completion: April, 1993 (start: April, 1991)
c. Study Director: K. Leser
Institute for Toxicology
Wuppertal, Germanyd. Substance and Dosage Form: Technical drug substance; incorporated into feed
e. Species and Strain of Animal: Rat (Wistar)
f. Number of Animals per Sex per Treatment Group: 50
g. Levels and Duration of Dosing: 0, 10, and 50 ppm for 24 months
h. Route of Administration: Oral
i. Parameters Tested and Results: Histopathological evaluation was limited only to the liver. A further 10 rats per group received the test substance in the same doses and were used for an interim necropsy after 12 months.
Inspection of Animals: Observations were conducted twice daily with a detailed inspection once a week. There were no treatment increases in the incidence of unusual findings.
Mortality: No influence of the test drug on mortality was suggested by the numbers of rats which died or by their distribution between the individual treatment groups.
Body Weights: Weights were recorded once a week with the treated animals comparable to the controls.
Food Consumption: Feed intake was determined once a week until Week 13 and then every 4 weeks. Quantities consumed were not substantially different from the values in the control rats.
Hematology: Samples were obtained on 10 rats per group after weeks 27, 79, and 104. The investigations yielded no evidence of damage to red or white blood cell parameters in either sex at any time.
Clinical Chemistries: Conducted at the intervals as per hematology investigations with no treatment-related changes.
Urinalysis: Performed on 10 rats per dose and per sex at the intervals stated for hematology investigations with no findings of toxicological significance.
Ophthalmological Investigations: The examination yielded no evidence of any treatment induced changes after 1 or 2 years of treatment.
Gross Pathology: There was no evidence of any treatment damage in the rats dying spontaneously, after 12 months or at the end of the study.
Organ Weights: No significant differences were seen in either absolute or relative organ weights.
Microscopic Pathology: Histological readings were conducted on one part of the left hepatic lobe and one part of the right hepatic lobe. The incidence and severity of bile duct hyperplasia in the treated female and male rats were comparable with the controls.
j. Conclusion: The Agency concluded that no apparent treatment-related effects were noted in Wistar rats administered 10 or 50 ppm enrofloxacin in the feed.
24. Pathology Working Group on a Two-Year Chronic Feeding Study with One-Year Interim Kill in Rats on the Compound BAY Vp 2674
a. Study Report Number: Not Applicable. This assessment by a Pathology Working Group was arranged and conducted at the request of the Agency. It included a review of the heart tissue from all of the rats of the chronic study conducted by Bayer (Report No. 74230).
b. Report Date: November, 1992
c. Study Director: W. Hall, V.M.D., Ph.D.
Pathology Associates, Inc.
Frederick, Marylandd. Conclusions: The endocardial neoplasms and Schwann cell-like hyperplasia were not considered by the Pathology Working Group to be related to treatment with the test article for the following reasons:
1) the incidence of endocardial neoplasia in male and female rats was not statistically significant (P>0.05) [the statistically positive trend in endocardial neoplasia in females was not considered to be biologically significant by the Pathology Working Group because of the low numbers (0/50 controls vs. 3/50 high-dose) and the reduced incidence in controls compared to historical data];
2) the increase in endocardial neoplasms seen in dosed male rats compared with controls did not increase with increasing dose;
3) the incidence of endocardial Schwann cell-like hyperplasia was low and did not support an increased incidence of endocardial neoplasia in high-dose rats [the statistically positive trend in Schwann cell-like hyperplasia in males and females was not considered to be biologically significant by the Pathology Working Group because of the low numbers (0/48 or 50 controls vs. 2/50 high dose)]; and
4) all other endocardial hyperplastic lesions were considered to be reactive in nature in response to an underlying myocardial lesion.
B. Safe Concentrations of Total Residue of Enrofloxacin
1. No-Observed-Effect-Levels (NOEL)
The Safe Concentrations (SC) of total residue of enrofloxacin were determined from the lowest NOEL in the most sensitive species tested in the various toxicologic studies conducted. Studies considered in establishing the Acceptable Daily Intake (ADI) are summarized in Table 6.1.
