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U.S. Department of Health and Human Services

Animal & Veterinary

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NADA 139-075 CYGRO; 1% Type A Medicated Article - original approval

Approval Date: February 2, 1989

I. GENERAL INFORMATION:

NADA 139-075
Sponsor: American Cyanamid Company
Generic Name: maduramicin ammonium
Trade Name: CYGRO; 1% Type A Medicated Article
Marketing Status: Over the Counter (OTC)

II. INDICATIONS FOR USE

For use in the manufacture of broiler chicken feeds. For the prevention of coccidiosis in broiler chickens caused by Eimeria acervulina, E. tenella, E. brunetti, E. maxima, E. necatrix and E. mivati.

III. DOSAGE FORM(S), ROUTE OF ADMINISTRATION AND RECOMMENDED DOSAGE

CYGRO® is a Type A Medicated Article (premix) containing 1% maduramicin ammonium active ingredient.

Dosage and Administration:

Thoroughly mix CYGRO 1% Type A Medicated Article into the feed at a rate of 1 to 1.2 lb per ton of broiler feed to obtain a level of 4.54 to 5.45 g/ton (5 ppm to 6 ppm) maduramicin ammonium in Type C medicated feed. Feed continuously as the sole ration to broiler chickens from one day of age until 5 days before slaughter.

IV. EFFECTIVENESS - Pivotal Studies

a) Establishment of Dose

i) Field Isolate Battery Trials

Seventy-three field isolates were evaluated in 41 battery trials totaling 4,305 cockerel chickens, to titrate various levels of maduramicin against single or mixed infections of Eimeria spp. recently collected from commercial poultry farms at various geographical locations in North America. Each sample was analyzed to determine the major and minor Eimeriasp. which were present. These trials were conducted by Dr. H. Berger at the Agricultural Research Center of American Cyanamid Company in Princeton, New Jersey.

Treatments consisted of noninfected, nonmedicated control (NNC), infected, nonmedicated control (INC) and infected, medicated treatments of 2, 4, 5, 6 or 7 ppm maduramicin. The treatments were replicated three times in each trial, with 5 birds per replicate. Ten-day-old broiler cockerels were infected with a pretitrated inoculum from the field samples which contained a single or mixed Eimeria spp. Weight gains for the 3 to 7 days postinfection, mortality (coccidiosis and noncoccidiosis related), and coccidial lesion scores on termination of the study were determined.

A summary of the data is given in Table 1.

The data were analyzed using Linear Plateau Model Analysis. The statistical summary of the 41 trials considered coccidial lesion scores 7 days after infection. Using these parameters, it can be concluded that maduramicin is an effective anticoccidial against the 6 pathogenic species of Eimeria named and that the optimum dosage range is 5 - 6 ppm.

(Eds. note: The following table consists of 9 columns.)

Table 1

                  Mean          ---------------Lesion Scores[3]-------------
             Weight Gain[2]    E.A.      E.M.     E.T.    E.B.    E.N.   E.Mi. Mortality[4]
Treatment[1]      (g)          (32)      (17)     (18)    (3)     (3)     (1)     (%)
NNC                127.91          --         --        --       --       --       --
INC                54.12           2.79       2.54      2.93     3.09     2.84     2.60     25.70
maduramicin        89.46           2.48       2.00      2.14     2.56     l. 80    2.07     1.37
ammonium (2 ppm)
maduramicin        112.94          1.88       1.54      1.27     1.44     1.64     1.73     0.00           
ammonium (4 ppm)
maduramicin        116.93          1.53       1.31      0.96     1.16     l. 36    1.00     0.00           
ammonium (5 ppm)                                                                                           
                                                                                                           
maduramicin        119.19          1.21       1.01      0.66     0.87     1.04     1.27     0.00           
ammonium (6 ppm)
maduramicin        117.89          1.04       0.96      0.49     1.16     1.33     1.47     0.00           
aminomum (7 ppm)

[1] NNC Is noninfected, nonmedicated control. INC is infected, nonmedicated control.

[2] Average weight gain (g) 3-7 days postinoculation.

[3] Lesion scores per bird 7 days after challenge. Maximum possible score is 4. The number in parentheses below each species indicate the number of each species isolated and tested (E.A., Eimeria acervulina/E. mivati/E. mitis complex; E. M., E. maxima; E.T., E. tenella; E.B, E. brunetti E.N, E. necatrix and E.Mi., E. mitis).

[4] Average % mortality due to coccidiosis in 19 of 41 studies that coccidiosis related mortality occurred in one or more treatments.

MAS#310
Rev. 7/14/87

4. Effectiveness (Con't)

b) Floor pen trials

i) Challenge studies

Summary:
Five challenge floor pen studies were conducted at various geographical locations in the U.S. to evaluate the anticoccidial effect of maduramicin in broiler chickens challenged with recently collected field isolates of Eimeria maxima, E. tenella, E. acervulina, E. necatrix, E. brunetti,and E. mivati. These birds were maintained under use conditions to provide a simulated field evaluation of the effectiveness of maduramicin.

Broiler chickens in the five studies were exposed to cocddial challenge at 14 days of age by feed and/or litter. Lesion scores were taken at 7 and 14 days postinfection (21 and 28 days of age).

In trials 1, 2 and 3, an infected, non-medicated control group was compared with maduramicin at four dosage levels (4, 5, 6 and 7 ppm). Trial 1 added an additional group of birds treated with maduramicin at 8 ppm, and trial 4 did not have a 7 ppm treatment.

The treated groups received anticoccidial medication in the diets from 0-44 days of age, followed by a 5-day drug withdrawal period on non-medicated feed prior to final weights. (These birds were discarded and did not enter the human food chain).

The data from these five (5) trials are summarized in Tables 2-6

In conclusion, maduramicin at 5 to 7 ppm in the diet effectively controlled coccidial challenge. The optimum dose range is concluded to be 5 to 6 ppm, since no significant improvement in lesion control was seen from 6 to 7 ppm.

(Eds. note: The following table consists of 16 columns.)

TABLE 2
TRIAL 1
Larry R, McDougald
University of Georgia
Athens, Ga, 30002
Number of birds fed CYGRO Premix: 1000
Number of Control Birds: 200
                                                                                                                                          AVERAGE             AVERAGE
                                                                                                                                          WEIGHT             FEED/GAIN
                                   ---------------------------AVERAGE LESION SCORE-----------------------------     TOTAL AVERAGE         GAIN (g)
                   % MORTALITY    UPPER INTESTINE     MIDDLE INTESTINE     LOWER INTESTINE           CECUM          LESION SCORE     0-44     0-49        0-44      0-49
TREATMENT           0-49 DAYS    21 Days   28 Days   21 days    28 days   21 days   28 days    21 days   28 days 21 days   28 days   days     days        days      days
Infected                                                                                                                                                                                                    
non-medicated             8.5         3.19        2.38        2.88        1.88        1.06        0.13        2.31        0.63      9.44        5.00        1525        1784        2.36        2.41        
maduramicin (4 ppm)       0.0         2.81        1.31        2.25        0.88        0.19        0.06        1.50        0.75      6.75        3.00        1627        1963        2.17        2.19        
maduramicin (5 ppm)       0.0         2.63        1.19        2.63        0.75        0.50        0.13        1.25        0.19      7.00        2.25        1723        2017        2.15        2.21        
maduramicin (6 ppm)       0.0         2.50        1.06        2.19        0.88        0.31        0.00        0.69        0.88      5.69        2.81        1675        2041        2.14        2.13        
maduramicin (7 ppm)       0.0         2.00        1.19        1.75        0.69        0.13        0.00        1.00        0.88      4.88        2.75        1694        2016        2.10        2.15        
maduramicin (8 ppm)       0.0         2.13        0.38        2.31        0.13        0.13        0.06        1.31        0.50      5.88        1.06        1679        1978        2.08        2.15      

