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U.S. Department of Health and Human Services

Animal & Veterinary

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NADA 141-069 FIRST GUARD STERILE POWDER - original approval

Approval Date: January 13, 1998

I. GENERAL INFORMATION:

NADA141-069
Sponsor:Alpharma, Inc.
One Executive Drive
Fort Lee, New Jersey 07024
Generic Name:colistimethate sodium
Trade Name:FIRST GUARD STERILE POWDER
Marketing Status:A prescription (Rx) product which carries the following caution statement "Federal (USA) law restricts this drug to use by or on the order of a licensed veterinarian."

 

II. INDICATIONS FOR USE

FIRST GUARD STERILE POWDER (colistimethate sodium) is indicated for use in 1- to 3-day-old chickens for the control of early mortality associated with Escherichia coli susceptible to colistin.

 

III. DOSAGE

A.DOSAGE FORMFIRST GUARD STERILE POWDER is available in 10 gram glass vials. Constituted product should be used within 28 days of preparation when stored at room temperature. Constitute with 62.5 mL sterile physiological saline or Sterile Water For Injection. Each mL of the resulting solution contains colistimethate sodium equivalent to 133 mg colistin activity.
B.ROUTE OF ADMINISTRATIONAdminister by subcutaneous injection in the neck region of 1- to 3-day-old chickens. A sterile needle and syringe or properly cleaned automatic injection type machine should be used.
C.RECOMMENDED DOSAGES:FIRST GUARD STERILE POWDER should be administered as a single treatment to 1- to 3-day-old chickens at a dosage of 0.2 mg per chick.

 

IV. EFFECTIVENESS

Pivotal Studies (5 trials)

A. Dose Determination (2 trials)

"Dose Determination Trials of AL-452 MS Injection for the Control of Colibacillosis in Day-Old Chicks"

  1. Type of Study: dose titration with induced infections
  2. Investigator:

    Jeffrey N. Davidson, D.V.M.
    Health Management Services.
    P. O. Box 2046
    Tulare, CA 93275

  3. General Design:

    1. Purpose: The purpose of these studies was to determine the optimum dose of colistimethate sodium (colistin activity) in the day-old chick for the control of early chick mortality associated with E. coli susceptible to colistin.
    2. Animals: Newly hatched (day-old) broiler chicks from a commercial hatchery; 60 chicks (30 males and 30 females) per treatment group per study.
    3. Controls: Two control groups were used in the study. One group, uninfected control, was used to demonstrate that the challenge, pure cultures of E. coli, negatively affected the second, infected control group. The infected control was challenged with the same culture as the treated groups, and was injected with 0.2 mL saline containing no drug.
    4. Diagnosis: Diagnosis was based on a minimum of three daily observations. Any birds found dead during these observations had swabs taken from pericardia and airsacs. Swabs were incubated for 24 hours on MacConkey's agar to determine the presence of E. coli.
    5. Dosage Form: The dosage form was a reconstituted sterile saline solution which delivered the appropriate dose of colistin activity in 0.2 mL.
    6. Route of Administration: Subcutaneously in the neck region.
    7. Dose: Single injections were given as shown in Table 4.1.

      Table 4.1. Doses of colistin (mg) per bird in Trials 1 and 2

      Trial 1Trial 2
      0.000.00
      0.050.10
      0.100.20
      0.200.30
      0.400.40
      0.600.50
    8. Test Duration: Chicks were observed for 14 days post-treatment.
    9. Pertinent Variables Measured: Mortality and Day 14 body weights were the primary study parameters.
  4. Results: The mortality results and Day 14 body weights are shown in Tables 4.2 and 4.3, respectively.