Table 6.1. No-Observed-Effect-Levels (NOEL) in toxicology studies for enrofloxacin
Study Study No-Observed-Effect Number Level (NOEL) Subchronic Oral Toxicity in Dogs No. 73775 3 mg/kg Chronic Toxicity and Carcinogenicity Study No. 74229 323 mg/kg in Mice Chronic Toxicity in Rats No. 74387 5.3 mg/kg Two-Generation Rat Reproduction Study No. 73892 125 mg/kg Embryotoxicity/Teratogenicity Study in the No. 73705 25 mg/kg RabbitThe Agency concluded that under the given exposure conditions in the two-year rodent studies, a significant increased frequency or distribution of tumors was not noted. The Subchronic Oral Toxicity Study in Dogs (Study No. 73775) was concluded to be the most appropriate study (based upon the lowest NOEL) to determine the Acceptable Daily Intake (ADI). The no-observed-effect-level (NOEL) for establishing the ADI is 3 mg/kg/day. 2. Calculation of the Acceptable Daily Intake (ADI) of Total Residue of EnrofloxacinLowest NOEL Acceptable Daily Intake (ADI) = --------------------------- Safety FactorA Safety Factor of 1000 was used because the NOEL was from a subchronic study.3 mg/kg of body weight/day The lowest NOEL is 3 mg/kg, so ADI = ---------------------------------- 1000 = 0.003 mg/kg of body weight/day3. Calculations of Safe Concentrations (SC): The calculation of the safe concentrations is based on the General Principles for Evaluating the Safety of Compounds used in Food-Producing Animals (FDA/CVM, revised July 1994).Safe Concentration (SC) = Acceptable Daily Intake (ADI) x Human Weight -------------------------------------------- Consumption ValueThe average human weight is approximated at 60 kg. The daily consumption values of edible tissues of poultry are approximated as 300 g for muscle, 100 g for liver, 50 g for fat, and 50 g for skin.SC (muscle) = .003 mg/kg bw/day x 60 kg body weight = 0.6 mcg/kg = 0.6 ppm -------------------------------------- 0.3 kg/day SC (liver) = .003 mg/kg bw/day x 60 kg body weight = 1.8 mcg/kg = 1.8 ppm -------------------------------------- 0.1 kg/day SC (fat) = .003 mg/kg bw/day x 60 kg body weight = 3.6 mcg/kg = 3.6 ppm -------------------------------------- 0.05 g/day SC (skin) = .003 mg/kg bw/day x 60 kg body weight = 3.6 mcg/kg = 3.6 ppm -------------------------------------- 0.05 g/dayTable 6.2. Safe Concentrations (SC) for total residues of enrofloxacin in edible tissues of poultry using the revised food consumption factors Edible Tissue Amount Consumed/Day Safe Concentration (SC) Muscle 300 g 0.6 ppm Liver 100 g 1.8 ppm Fat/Skin 50 g 3.6 ppmC. Total Residue Depletion and Metabolism Studies in Chickens and Turkeys;Bayer Report No.s: 106543-1, 106543, and 106753 for studies in chickens;
106544-1, 106544, and 106754 for studies in turkeys
Investigator(s): Dr. Abraham E. Mathew
Bayer Research Park
Stilwell, Kansas 66085Animals Used: Broiler Chickens (Peterson Hubbard), 22 days old at time of dosing; Turkeys (Nicholas Large Breasted), 11 weeks old at time of dosing
Drug was administered via oral gavage, three times a day, to chickens and turkeys for seven consecutive days at a dose rate equivalent to 50 ppm enrofloxacin in the drinking water. 14C-Enrofloxacin was used for the studies. The label was at a position (C-4 position of the quinolone ring) that is not susceptible to metabolism.
Sacrifice Intervals: Chickens: 6 (0-day), 10, 15, and 24 hours after the final dose. Turkeys: 6 (0-day), 10, 15, and 24 hours after the final dose.
Tissues Collected: Liver, muscle, and skin with adhering fat from at least eight (4 males, 4 females) chickens or at least four (2 males, 2 females) turkeys at each interval.
1. Total Residue Depletion in Chickens
Tissue samples from each of the sacrifice intervals were assayed for radioactivity to determine the [14C] total residue depletion. Mean concentrations of radioactivity (total residues) are summarized in Table 6.3.