COMMENTS: Only coccidiosis related mortality is shown Average lesion scores have a maximum score of 4.00 Total average lesion scores have a maximum score of 16. Challenged with Eimeria field strains of E. tenella, E. necatrix, E. maxima, E. brtunetti, E. acervulina, and E. mivatiat 14 days of age.

(Eds. note: The following table consists of 16 columns.)

TABLE 3
TRIAL 2
Carey L. Quarles
Colorado Quality Research
Ft. Collins, CO 80524
Number of birds fed CYGRO Premix: 600
Number of Control Birds: 150
                                                                                                                                              AVERAGE             AVERAGE
                                                                                                                                              WEIGHT             FEED/GAIN
                                   ----------------------------AVERAGE LESION SCORE------------------------              TOTAL AVERAGE        GAIN (g)
                   % MORTALITY    UPPER INTESTINE      MIDDLE INTESTINE     LOWER INTESTINE            CECUM             LESION SCORE     0-44      0-49      0-44     0-49
TREATMENT           0-49 DAYS    21 Days   28 Days    21 days    28 days   21 days   28 days   21 days   28 days    21 days     28 days   days      days     days      days
Infected
non-medicated            60.0         2.58        1.42        2.58        0.75        1.42        0.17        3.33        1.00        9.9           3.3         1285        1495        3.37        3.35        
maduramicin (4 ppm)       0.0         1.67        0.25        0.58        0.00        0.00        0.00        1.50        0.08        3.8           0.3         1481        1728        2.05        2.09        
maduramicin (5 ppm)       0.0         0.83        0.17        0.33        0.00        0.25        0.08        0.50        0.00        1.9           0.3         1515        1718        2.05        2.10        
maduramicin (6 ppm)       0.0         0.17        0.08        0.08        0.00        0.00        0.00        0.42        0.00        0.7           0.8         1450        1752        2.08        2.06        
maduramicin (7 ppm)       0.6         0.17        0.00        0.25        0.00        0.00        0.00        0.00        0.00        0.4           0.0         1393        1675        2.12        2.11        
COMMENTS:

Only coccidiosis related mortality is shown Average lesion scores have a maximum score of 4.00 Total average lesion scores have a maximum of 16. Challenged with Eimeria field strains of E. tenella, E. Necatrix, E. maxima, E. brunetti, E. acervulina, and E. mivati at 14 days of age.

(Eds. note: The following table consists of 16 columns.)

TABLE 4
TRIAL 3
S. A. Edgar
Auburn University
Auburn, AL 36849
Number of birds fed OYGRO Premix: 800
Number of Control Birds: 200                                                                                                                                             AVERAGE            AVERAGE
                                                                                                                                             WEIGHT             FEED/GAIN
                                   ------------------------------AVERAGE LESION SCORE---------------------           TOTAL AVERAGE           GAIN (g)
                     % MORTALITY  UPPER INTESTINE     MIDDLE INTESTINE     LOWER INTESTINE         CECUM             LESION SCORE        0-44       0-49      0-44      0-49
TREATMENT            0-49 DAYS   21 Days   28 Days   21 days    28 days   21 days    28 days   21 days   28 days     AIC        ACC      days       days      days      days
Infected
non-medicated             30.5        0.84        0.09        0.56        0.06        0.31        0.00        2.59        0.03          1.81        2.59        1403        1693        2.24        2.23        
maduramicin (4 ppm)       10.0        0.72        0.28        0.16        0.06        0.06        0.16        1.25        0.06          0.94        1.25        1544        1793        2.00        2.09        
maduramicin (5 ppm)        4.0        0.47        0.47        0.00        0.25        0.19        0.22        0.97        0.09          0.69        0.97        1571        1839        1.98        2.02        
maduramicin (6 ppm)        4.0        0.59        0.13        0.38        0.06        0.13        0.06        0.53        0.13          1.19        0.53        1557        1834        1.94        2.00        
maduramicin (7 ppm)       13.0        1.16        0.16        0.34        0.00        0.16        0.09        0.94        0.13          1.75        0.94        1589        1784        1.98        2.10      

COMMENTS: Only coccidiosis related mortality is shown AIC is acute intestinal coccidiosis and ACC is acute cecal coccidiosis lesion scores/bird at 21 days of age. Average lesion scores have a maximum score of 4.00 The maximum possible score is 4 for ACC and 12 for AIC. Challenged with Eimeria field strains of E. tenella, E. necatrix, E. maxima, E. brunetti, E. acervulina and E. mivati at 14 days of age.

(Eds. note: The following table consists of 16 columns.)

TABLE 5
TRIAL 4
T. J. Kennedy
A.E.F. Research. Inc.
Waunakee. WI 53597
Number of birds fed CYGRO Premix: 600
Number of Control Birds: 200                                                                                                                                               AVERAGE            AVERAGE
                                                                                                                                               WEIGHT            FEED/GAIN
                                     ----------------------------AVERAGE LESION SCORE-----------------------          TOTAL AVERAGE            GAIN
                    % MORTALITY    UPPER INTESTINE    MIDDLE INTESTINE      LOWER INTESTINE           CECUM           LESION SCORE        0-44      0-49      0-44      0-49
TREATMENT            0-49 DAYS    21 Days   28 Days   21 days   28 days    21 days    28 days   21 days    28 days  21 days   28 days     days      days      days      days
Infected
non-medicated              31.5         3.18        1.68        2.73        1.90        2.10        0.95        4.00        0.70        12.0        5.2         1750        1960        2.76        2.86        
maduramicin (4 ppm)         1.0         2.10        0.78        1.95        1.03        0.70        0.70        2.70        0.35        7.4         2.9         1810        2000        2.32        2.50        
maduramicin (5 ppm)         0.0         1.95        0.33        1.63        0.70        0.45        0.28        1.90        0.00        5.9         1.3         1830        2020        2.24        2.44        
maduramicin (6 ppm)         0.0         1.65        0.28        1.40        0.60        0.28        0.33        1.33        0.08        4.6         1.3         1800        2000        2.23        2.41      

COMMENTS: Only coccidiosis related mortality is shown Average lesion scores have a maximum score of 4.00 Total average lesion scores have a maximum score of 16. Challenged with Eimeria field strains of E. tenella, E. necatrix, E. maxima, E. brunetti, E. acervulina, and E. mivati at 14 days of age.