    Table 4.2. Mean percent mortality: Trials 1 and 2

     Mean Percent Mortality
    Dose colistin (mg/chick)Trial 1Trial 2
    085.291.3
    0.0534.0---
    0.134.921.6
    0.223.211.1
    0.3---5.0
    0.419.86.2
    0.5---4.7
    0.619.5---
    Table 4.3. Mean Day 14 body weights: Trials 1 and 2
     Mean Weight (grams)
    Dose colistin (mg/chick)Trial 1Trial 2
    0298157
    0.05272---
    0.1299298
    0.2299300
    0.3---301
    0.4299293
    0.5---307
    0.6320---
  5. Statistical Analysis: An analysis of variance was conducted for each set of data presented in Tables 4.2 and 4.3. The means for the treatment groups were then used to investigate the dose response obtaining a best fitting model from the class of polynomial and linear plateau models. A one-sided t-test on the means of the control group and the smallest non-zero level group was used to obtain the lowest statistically effective level. Each model was used to determine the largest statistically significant effective level. These two levels together give the following statistically significant (P < 0.05) effective ranges or levels:

    Trial 1: % Mortality--0.05 to 0.2 Body Weight--none

    Trial 2: % Mortality--0.1 to 0.13 Body Weight--0.1

  6. Adverse Reactions: No adverse reactions were noted.
  7. Conclusions: The dose titration studies support a dose of 0.2 mg colistin activity per chick as the optimum dose.

B. Dose Confirmation/Field Trials (3 trials)

"The Efficacy of AL-452 MS as an Injection for the Control of Colibacillosis in 1- to 3-Day-Old Chickens"

  1. Type of Study: Dose confirmation under clinical (field) conditions
  2. Investigators:

    Jeffrey N. Davidson, D.V.M.
    Health Management Services
    P.O. Box 2046
    Tulare, California 93275

    Carey L. Quarles, Ph.D.
    Colorado Quality Research, Inc.
    1401 Duffy Drive, Suite 700
    Fort Collins, Colorado 80524

    James L. McNaughton, Ph.D.
    PARC Institute, Inc.
    30 N. Harrison Street
    P.O. Box 1161
    Easton, Maryland 21601

  3. General Design:

    1. Purpose: The purpose of these studies was to confirm that injection of 0.2 mg colistin activity (given as colistimethate sodium) per chick controls early chick mortality associated with E. coli susceptible to colistin in 1- to 3-day-old chickens exposed to natural infections under field conditions.
    2. Animals: Newly hatched (1- to 3-day-old) broiler chicks from a commercial hatchery; mixed sexes; approximately 2,200 to 2,960 per trial site (1,100 to 1,480 per treatment group per site)
    3. Control Group: A negative control group (no colistimethate sodium administered) was used.
    4. Diagnosis: Diagnosis was based on twice daily observations. Any birds found dead during these observations were necropsied and pericardia, livers, airsacs and yolk sacs (if present) were sampled with a sterile cotton swab. Swabs were incubated on MacConkey's agar for 24 hours to determine the presence of E. coli. Additionally, necropsied birds were scored for airsac, liver and pericardial lesions and given a combined score, using a 0-3 scoring system (0 = no lesion; 1 = mild to moderate airsacculitis; 2 = moderate airsacculitis and mild to moderate pericarditis and/or perihepatitis; 3 = severe airsacculitis and moderate to severe pericarditis and/or perihepatitis).
    5. Dosage Form: FIRST GUARDÔ STERILE POWDER was reconstituted with sterile diluent to form a concentrated solution (133.33 mg colistin activity/mL). The concentrated solution was further diluted with diluent to a concentration of 1 mg colistin activity/mL in order to deliver the appropriate dose (0.2 mg colistin activity) in 0.2 mL.
    6. Route of Administration: Subcutaneously in the neck region.
    7. Dose: Single injections of 0.2 mL, containing 0.2 mg colistin activity, were given to each chick.
    8. Test Duration: Chicks were observed for 14 days post-treatment.
    9. Pertinent Variables Measured: Mortality and mean organ lesion scores were the primary study parameters.
  4. Results: The mean percent mortality and mean lesion scores are shown in Table 4.4.