Table 6.3. Mean concentrations (ppm ± standard deviation) of radioactive total residues in tissues of chickens following 7 days gavage with 14C-enrofloxacin at a dose equivalent to 50 ppm in drinking water ad libitum
Sacrifice Interval (Hours Post Final Dose) Tissue 6 (0-Day) 10 15 24 Liver 5.414 (±2.557) 0.675 (±0.260) 0.252 (±0.073) 0.089 (±0.036) Muscle 1.015 (±0.655) 0.107 (±0.046) 0.032 (±0.012) 0.009 (±0.002) Fat/Skin 0.531 (±0.352) 0.086 (±0.023) 0.039 (±0.036) 0.012 (±0.002)2. Metabolism of 14C-Enrofloxacin in Chickens Samples of the edible tissues of males and females from the 0-day (6 hours following the administration of the final dose) sacrifice interval were analyzed by high-performance liquid chromatography (HPLC), and the parent compound and 3 metabolites were found. Approximately 95% to 99% of the total radioactive residues was extracted from the 0-day liver, muscle, and skin with adhering fat tissues. The only significant (>10% of the total radioactive residues) compounds found were enrofloxacin and ciprofloxacin (the N-deethylated enrofloxacin).Table 6.4. Percentage of total radioactive residues (TRR) in edible tissues of chickens
Tissue Compound Sex Liver Muscle Fat/Skin enrofloxacin male 48 94 96 female 57 94 69 ciprofloxacin male 32 4 nd* female 24 4 13 *nd = not detected3. Total Residue Depletion in TurkeysTissue samples from each of the sacrifice intervals were assayed for radioactivity to determine the [14C] total residue depletion. The results are summarized in Table 6.5.
Table 6.5. Mean concentrations (ppm standard deviation) of radioactive total residues in tissues of turkeys following 7 days gavage with 14C-enrofloxacin at a dose equivalent to 50 ppm in drinking water ad libitum
Sacrifice Interval (Hours Post Final Dose) Tissue 6 (0-Day) 10 15 24 Liver 8.882 (±2.415) 7.028 (±0.679) 4.215 (±0.966) 3.047 (±0.529) Muscle 2.335 (±0.519) 1.432 (±0.183) 0.798 (±0.150) 0.432 (±0.042) Fat/Skin 0.949 (±0.225) 0.570 (±0.116) 0.362 (±0.049) 0.265 (±0.049)4. Metabolism of 14C-Enrofloxacin in Turkeys Samples of the edible tissues of a male and a female from the 0-day (6 hours following the administration of the final dose) sacrifice interval were analyzed by HPLC, and the parent compound and 3 metabolites were found. Approximately 97% to 99% of the total radioactive residues was extracted from the 0-day liver, muscle, and skin with adhering fat tissues. The only significant (>10% of the total radioactive residues) compounds found were enrofloxacin and ciprofloxacin (the N-deethylated enrofloxacin). Results are summarized in Table 6.6.Table 6.6. Percentage of total radioactive residues (TRR) in edible tissues of turkeys
Tissue Compound Sex Liver Muscle Fat/Skin enrofloxacin male 60 99 97 female 58 99 94 ciprofloxacin male 13 nd* nd* female 13 nd* nd* *nd = not detectedD. Comparative Metabolism in the RatBayer Report No. 106547: Wistar Furth rats (WF/NHsd: males and nulliparous females) were treated with five daily oral gavage doses of [14C] enrofloxacin at a rate of 15 mg/kg/day, and urine was collected. The urine samples were assayed by HPLC. All four enrofloxacin-related residues detected in the edible tissues of broiler chickens and turkeys were present in the urine of rats ( see Table 6.7). Therefore, the rats used in the toxicity tests were exposed to all of the enrofloxacin metabolites observed in edible tissues of poultry. Results are summarized in Table 6.7.
Table 6.7. Percent of total radioactivity of enrofloxacin-related compounds in rat urine
Compound Percent of Total Radioactivity in Rat Urine enrofloxacin 17 ciprofloxacin 31 oxociprofloxacin 5 enro-conjugate (enro amide) 23 dioxociprofloxacin 9 desethylene ciprofloxacin 3 desethylene enrofloxacin 2 N-formyl ciprofloxacin <2 oxoenrofloxacin <2 hydroxy oxoenrofloxacin 3E. Determination of the Target Tissue and the Marker Residue Using the data in the radiolabeled residue studies summarized in Section C, liver was assigned as the target tissue and parent enrofloxacin was assigned as the marker residue for both chickens and turkeys treated with enrofloxacin.1. Target Tissue Determination
In Bayer Report No. 106543, it was determined that the amount of time required for the average total 14C-residue level in edible chicken tissues to deplete to the safe concentration was less than 10 hours for liver and muscle and 6 hours for skin with adhering fat. Total residue levels in chicken muscle and liver depleted to the safe concentrations at approximately the same time. Muscle was selected as the target tissue for chickens, because muscle is an easier matrix to analyze enrofloxacin residues than liver.