(Eds. note: The following table consists of 16 columns.)

TABLE 6
TRIAL 5
J. D. Kobland
Agricultural Research Center
American Cyanamid Company
Princeton, NJ 08540
Number of birds fed CYGRO Premix: 1408
Number of control birds: 352                                                                                                                                            AVERAGE         AVERAGE
                                                                                                                                            WEIGHT          FEED/GAIN
                                   ---------------------------AVERAGE LESION SCORE------------------------            TOTAL AVERAGE         GAIN(g)
                  % MORTALITY     UPPER INTESTINE     MIDDLE INTESTINE    LOWER INTESTINE           CECUM            LESION SCORE      0-44       0-49     0-44       0-49
TREATMENT          0-49 DAYS    21 Days    28 Days   21 days    28 days  21 days    28 days   21 days    28 days  21 days    28 days   days       days     days       days
Infected
non-medicated            53.7         1.81        0.00        2.75        0.00        2.19        0.00        3.00        0.19        9.75        0.19        1473.1      1784.1      2.51        2.44        
maduramicin (4 ppm)       1.4         2.06        0.00        1.94        0.00        0.88        0.00        2.50        0.06        7.38        0.06        1802.9      2071.0      1.84        1.92        
maduramicin (5 ppm)       0.9         0.94        0.00        2.13        0.00        0.56        0.00        1.69        0.00        5.32        0.00        1786.2      2104.2      1.85        1.92        
maduramicin (6 ppm)       0.3         0.38        0.06        0.69        0.00        0.00        0.00        0.69        0.00        1.42        0.06        1784.3      2107.7      1.84        1.89        
maduramicin (7 ppm)       0.9         1.00        0.13        1.69        0.00        0.19        0.00        0.31        0.00        3.19        0.13        1700.8      2039.7      1.87        1.88      

COMMENTS: Only coccidiosis related mortality is shown Average lesion scores have a maximum score of 3.00 Total average lesion scores have a marlmum score of 12. Challenged with Eimeria field strains of E. tenella, E. necatrix, E. brunetti, E. acervulina, and E. mivati at 14 days of age.

V. ANIMAL SAFETY

- Pivotal Studies

a) Target Animal (broiler chicken) Safety

i) Floor Pen Safety Study in Broiler Chickens

Investigator:

P.E. Gingher
Agricultural Research Center
American Cyanamid Company
Princeton, New Jersey 08540

In this pivotal study broiler chickens were grown to market age (49 days) under simulated commercial conditions and fed dietary levels of 0, 5, 6, 7.5, 9 and 15 ppm of maduramicin ammonium. Medication was fed from 0-44 days and followed with a 5-day medication withdrawal period. There were 8 replicates of 100 chickens of equal sex in each group.

Parameters for determining the safety of maduramicin ammonium in broiler diets were growth, feed efficiency, mortality, hematology (hemoglobin, hematocrit, RBC and clotting times) and histopathology of various body organs and tissues as well as effects on pigmentation and feathering.

The Linear Plateau Model analysis of 49-day weight gains indicated that in the absence of coccidiosis there was a significant dose related decrease in weight gains (P < 0.05).

Feed efficiency, as determined by feed gain ratios was comparable for all groups at the end of 49 days, except that the 15 ppm level of maduramicin ammonium significantly impaired feed efficiency during the medication period.

Percent mortality for the 9 and 15 ppm groups was higher than for all other groups by 49 days.

There were no effects on hemoglobin, hematocrit or red blood cell counts in males or females fed maduramicin ammonium for 44-45 days at levels up to 7.5 ppm. Reduced blood values were seen in males at the 9 and 15 ppm levels andfemales at 15 ppm at the 44-45 day period.

Blood clotting times ranged from a low of 145 to a high of 1355 seconds, about the normal variation associated with chicken blood. There were no apparent trends with regard to treatment at the 44-45 day sampling.

Histopathology of various tissues indicated that changes ascribable to maduramicin ammonium were apparent in the bursa of Fabricius and thymus. Partial atrophy of the bursa of Fabricius and of the thymus was an observable effect at 9 ppm (earliest) and 15.0 ppm (highest frequency). No other treatment related microscopic changes were observed. There was no adverse drug related effect on pigmentation. At levels above 5 ppm there were reduced floor feathers at the end of the experiment which is indicative of feather eating by broilers.

The production data from the trial can be summarized as follows:

(Eds. note: The following table consists of 7 columns.)

Table 7
               Weight Gain                                   Avg.
             Average (g/bird)        Feed Gain           % Mortality
Treatment  0-44 Days 0-49 Days  0-44 Days 0-49 Days  0-44 Days 0-49 Days
Nonmedicated  1867.1      2168.6      1.96        2.04        2.3         2.4
maduramicin   1807.3      2092.4      1.97        2.05        1.9         1.9
ammonium
(5 ppm)
maduramicin   1738.8      2018.5      1.99        2.05        2.6         3.4
ammonium
(6 ppm)
maduramicin   1686.4      1967.2      2.01        2.06        2.1         2.4
ammonium
(7.5 ppm)
maduramicin   1629.0      1962.8      1.98        2.01        2.4         3.0         
ammonium
(9.0 ppm)
maduramicin   980.4       1317.2      2.12        2.00        4.5         6.6         
ammonium
(15.0 ppm)

ii. Immunocompetency Study

In order to confirm that there was no biological significance to the histological changes observed in the bursa of Fabricus and thymus in the Floor Pen Safety study an additional study was conducted to determine immunocompetency of broiler chickens fed dietary concentration of maduramicin ammonium at 5, 7, 10 or 15 ppm in the starter diets during the first 3 weeks followed by 5 - 7 ppm of maduramicin ammonium during the remaining study period of 22 - 41 days. Since the Newcastle Disease (ND) involves both the humoral and cell-mediated immunity of chickens(1) the ND vaccine was selected to test the immunocompetency of maduramicin- medicated chickens. Newcastle Disease vaccine was given in drinking water at 7 and 21 days of age. A group of chickens served as the nonmedicated, nonvaccinated control and another group served as the nonmedicated, vaccinated control. At 31 days of age, all birds were challenged with a pretitrated dose of ND virus. Blood samples were taken from each bird at 7, 21 and 31 days of age for hemaglutination inhibition (HI) test. Mortality, morbidity, weight gain and feed intake were measured during the ND challenge period.

The HI titers at 21 and 31 days show that maduramicin ammonium medicated birds developed good primary and secondary antibody response to ND vaccine and their titers were comparable to those of the nonmedicated, vaccinated chickens. The mortality due to Newcastle disease virus challenge in the nonvaccinated controls was 12/15 (80%). One bird died prior to challenge. Neither mortality nor morbitity was observed in any of the vaccinated chickens.

It is concluded that the immunocompetency of chickens treated with maduramicin ammonium as high as 15 ppm for 3 weeks followed by 7 ppm for 3 more weeks was not compromised.

Data from the study are summarized in tables 8-9 on the following page. There were 16 chickens per group with 4 replicates of 2 males and 2 females per replicate.

(1) Y. E. Perey, G.B. Cleland and P.B. Dent, Am. J.. Vet. Res., 36, (1975): 513-517.