In Bayer Report No. 106544, it was determined that the amount of time required for the average total 14C-residue level in edible turkey tissues to deplete to the safe concentration was less than 24 hours for muscle and 6 hours for skin with adhering fat. Although total residue levels in liver did not deplete to the safe concentration within 24 hours, muscle was selected as the target tissue for turkeys based on the data obtained in the marker residue depletion study (Section G) and the regulatory method developed to determine enrofloxacin in poultry muscle. In addition, a marker residue tolerance can be established in muscle to monitor the safe concentration for liver residues.
2. Marker Residue Determination
HPLC analysis of the chicken and turkey muscle samples collected at all intervals after the administration of the final enrofloxacin dose indicated that unchanged enrofloxacin comprised the major portion of the extractable residues (Bayer Report Nos. 106753 and 106754). Thus, parent enrofloxacin was selected as the marker substance in poultry muscle.
F. Determination of the Tolerance for Enrofloxacin in Poultry Muscle
Using the determinative procedure for enrofloxacin in poultry muscle (Section G of this document), the enrofloxacin content in the muscle samples of poultry treated with 14C-enrofloxacin (Section C, above) was measured. Based on these assay results, a tolerance of 0.3 ppm enrofloxacin was assigned for both chickens and turkeys.
1. Tolerance for Enrofloxacin in Chicken Muscle
In Bayer Report No. 106753, muscle samples from 6 chickens (3 males, 3 females) for each of 6-, 10-, and 15-hour sacrifice intervals were used in the quantitation of radiolabeled enrofloxacin. In order to correlate the total 14C-residue levels in liver and muscle with radiolabeled parent enrofloxacin (the marker residue) in muscle of dosed chickens, average residue values from each set of 6 animals at each sacrifice interval were calculated as shown below:
Table 6.8. Mean concentrations (ppm) of total enrofloxacin residues in the livers and muscle of chickens following 7 days gavage with 14C-enrofloxacin at a dose equivalent to 50 ppm in drinking water ad libitum
Sacrifice Total [14C] Residues (ppm)a Parent Enrofloxacin in Muscle (ppm)b Interval Liver Muscle (Hours) 6 5.276 0.864 0.752 10 0.709 0.112 0.099 15 0.273 0.035 0.028 a Individual data taken from the total radioactive residue depletion study report (Bayer Report No. 106543). b Individual data taken from the metabolism study report (Bayer Report No. 106753).From plots of the above average values, a tolerance value in muscle of approximately 0.3 ppm was obtained when the chicken liver TRR (total radioactive residues) cross the safe concentration of 1.8 ppm.2. Tolerance for Enrofloxacin in Turkey Muscle
The total radioactive residue depletion study report (Bayer Report No. 106544) suggested that the TRR in turkey liver deplete to the safe concentration of 1.8 ppm after the muscle residues cross its safe concentration of 0.6 ppm. Therefore, the tolerance would be the concentration of parent enrofloxacin (marker residue) in turkey muscle (target tissue) when the total residue level in turkey liver (the slowest-depleting tissue) reaches the safe concentration of 1.8 ppm. Since the total radioactive residue depletion study did not continue until the average enrofloxacin-related residues in turkey liver decreased to below the safe concentration, the following procedures were employed in the estimation of a tolerance value for parent enrofloxacin in turkey muscle.
a) Based on the average of the percent of total radioactivity calculations presented in Bayer Report No. 106546 for enrofloxacin in liver, 59%, 55%, and 46% of total radioactivities measured in turkey livers were due to enrofloxacin at 6-, 12-, and 24-hour sacrifice intervals, respectively. During the marker residue depletion study under field conditions (Bayer Report No. 106542), unlabeled enrofloxacin concentrations of 2.37 ppm, 1.31 ppm, and 0.40 ppm were measured in the liver of one turkey at 6, 12, and 24 hours, respectively, after removal of the medicated drinking water (Bayer Report No. 106546). The liver marker residue concentrations were divided by the fraction of marker residue in total residues to estimate the TRR levels of 4.02 ppm, 2.38 ppm, and 0.87 ppm in turkey liver at 6-, 12-, and 24-hour sacrifice intervals, respectively.
b) The marker residue concentrations of 0.72 ppm, 0.40 ppm, and 0.11 ppm were determined at sacrifice intervals of 6, 12, and 24 hours, respectively, in the muscle of the same turkeys from which liver marker residues were quantitated (Bayer Report No. 106546). These muscle marker residue values and the estimated liver TRR values in a) were then plotted.