(Eds. note: The following table consists of 10 columns.)

Table 8 Summary of Body Weight, Weight Gain and Feed Intake of Birds Fed Maduramicin Ammonium for 41 days

         Maduramicin (ppm)
       Days   Days      ND       Avg.Wt.(g/)Bird     Feed Intake (g)/Bird/Day
Group  1-21   22-41     Vac.    31      41    Gain    0-31    0-41   31-41
  A      0        0        No       1031     1212     181      54       47       22
  B      0        0        Yes      1090     1630     541      65       69       80
  C      5        5        Yes      1033     1463     430      60       67       88
  D      7        7        Yes      1084     1511     427      52       59       81
  E     10        7        Yes      1029     1583     554      59       60       63
  F     15        7        Yes       912     1458     559      47       54       76

(Eds. note: The following table consists of 8 columns.)

Table 9

Hemaglutination Inhibition (HI) Titers at 7, 21 and 31 Days of Age and Mortality (%) After Vital Challenge Maduramicin (ppm) % Days Days ND Mean HI Titers at Days Mortality Group 1-21 22-41 Vac Day 7(1) Day 21(2) Day 31(2) 31-41 A 0 0 No - 0 0 80 B 0 0 Yes 0.88 5.13 21.9 0 C 5 5 Yes 0.67 3.63 20.0 0 D 7 7 Yes 0.17 3.80 17.4 0 E 10 7 Yes 0.38 4.79 17.4 0 F 15 7 Yes 1.38 4.17 29.5 0 (l)arithmetic mean (2)geometric mean iii) Non-Challenge Studies Summary:

Five pivotal non-challenge floor pen studies were conducted in various geographical locations in the U.S. to demonstrate the safety and dose range of maduramicin in broiler chickens under simulated commercial conditions, in the absence of coccidiosis. Maduramicin was examined at four dosage levels (5, 6, 7 and 8 ppm).

The primary parameters for measuring safety and efficacy of maduramicin were weight gain, feed efficiency and mortality.

The various treatment groups, except the non-medicated control group, received anticoccidial medication in the diets from 0-44 days of age, followed by a 5-day withdrawal period on non-medicated feed.

The body weight, feed efficiency and mortality of broiler chickens receiving maduramicin at various levels, was compared to the non-medicated control group. There were no significant differences in feed efficiency or mortality at 49 days of age between broilers receiving either of the four levels of maduramicin (5, 6, 7 and 8 ppm) and the non- medicated control group. A dose related adverse affect on weight gain was seen. Doses greater than 6 ppm showed a statistically significant decrease in weight gain as compared to the non-medicated control group.

Other observations included mortality, feed consumption, pigmentation and feathering. Feed consumption, pigmentation and feathering were normal in all trials. In trials 1 and 4 which were conducted in the same house, increased litter moisture was noted in the CYGRO pens.

Each trial is summarized as follows:

Trial l

Investigator:

P.E. Gingher
Agricultural Research Center
American Cyanamid Company
Princeton, New Elersey 08540

Number of birds fed CYGRO® Premix: 2400
Number of control birds: 600

The data from the trial is summarized as follows:

(Eds. note: The following table consists of 7 columns.)

Table 10

                                      Avg. Weight
               % Mortality(1)          Gain (g)           Avg. Feed/Gain
Treatment   0-44 Days  0-49 Days 0-44 Days  0-49 Days  0-44 Days  0-49 Days
Nonmedicated     3.7         4.0         1841        2141        1.87        1.95
CYGRO (5 ppm)    3.7         3.8         1805        2117        1.86        1.93
CYGRO (6 ppm)    3.7         3.0         1767        2090        1.88        1.93
CYGRO (7 ppm)    2.5         2.5         1712        2047        1.89        1.93
CYGRO (8 ppm)    1.2         1.2         1645        1990        1.92        1.94
Comments: (1) Total mortality shown

Trial 2

Investigator:

P. M. Waldroup
University of Arkansas
Fayetteville, AR 72701

Number of birds fed CYGRO Premix: 1680
Number of control birds: 420

The data from trial 2 is summarized as follows:

(Eds. note: The following table consists of 6 columns.)

Table 11

                                Av. Weight              Avg. Feed/
           %Mortality(1)         Gain (g)                  Gain 
Treatment    0-49 Days    0-44 Days    0-49 Days   0-44 Days   0-49 Days
Nonmedicated     8.51          1486          1759          1.84          1.92          
CYGRO (5 ppm)    6.37          1514          1797          1.88          1.95          
CYGRO (6ppm)     6.37          1472          1748          1.83          1.92          
CYGRO (7ppm)     1.88          1497          1777          1.86          1.93          
CYGRO (8ppm)     6.15          1406          1707          1.85          1.86          
Comments: (1) Total mortality shown

Trial 3

Investigator:

Carey Quarles
Colorado Quality Research
Ft. Collins, CO 80524

Number of birds fed CYGRO® Premix: 1200
Number of control birds: 300

The data from trial 3 is summarized as follows:

(Eds. note: The following table consists of 6 columns.)

Table 12

                           
          % Mortality(1)   Avg. Weight Gain (g)       Avg. Feed/Gain
Treatment   0-49 Days     0-44 Days    0-49 Days   0-44 Days   0-49 Days
Nonmedicated     3.0            1644          1904          1.81          1.89          
CYGRO (5ppm)     2.3            1619          1929          1.80          1.87          
CYGRO (6ppm)     4.7            1610          1924          1.77          1.86          
CYGRO (7ppm)     3.4            1565          1918          1.79          1.86          
CYGRO (8ppm)     2.7            1428          1829          1.86          1.87          
Comments: (1) Total mortality shown

Trial 4

Investigator:

P.E. Gingher
Agricultural Research Center
American Cyanamid Company
Princeton, New Jersey 08540

Number of birds fed CYGRO® Premix: 2400
Number of control birds: 600

The data from trial 4 is summarized as follows:

(Eds. note: The following table consists of 7 columns.)

Table 13

                                        Avg.Weight               Avg.
                 Mortality(1)            Gain(g)              Feed/Gain
Treatment    0-44 Days  0-49 Days  0-44 Days  0-49 Days  0-44 Days  0-49 Days
Nonmedicated      2.7          2.8         1862        2189        1.88        1.94
CYGRO (5 ppm)     2.2          2.5         1849        2158        1.85        1.91
CYGRO (6 ppm)     2.0          2.0         1828        2139        1.86        1.92
CYGRO (7 ppm)     3.5          3.7         1790        2119        1.86        1.91
CYGRO (8 ppm)     3.3          3.3         1748        2086        1.83        1.87
Comments: (1) Total mortality shown

Trial 5

Investigator:

J. F. Ort
North Carolina State University
Raleigh, N.C. 27695

Number of birds fed CYGRO Premix: 1920
Number of control birds: 480

The data from the trial 5 is summarized as follows:

(Eds. note: The following table consists of 7 columns.)

Table 14

                                      Avg. Weight           Avg. Feed/                
               %Mortality(1)            Gain (g)               Gain                      
Treatment   0-44 Days  0-49 Days  0-44 Days  0-49 Days  0-44 Days  0-49 Days
Nonmedicated     6.88        8.75        1807        1981        1.74        2.01        
CYGRO (5ppm)     11.25       11.67       1756        1983        1.77        l.98        
CYGRO (6ppm)     6.46        8.33        1723        2018        1.76        1.90        
CYGRO (7ppm)     3.75        5.63        1668        1969        1.82        1.97        
CYGRO (8ppm)     4.58        6.25        1614        1919        1.86        1.99        
Comments: (1) Total mortality shown

In summary, it can be concluded from the data in these nonchallenge floor pen studies that in the absence of coccidiosis, a significant dose related adverse effect on growth with no improvement in feed efficiency was seen starting at 6 ppm maduramicin ammonium. No significant adverse effect on growth was seen at 5 ppm maduramicin ammonium.

iv. Clinical Studies - Commercial Scale Field Trials - Corroborative Studies

Summary:

Four field trials were conducted in the U.S. to evaluate the safety of maduramicin ammonium at 5 ppm in large scale broiler production.

A total of 67,941 broiler chickens were reared under commercial conditions with maduramicin ammoniurn included in the ration at the 5 ppm level from 1-day-old to seven days before slaughter. Parameters measured were: observations for clinical coccidiosis, mortality, live bird weight and feed conversion. Any possible adverse drug effects on feathering or litter condition were also monitored.

The performance of broilers in the four trials was as follows:

(Eds. note: The following table consists of 5 columns.)

Table 15

            PROCESSING    AVERAGE LIVE       FEED
LOCATION    AGE (DAYS)    WEIGHT (LB)     CONVERSION    MORTALITY(%)
   NC             46            4.09              1.86            3.44              
   MD             52            4.58              1.95            13.79*            
   GA             49            4.58              2.01            6.48              
   AR             42            2.97              1.95            2.16              
*Mortality primarily due to vaccine reaction.

No sign of drug related toxicity or clinical coccidiosis was observed throughout the four trials. Litter condition and feathering were normal.

These reports further support the safety and effectiveness of maduramicin at 5 ppm in the complete feed of broiler chickens when fed under commercial conditions.

                                       No. of Birds
Name and Address of Investigator    Fed CYGRO
1.  Tom Hester                           16,320 
    B & L Feeds 
    Morganton, NC
2.  Tom Holder                           10,700 
    Perdue Farms, Inc.
    Salisbury, MD
3.  Birch McMurray                       20,921
    Seaboard Farms 
    Athens, GA
4.  John D. Story                        20,000
    Campbells Institute
      of Research & Technology
    Farmington, AR

Four field trials were conducted in the U.S. to evaluate the safety of maduramicin at 7 ppm in large scale broiler production.

A total of 85,572 broiler chickens were reared under commercial conditions with maduramicin included ln the ration of the 7 ppm level from 1-day-old to nine to twelve days before slaughter. Parameters measured were: observations for clinical coccidiosis, mortality, live bird weight and feed conversion. Any possible adverse drug effects on feathering or wet litter were also monitored.

The performance of broilers in the four trials were as follows:

(Eds. note: The following table consists of 5 columns.)

Table 16

              PROCESSING    AVERAGE LIVE       FEED
LOCATION      AGE (DAYS)    WEIGHT (LB)      CONVERSION    MORTALITY(%)
   NC              47              3.88               1.91            4.67
   GA              52              3.97               2.03            2.04
   AL              50              3.88               1.87            1.67
   AL              42              3.67               1.94            3.24

No sign of drug related toxicity or clinical coccidiosis was observed throughout the four trials. Litter condition and feathering were normal.

These reports support the safety and effectiveness of maduramicin at 7 ppm in the complete feed of broiler chickens when fed under commercial conditions.

                                    No. of Birds
Name and Address of Investigator    Fed CYGRO
1.  Tom Hester                            16,320 
    B & L Feeds
    Morganton, NC
2.  Birch McMurray                        21,300
    Seaboard Farms
    Athens, GA
3.  Gerald Bailey                         21,352
    Goldkist Poultry
    Cullman, AL
4.  Phil Dorr                             26,600
    Con Agra Poultry
    El Dorado, AR

v. Additional Corroborative Information Concerning Animal Safety

CYGRO maduramicin ammonium has been approved and marketed internationally since 1984. It is currently marketed in 28 countries throughout the world, including most of the major broiler producing countries. Approximately 1.5-2.0 billion broilers per year are currently receiving CYGRO maduramicin ammonium. No adverse commercial reports attributable to CYGRO maduramicin ammonium including unacceptable litter condition, disease outbreaks or poor featbering have been received.

In addition, to determine the effect, if any, of maduramicin ammonium on fecal moisture, carefully controlled battery experiments were conducted by Dr. Carey Quarles of Colorado Quality Research, Fort Collins, CO. These studies of 4 weeks duration with 5 different U.S. broiler diets showed that CYGRO maduramicin ammonium caused no increase in water consumption or fecal moisture. These data are shown in the following Table 17.

(Eds. note: The following table consists of 4 columns.)

Table 17 Fecal Moisture and Total Water Consumption at 28 days of Age Study A-84-30

           Level of        Fecal       Total 28 day
         maduramicin      Moisture        Water
Diet        (ppm)           (%)        Consumption (ml)
1               0               65              2302
2               0               71              2293
3               0               68              2269
4               0               71              2285
5               0               71              2282
1               5               70              2318
2               5               68              2291
3               5               70              2345
4               5               67              2286
5               5               69              2299

Additionally, a series of both floor pen and field trials with either 5 or 7 ppm CYGRO maduramicin ammonium in combination with various antibiotics and/or roxarsone have been conducted in support of combination NADAs. This information has been submitted under the following NADAs: 140-821, 140-822, 140-823, 140-899, 140-902 and 140888. Nearly 1 million birds were involved in these studies. No adverse commercial effects attributable to CYGRO maduramicin ammonium on litter conditions, disease outbreaks or feathering were observed in any of these studies.

b) Effects on microbial populations

i) Antimicrobial Resistance

Investigator:

D. Fagerberg
CARE, Inc.
Fort Collins, CO

A study was conducted to evaluate the effect of maduramicin ammonium fed ad libitumto chickens at 7 ppm on antimicrobial resistance of indigenous coliforms. Fecal samples of medicated and non-medicated birds were collected prior to treatment initiation for base-lines and weekly thereafter during an eight week treatment period. Ten coliform isolates per bird per sampling were tested for susceptibility to amikacin, ampicillin, carbenicillin, cefoxitin chloramphenicol, gentamicin kanamycin, nalidixic acid, streptomycin, sulfadiazine, tetracycline and trimethoprim/sulfamethoxazole.