From these plots, a tolerance value of approximately 0.3 ppm enrofloxacin marker residue in turkey muscle was obtained when the estimated turkey liver TRR (total radioactive residues) cross the safe concentration of 1.8 ppm.
G. Determination of the Withdrawal Time
1. Bayer Report Nos.: 106541 for the marker residue depletion study in chickens;
106542 for the marker residue depletion study in turkeys
2. Investigator(s): Dr. Abraham E. Mathew
Bayer Research Park
Stilwell, Kansas 660853. Test Substance: 3.23% enrofloxacin solution prepared under Good Manufacturing Practices
4. Dose: 50 ppm enrofloxacin in the drinking water for seven days
5. Route of Administration: Oral in drinking water.
6. Species: Chickens (Peterson Arbor Acres), 24 days old at time of dosing;
Turkeys (Large White Nicholas), 10 weeks old at time of dosing
7. Sacrifice Intervals: Chickens: 6 (0-day), 12, 18, and 48 hours after the final dose.
Turkeys: 6 (0-day), 12, 18, 24, and 48 hours after the final dose.
8. Tissue Collected: Muscle from five (3 males/2 females or 2 males/3 females) chickens or turkeys at each interval.
9. Results: The muscle samples from the chicken and turkey studies were analyzed using the determinative procedure (Section H, below) to follow the depletion of enrofloxacin from the muscle of chickens and turkeys treated with enrofloxacin 3.23% concentrate in drinking water at the maximum proposed label use rate. The average residue levels are presented in Table 6.9.
Table 6.9. Mean concentration of enrofloxacin ( standard deviation) in chicken and turkey muscle following 7 days administration of enrofloxacin at 50 ppm in drinking water ad libitum
Species Withdrawal Interval (hr) Enrofloxacin Concentration (ppm) Chicken 6 (0-day) 0.885 (±0.176) 12 0.537 (±0.112) 18 0.313 (±0.099) 48 0.043 (±0.013) Turkey 6 (0-day) 0.906 (±0.160) 12 0.450 (±0.055) 18 0.290 (±0.118) 24 0.196 (±0.013) 48 0.052 (±0.007)The residue data were statistically analyzed using Center for Veterinary Medicine's criteria to determine, with 95% confidence, the length of time required for enrofloxacin residues to deplete to the tolerance (0.3 ppm) in 99% of all medicated chickens and turkeys. The withdrawal interval was determined to be 2 days for chickens and turkeys treated with 3.23% enrofloxacin concentrate in the drinking water at 50 ppm for seven days.
H. Regulatory Method
1. Analytical Method for the Determination and Confirmation of Enrofloxacin in Poultry Muscle
The determinative and confirmatory procedures for measuring enrofloxacin residues in treated chickens and turkeys consists of the extraction of the parent drug from muscle tissue. The quantitation of the enrofloxacin residue in the extract is by high performance liquid chromatography (HPLC) and fluorescence detection, and the confirmation of identity is by tandem liquid chromatography and mass spectrometry.
In the determinative procedure, ground muscle tissue is homogenized with ethanol:water:acetic acid (98:1:1) to extract the enrofloxacin residues. The homogenate is centrifuged and the supernatant collected. The enrofloxacin residues are isolated using a strong cation exchange solid phase extraction (SPE) column. The SPE column is washed with methanol and water, and the enrofloxacin residues eluted with concentrated aqueous ammonium hydroxide:methanol (1:3). The volume of the eluate is adjusted to 10 mL and a portion is evaporated to dryness and redissolved in the mobile phase. The solution is analyzed by HPLC, using a reversed phase C8 analytical column under isocratic conditions with excitation at 280 nm and measuring the fluorescence at 440 nm. The limit of quantitation for the determinative procedure is approximately 0.01 ppm.
In the confirmatory procedure, a sample is processed as above, through the point at which the residue is eluted from the SPE column and adjusted to a final volume of 10 mL. A portion of the extract is dried and dissolved in the mobile phase. The procedure uses HPLC for separation and electrospray mass spectrometry/mass spectrometry (ESI/MS/MS) with selective ion monitoring (SIM) to monitor three major product ions characteristic of enrofloxacin.
2. Method Validation
A method trial of the determinative and confirmatory procedures was satisfactorily completed by FDA.