Essentially no antimicrobial resistance was found among indigenous fecal coliforms of birds in either the non-medicated or the maduramicin treated group. Therefore, maduramicin ammonium had no effect on coliform antimicrobial resistance in poultry when administered at 7 ppm in the diet.

ii) Salmonella Shedding

Investigator:

D. Fagerberg
CARE, Inc.
Fort Collins, CO

A study was conducted to evaluate the effect of maduramicin ammonium fed to chickens at 7 ppm on Salmonella typhimuriumshedding (quantity, prevalence and duration), antimicrobial resistance and tissue levels. Fecal samples of medicated and nonmedicated birds were collected prior to treatment initiation to assure freedom from indigenous salmonellae. Chicks were orally inoculated with Salmonella typhimuriumat 11 and 12 days of age. Medicated diet was initiated to the appropriate treatment group five days after the final challenge and continued throughout the remainder of the eight week study. Fecal samples were collected 14 times during the 56 day treatment period and were directcounted for quantities of test salmonellae and enriched for detection of non-countable levels of test salmonellae. From ten of the post-challenge sampling periods, five S. typhumuriumisolates per bird were tested by a microdilution, MIC method for susceptibility to amikacin, ampicillin, carbenicillin, cefoxitin, chloramphenicol, gentamicin, kanamycin, nalidixic acid, streptomycin, sulfadiazine, tetracycline and trimethoprim/sulfamethoxazole. All Salmonella-challenged birds were necropsied at time of death or at study end, and samples of liver, spleen, colon contents and cecal contents were analyzed for presence of test salmonella.

Maduramicin ammonium had no adverse effect on the quantity, prevalence or duration of salmonella shedding, nor on antimicrobial resistance of the salmonella, nor did it have any effect on the presence of test salmonella in tissues.

VI. HUMAN SAFETY:

a. Toxicity tests

i) Acute toxicity tests

Acute toxicology:

Rabbit eye irritation-maduramicin technical

Six male rabbits received 0.1 g of the test material in the conjunctival sac of one eye. Within 24 hours signs of severe conjunctival and corneal irritation were apparent in all animals. These animals all died by day 7. Three other rabbits received the test material followed by a 20 second rinse immediately after instillation. Only mild signs of conjunctival irritation which reversed by 3 days were observed.

Rabbit skin irritation-maduramicin technical

Six male rabbits were patched with 0.2 g of the test material as an aqueous paste. The occluded patches were held in place for 24 hours, then removed and the skin was examined for signs of irritation. The primary irritation score which resulted was 2.9. Two rabbits died 72 hours after application. The material produces both moderate erythema and edema upon skin application.

Rabbit eye irritation-maduramicin in benzyl alcohol

Six rabbits were dosed with 0.1 ml of this mixture instilled into the conjunctival sac. All eyes were rinsed after 24 hours. Results showed that the product was irritating to the cornea, iris and conjunctivae of most animals. All animals had died by day 4.

American Cyanamid Report A-83-10:

The following studies were conducted with the 1% premix formulation which is the subject of this NADA with the exception that the final premix contained approximately 5% corn oil.

Rabbit eye irritation-premix

A standard dose (0.l g) of formulation was instilled into the conjuctival sac of 9 rabbits. After 20 seconds the eyes of three of the rabbits were rinsed thoroughly, white the other 6 rabbits' eyes were washed after 24 hours. The animals whose eyes had been washed after 20 seconds showed no signs of irritation. The animals receiving the 24 hour rinse showed corneal and conjunctival irritation as well as iritis. All eyes returned to normal before l4 days.

Rabbit skin irritation-premix

A standard dose (0.5 g) of the product as an aqueous paste was held under an impervious patch for 24 hours on 6 rabbits and the skin was examined and scored at 24 and 72 hours. The primary irritation score was 2.0. Moderate erythema and mild edema were observed which was more pronounced at the abraded sites.

ii. Subchronic Toxicology:

American Cyanamid Report L-1966B: Rat 28-day feeding study-maduramicin technical

Five rats/sex/group were fed diets containing test material at concentrations of 4, 6 and 8 ppm for 28 consecutive days. A control group of 10 rats/sex received basal diet at the same time. No signs of toxicity were observed at any treatment level over the course of the study. The no observable effect level, therefore, was considered to be > 8 ppm, equivalent to 0.87 mg/kg maduramicin body weight.

American Cyanamid Report L-1983: Rat 13-week feeding study-maduramicin technical

Thirty rats/sex/group were fed diets containing maduramicin at levels of 0, 1, 3, 5, and 7 ppm for 13 weeks. A significant decrease in body weight over the course of the study was observed for males receiving 7 ppm. Males receiving 5 and 7 ppm also showed significantly increased relative heart weights as compared to the controls. For these reasons the no effect level was considered to be 3 ppm (0.21 mg/kg).

American Cyanamid Report L-l984: Dog 28-day feeding study-maduramicin technical

Two dogs/sex were fed diets containing 0, 6, 12, 24 and 48 ppm maduramicin for 28 days. No effects were observed in any parameters measured in any animals receiving 12 ppm or less. The only effect observed in animals receiving 24 ppm was an intermittent hindlimb weakness/stiffness observed in 2/4 dogs. The effect was transient and was not seen after 16 days of test material administration. At 48 ppm ataxia which progressed to hindlimb paralysis was seen in all dogs. Atrophy, focal degeneration and diffuse sarcolemmal proliferation of skeletal muscle were observed microscopically in these dogs. These changes were not observed in the animals receiving 24 ppm. The no effect level was considered to be 12 ppm.

BioResearch Laboratories Report 50716-maduamicin technical

A range finding study to determine doses to be used in the definitive cardiovascular screening of maduramicin in the beagle dog following single oral or intravenous administration.

Ten dogs (5 male, 5 females) received incremental doses of maduramicin in ethyl alcohol at about 1 hour intervals in order to find a dose which would produce an effect and one which would produce no effect. Following each dose all dogs were monitored for heart rate, respiration rate, blood pressure, left ventricular pressure, dp/dt, aortic pressure and cardiac output. Coronary blood flow was determined at termination. The time course of the response was determined also. In addition, eight (4 male and 4 female) dogs were fed the test material in a ball of ground meat and the time course of the response was again determined measuring the same parameters. The results showed that after intravenous administration the apparent response was over in about l hour, and after oral administration the first signs of response could be observed after about 2 hours. Therefore, the dose levels chosen for intravenous dosing in the main study were 0.05, 0.20 and 0.60 mg/kg, and for oral dosing 0.5, 2.0 and 6.0 mg/kg. The optimum time to begin data collection in orally dosed dogs appeared to be l l/2 hours post-dose. The most sensitive parameters measured appeared to be left ventricular and aortic pressure.

BioResearch Laboratories Report 50724-maduramicin technical

Cardiovascular profile of maduramicin in the beagle dog following single oral or intravenous administration.

Three dogs/sex/dose group were administered test material at dose levels of 0.05, 0.20, 0.60 mg/kg (intravenous), and 0.5, 2.0 and 6.0 mg/l (oral) in a single dose and various cardiovascular parameters were measured. Intravenous injection of the test material caused an increase in heart rate, left ventricular pressure, systolic aortic pressure and dp/dt in dogs given a single dose of 0.60 mg/kg. No significant cardiovascular effect was observed following injection of 0.20 mg/kg. None of these changes was detected consistently in dogs given the test material orally at dose levels up to ten times higher than the IV dose.

Lederle Laboratories Report 36624-maduramicin technical

Cardiovascular screening profile

The effects of the test material on parameters such as blood pressure, urine output and renin production were studied in rats.

Isolated heart muscle was examined to determine the effect of perfusion of test material on contractile force, coronary vasodilatation, etc. Many other cardiovascular parameters were examined in this study. Results showed that there was nothing remarkable in the cardiovascular screening profile of maduramicin.

American Cyanamid Report C-84-1-maduramicin technical

Changes in heart rate, ECG measurements and amount of drug in urine measured at different intervals from dogs injected intravenously with maduramicin.

The purpose of this experiment was to measure heart rate changes, electrocardiographic recordings and maduramicin residues in urine of conscious trained beagle dogs intravenously injected with maduramicin in order to find one or several parameters which can be used for monitoring the degree of accidental human exposure to maduramicin.

The results of this experiment indicate that the-analysis for determination of maduramicin residues in the urine is the best parameter which can be used for monitoring the degree of accidental human exposure to maduramicin since electrocardiographic changes are very slight or not existent.

iii. Teratology and Reproductive Toxicology

Toxigenics Study 450-1252:
Teratology pilot study in albino rats with with maduramicin-maduramicin technical

Five females/group were administered the test material in corn oil on gestation days 6 through 15 at dosage levels of 0 (corn oil only), 1, 5, 10 or 15 mg/kg maduramicin. All females receiving 5, 10 or 15 mg/kg died during the dosing period. Animals receiving 1 mg/kg showed no signs of toxicity. From the study dose levels of 0.1, 1.0 and 3.0 mg/kg were chosen for the definitive study.

Toxigenics Study 450-1253:
Teratology study in albino rats with maduramicin-maduramicin technical

Twenty-five virgin female rats/group were mated and treated with test material via gavage on gestation days 6 through 15 at dose levels of 0, 0.3, 1.0 or 3.0 mg/kg/day. All females treated with 3.0 mg/kg died prior to gestation day 20. Animals treated with lower dosages showed no signs of maternal toxicity, and offspring of these two groups of females showed no signs of teratogenicity.

Hazelton Laboratories study 6123-110:
Three generation reproduction study with maduramicin in rats-maduramicin technical

Groups of 12 male and 24 female immature rats were fed diets containing 0, 1, 3 or 5 ppm maduramicin for 71 days. They were then mated twice to produce the F1a and F1b litters. After a minimum of 100 days of exposure to the same diets as the parents, the breeding program was repeated with F1b animals to produce the F2a and F2b pups and with F2b animals to produce the F3a and F3b pups. The only finding was slight inconsistent body weight changes in adults and pups which did not interfere with development and had no adverse effects on the reproductive function of either sex in any of the 3 generations.

iv. Chronic Toxicology

BioResearch Laboratories Project 81603:
A one-year dietary toxicity study in dogs with maduramicin-technical

No observable effects were seen in dogs administered 6 ppm or less for one year. Target organs seen in higher dose groups were the heart and the eye.

Five beagles/sex/group were fed diets containing maduramicin at levels of 0, 3, 6, 12 and 24 ppm for one year. Two dogs receiving 24 ppm died during the course of the study. In both cases the cause of death was considered to be congestive heart failure. Electrocardiograms showed that a significant increase in heart rate was observed in surviving dogs receiving 24 ppm after l year of study. At necropsy, a slight increase was seen in the mean (absolute and relative) heart weights of high dose group dogs, probably related to the ventricular dilation seen on gross examination. Subsequent microscopic examination of the heart tissue showed no histological changes that could definitely be attributed to test material administration. Eye lesions were observed in two females receiving 12 ppm and three males and five females receiving 24 ppm. In-life examinations showed the presence of what appeared to be reversible lesions described as multifocal lesions appearing as water droplets and hyperreflectivity. Histopathological examination of the eye confirmed retinal.atrophy in dogs showing hyperreflectivity. The no observable effect level was 6 ppm (0.204 mg/kg).

v. Genetic Toxicology Summary

A battery of in vitro and in vivo tests directed at assessing mutagenic/carcinogenic potential (in vitro) and to confirm presence/absence of mutagenic activity (in vivo) of maduramicin have been undertaken. Such systems are thought to possess high predictive accuracy for carcinogenicity and hence constitute a useful tool to obtain data which can be used together with our toxicological data for the evaluation of the safety of the product.

The in vitroand in vivotests completed with maduramicin technical are:

  1. A bacterial/microsome reverse mutation test (Ames).
  2. A Chinese hamster ovary/hypoxanthine guanine phosphoribosyl transferase test (CHO/HGPRT).
  3. In vitro chromosome aberration analysis in Chinese hamster ovary cells (CHO).
  4. In vivo bone marrow cytogenetic rat metaphase analysis.
  5. Unscheduled DNA Synthesis in rat primary hepatocytes.

Ames Test

In the Ames test, maduramicln was tested using six bacterial strains (S. TyphimuriumTA-98, TA-100, TA-1535, TA-1537 and TA-1538 and E. coliWP-2 uvrA-) in both the presence and absence of arochlor 1254 - induced rat liver S-9 homogenate. The dose levels in the plate assay ranged from 15.8 to 5000 mcg/plate; the maximum dose level tested for each strain was limited by solubility or cytotoxicity. In addition, a disc test was performed. Non-mutagenic responses were obtained for all strains at all dose levels.

CHO/HGPRT Assay

In the CHO/HGPRT assay, maduramicin again was tested in both the presence and absence of S-9. The highest dose level was chosen based on cytotoxicity. The dose levels tested ranged from 1 to 100 mcg/ml in the presence of S-9 and from 10 to 150 mcg/ml in the absence of 5-9. Non-mutagenic responses were again obtained at all dose levels.

Unscheduled DNA Synthesis in Rat Primary Hepatocytes

In this in vitro unscheduled DNA Synthesis Test maduramicin was tested using rat primary hepatocytes. Based on the results of an initial toxicity test, the test article was tested at live dose levels ranging from 0.10 to 4.0 mg/ml.

The results of the UDS assay indicate that under the test conditions, the test article did not cause a significant increase in the number of labelled nuclear grain counts and therefore is considered negative in this study.

CHO/Chromosome Aberration Assay

In the in vitro CHO/Chromosome aberration assay, the highest dose levels were again limited by cytotoxicity. Maduramicin was tested in the presence and absence of S-9 with cultures harvested at two time points, 6-8 hours and 14-18 hours after treatment. Uniformly non-mutagenic results were obtained for the 6-8 hour sampling time. For the 14-18 hours sampling time, chromosomal breakage was observed at the high dose level (100 mcg/ml) without S-9, and at the middle (50 mcg/ml) and high (100 mcg/ml) dose levels in the presence of S-9. The response at the middle dose level was greater than the response at the high dose level.