3. Display of the Method
The validated regulatory analytical method for enrofloxacin (marker residue) in poultry muscle is on display in the Dockets Management Branch (HFA-305), Parklawn Building (Room 1-23), 12420 Parklawn Drive, Rockville, MD 20857. It is attached to this FOI Summary.
I. User Safety
User safety concerns associated with enrofloxacin have been satisfactorily addressed by establishing label warnings. In addition, a toll-free number is available on the label which users can call to report adverse events or to obtain Material Safety Data Sheets.
VII. AGENCY CONCLUSIONS
The data submitted in support of this NADA satisfy the requirements of Section 512 of the Federal Food, Drug, and Cosmetic Act and 21 CFR Part 514 of the implementing regulations. The data demonstrate that BAYTRIL® (enrofloxacin) 3.23% Concentrate Antimicrobial Solution, a fluoroquinolone antibiotic, when administered ad libitum in the drinking water at a concentration of 25 to 50 ppm to chickens and turkeys for 3 to 7 days, is safe and effective for the control of mortality associated with E. coli susceptible to enrofloxacin in chickens and for the control of mortality associated with E. coli and P. multocida (fowl cholera) susceptible to enrofloxacin in turkeys.
Based on a battery of toxicology tests, the safe concentrations for total enrofloxacin-related residues are 0.6 ppm in muscle, 1.8 ppm in liver, and 3.6 ppm in skin with adhering fat. Based on metabolism studies in chickens and turkeys and the measurement of unchanged enrofloxacin in turkey liver, a tolerance (Rm) of 0.3 ppm for the marker residue, parent enrofloxacin, has been established in chicken and turkey muscle. The tolerance (Rm) refers to the residue measured by the regulatory method described herein.
A pre-slaughter withdrawal period of 2 days for both species was calculated from the results of the marker residue depletion studies in chickens and turkeys, following the oral administration of Baytril® 3.23% Concentrate Antimicrobial Solution. The 2-day withdrawal time was based on a statistical analysis of the depletion data, using an upper tolerance limit containing 99 percent of the population with a 95 percent confidence limit.
Labeling restricts this drug to use by or on order of a licensed veterinarian. This decision was based on the following factors: a) adequate directions cannot be written to enable lay persons to appropriately diagnose and subsequently use this product to control mortality associated with E. coli or P. multocida organisms, (b) restricting this drug to use by or on order of a licensed veterinarian should help prevent indiscriminate use which could result in violative tissue residues, and (c) the rate of emergence of enrofloxacin-resistant organisms may be reduced by the involvement of veterinarians in product use.
Public health concerns associated with potential increases in antimicrobial resistance have been satisfactorily addressed by establishing conditions of use intended to minimize inappropriate use of this product and to minimize excretion of drug and drug-resistant zoonotic pathogens into the environment. In addition, the sponsor has agreed to participate in an antimicrobial resistance surveillance program.
The agency has carefully considered the potential environmental effects of this action and has concluded that the action will not have a significant impact on the human environment and that an environmental impact statement is not required. The agency's finding of no significant impact (FONSI) and the evidence supporting that finding are contained in an environmental assessment, which may be seen in the Docket Management Branch (HFA- 305), Parklawn Building (Room 1-23), 12420 Parklawn Dr., Rockville, Maryland 20857.
Under section 512(c)(2)(F)(ii) of the Federal Food, Drug, and Cosmetic Act, this approval for food producing animals qualifies for THREE years of marketing exclusivity beginning on the date of approval because the application contains reports of new clinical or field investigations and new human food safety studies essential to the approval of the application and conducted or sponsored by the applicant.
BAYTRIL® 3.23% Concentrate Antimicrobial Solution is under patent numbers U.S. 4,670,444, and 5,077,429, expiring June 2, 2004, and December 31, 2008, respectively.
VIII. APPROVED LABELING
A copy of the draft facsimile labeling is attached to this document.
A. Baytril® 3.23% Concentrate Antimicrobial Solution Quart Sleeve Label
B. Baytril® 3.23% Concentrate Antimicrobial Solution Quart Shipper Stencil Label
C. Baytril® 3.23% Concentrate Antimicrobial Solution Gallon Sleeve Label
D. Baytril® 3.23% Concentrate Antimicrobial Solution Gallon Shipper Stencil Label
E. Baytril® 3.23% Concentrate Antimicrobial Solution Package Insert
Copies of these labels and the appendix may be obtained by writing to the:
Freedom of Information Office
Center for Veterinary Medicine, FDA
7500 Standish Place
Rockville, MD 20855