Since the induction of chromosomal aberrations was the only positive response in an in vitromutagenicity test battery, and since in vivo systems provide closeness to the physiological and biological complexities of the intact mammalian organism, it was decided to test CYGRO for its clastogenic potential using rat bone marrow metaphase analysis system. In this in vivostudy, CYGRO was tested at intraperitoneally (ip) administered doses of 0.9, 4.5 and 9 mg/kg; 9 mg/kg was the maximum tolerated dose permitting adequate metaphase cells for analysis. Bone marrow cells were harvested at 6, 24 and 48 hours after chemical injection for chromosomal aberration analysis. Negative results were obtained at all dose levels.

Thus positive results obtained under in vitroconditions are not corroborated by in vivo evaluation. Also, the in vivo test was performed at considerably higher doses than could result from potential human exposure. Greater reliance is placed on results obtained in living animals and it is concluded that CYGRO does not have genotoxic potential using chromosome breakage as a genetic endpoint.

b. Safe Concentration of Residues
The dog was the most sensitive species with a no-effect level of 0.204 mg/kg/day. Applying a 100-fold safety factor, the safe concentration of residues is as follows:

Calculation of Maduramicin Ammonium Acceptable Daily, Intake (ADI)

ADI
= 0.204 mg/kg/day (lowest NOEL) / 100 fold safety factor = .00204 mg/kg/day

ppm in muscle
= ADI x 60 kg (weight of average human) / 0.5 kg/day (daily consumption of meat) 
= .00204 mg/kg/day x 60 kg / 0.5 kg/day = 0.240 ppm or 240 ppb

(Eds. note: The following table consists of 3 columns.)

Safe concentrations of residues in edible tissues other than muscle:

            Consumption       Safe
Tissue        Factor     Concentration
Chicken Liver   3              720 ppb
Chicken Skin    2              480 ppb
Chicken Fat     2              480 ppb

c. Metabolism and Total Residue Depletion Studies, Demonstration of Withdrawal Time

In study PD-M Volume 21-1, six Hubbard x Hubbard broilers were offered feed medicated with 5.5 ppm carbon-l4 labeled maduramicin ammonium ad libitum for 7 consecutive days. The animals were sacrificed on the 0 day of withdrawal and the appropriate tissues were excised. The total carbon-l4 residue levels of the tissues are given below:

(Eds. note: The following table consists of 2 columns.)

 

               Total Carbon-14
Tissue          Content (ppm)
Fat                    1.19
Liver                  0.67
Skin                   0.49
Kidney                 0.22
Muscle                 0.08

Extracts of each tissue were subjected to two-dimensional TLC. Maduramicin ammonium was the most abundant residue found in each tissue. In fat, parent maduramicin ammonium represented 91.9% of the total residue. 6.4% of the total residue was observed to co-migrate in the TLC system with the beta-isomer of maduramicin ammonium. The remaining 1.7% consisted of unidentified minor components. The beta-derivative is produced by O-demethylation of a methoxy group in the A ring of maduramicin ammonium.

This metabolite would not be expected to produce toxicity different from that observed with the parent drug. Therefore, separate toxicity testing of the metabolite was not required.

In Study A-87-44, male and female broiler chickens were fed diets containing 6 ppm maduramicin ammonium for 44 days. The birds were sacrificed at 0, 1, 2, 3, 4 and 5 days withdrawal and the abdominal fat analyzed using the HPLC/F method for maduramicin ammonium residues. The results were as follows:

(Eds. note: The following table consists of 2 columns.)

Withdrawal Period             Range of
     in Days              Assay Values (ppm)
         0                        1.01-1.18
         1                        0.57-0.74
         2                        0.31-0.36
         3                        0.11-0.23
         4                        < 0.10-0.26
         5                        < 0.10-0.11


Limit of measurement = 0.10 ppm

In Study L-2295, carbon-14 labeled maduramicin ammonium was administered to broilers at a concentration of 6 ppm for seven consecutive days. At the conclusion of the medication period, chickens were sacrificed on 0, 1, 3 and 5 days withdrawal. Tissues were collected at time of sacrifice. Abdominal fat was analyzed via combustion analysis for total radioactivity with the following results:

(Eds. note: The following table consists of 2 columns.)

                              Total Radioactivity
Withdrawal Period            (mean ppm maduramicin
     in Days                 ammonium equivalents)
        0                               1.409
        1                               0.771
        3                               0.206
        5                               0.102

The results of the HPLC analyses obtained from Study A-87-44 were compared with those of combustion analyses from Study L-2295. At 0, 1, 3 and 5 days of withdrawal, parent maduramicin ammonium was 78%, 85%, 80% and 81% respectively, of the total residue.

Based on parent maduramicin ammonium being 80% of the total residue in fat (safe concentration, 480 ppb) as measured by the HPLC/F method, an R(m) of 380 ppb has been established for the marker residue maduramicin ammonium, in fat, the target tissue.

The HPLC data from Study A-87-44 was subjected to a statistical analysis (99% tolerance limit with 95% confidence) to determine the minimum withdrawal period. Based on this, a minimum withdrawal period of 4 days was calculated. It should be pointed out, however, that target animal safety and efficacy considerations will limit the withdrawal period to 5 days.

d. Regulatory Methods
The determinative method for quantitating residues of maduramicin in chicken fat is based on high pressure liquid chromatography. The confirmatory assay is a Liquid chromatography/mass spectrometry/mass spectrometry procedure.

The determinative and confirmatory methods were validated satisfactorily by FDA and USDA laboratories. The validated regulatory methods are filed in the Food Additives Manual on display in FDA's Freedom of Information Public Room (Room 12A30) 5600 Fishers Lane, Rockville, Maryland 20857.

VII. AGENCY CONCLUSIONS

The data submitted in support of this NADA satisfy the requirements of section 512 of the Act and demonstrate that maduramicin ammonium (CYGRO) at a concentration ranging from 5 ppm to 6 ppm in finished feed is safe and effective when fed to broiler chickens for the indications stated in the product labeling.

Maduramicin ammonium has been subjected to a threshold assessment in accordance with the procedures contained in the agency's "Guideline for Threshold Assessment" dated September, 1986. The submitted data are adequate to satisfy the Agency's food safety requirements. The safe concentration for total residues in noncooked edible chicken tissues are: 720 ppb in liver; 480 ppb in skin and 480 ppb in fat.

Based on parent maduramicin ammonium being 80% of total residue in fat, the target tissue, an Rm of 380 ppb has been established for the marker residue. The data supports a minimum withdrawal time of 4 days. All studies conducted to document efficacy of the drug product included a 5-day withdrawal period. This provides the basis for the established withdrawal time.

Proper use by non-veterinarians can be expected because poultry producers routinely use medicated feed containing an animal drug for the prevention of coccidiosis in broiler chickens. Directions are clearly written and there is reasonable certainty that the conditions of use, including mixing directions, on the label can and will be followed by the producer. The Agency has concluded that this product can be approved for over-the-counter use.

VIII. LABELING (Attached)

  1. Package Label, Type C Broiler Feed, Medicated
  2. Package Label, Type A Medicated Article

Copies of these labels may be obtained by writing to the:

Food and Drug Administration
Freedom of Information Staff (HFI-35)
5600 Fishers Lane
Rockville, MD 20857

Or requests may be sent via fax to: (301) 443-1726. If there are problems sending a fax, call (301) 443-2414.