    Table 4.4. Mean percent mortality and organ lesion score

    ParameterControlTreated
    Total mortality, %10.015.20
    Culture positive mortality, %9.183.09
    Mean organ lesion score1.730.71
  5. Statistical Analysis:

    A test of homogeneity of variances from the three trials was conducted from which it was concluded that the sample variances were from populations with a common variance. Thus, data from the three trials were pooled. Percent mortality data were transformed by arcsine square-root transformation prior to analysis using analysis of variance. Treatment effects on lesion scores were analyzed by ordinal data analysis, using the Mann-Whitney Rank Sum test for non-parametric data. Treatment effects versus controls were significant for all parameters, and are summarized in Table 4.5.

    Table 4.5. Probability that FIRST GUARDÔ STERILE POWDER treatment is significant

    ParameterSignificance
    Total mortality, %P < 0.05
    Culture positive mortality, %P < 0.0001
    Mean organ lesion scoreP < 0.0001
  6. Adverse Reactions: No adverse reactions were noted.
  7. Conclusions: These studies support a label claim for colistimethate sodium for the control of early mortality associated with E. coli susceptible to colistin at a dose of 0.2 mg colistin activity per chick.

 

V. ANIMAL SAFETY

"Target Animal Safety of AL-452 MS in 1- to 3-Day-Old Chickens"

A. Type of Study: Acute toxicity

B. Investigator:

Jeffrey N. Davidson, D.V.M.
Health Management Services.
P. O. Box 2046
Tulare, CA 93275

C. General Design:

  1. Purpose: The purpose was to determine the safety of colistimethate sodium in the day-old chick when injected at 1X, 3X, and 10X the recommended use level.
  2. Animals: Newly hatched (day-old) Hubbard x Hubbard chicks, weighing 45 to 48 grams at start of study; four treatment groups of 20/sex/group
  3. Control Group: A negative control was included. These birds received a single subcutaneous injection of 0.25 mL physiologic saline.
  4. Doses: 0, 0.2, 0.6, or 2.0 mg/chick (representing 0, 1X, 3X, and 10X the recommended label dose)
  5. Route of Administration: Subcutaneous injection
  6. Observation Period: 7 days
  7. Pertinent Variables Measured: Body weights (birds weighed on Days 1, 4, and 7), feed intake (Days 1-7) and clinical measurements including blood clotting time, total blood cells, packed cell volume, and hemoglobin. In addition, all chicks not used for clinical evaluation were necropsied and examined for gross pathology. Tissues were examined histologically.

D. Results: At 0.2 and 0.6 mg colistin per chick (1X and 3X the label dose), colistimethate sodium injection had no detrimental effects on chick body weight, feed intake, or clinical parameters. Birds receiving 10X the recommended use level consumed less feed and also showed a transitory decrease in weight gain as compared to the control birds. Two birds in the 10X group died the day after dosing. The cause of death for these two birds was not determined.

E. Conclusions: Colistimethate sodium is safe when administered to chicks as a single subcutaneous injection at the recommended label dose of 0.2 mg/chick.

 

VI. HUMAN SAFETY

A. Toxicity Studies:

  1. Title: Mutagenicity Test on AL-452 MS in the Rat Primary Hepatocyte Unscheduled DNA Synthesis

    Report Number: 9891-0-447

    Initiation Date: May 27, 1987

    Completion Date: September 11, 1987

    Study Director: Maria A. Cifone, Ph.D.

    Testing Laboratory:

    Hazelton Laboratories America, Inc.
    5516 Nicholson Lane
    Kensington, Maryland 20895

    Test Substance/Dosage Form: Colistimethate sodium powder, dissolved in Williams' Medium E plus 1% serum immediately before use

    Species/Strain: hepatocytes were obtained from Fisher 344 rats

    Number of Animals: not applicable

    Drug Levels Tested and Duration of Dosing: 0.5, 1.0, 2.5, 5.0, 10.0, 25.0, and 50.0 µg/mL plus controls (positive and negative)

    Route of Administration: not applicable

    Parameters Tested: unscheduled DNA synthesis (UDS)

    Significant Toxicities Observed: Concentrations up to 50 µg/mL did not cause increased UDS, while the positive control (2-acetyl aminofluorene, 0.10 µg/mL) did.

    No-Observed-Effect Level: not applicable

    Statistical Analysis: not applicable

    Conclusions: The test article does not induce UDS under the test conditions employed.

  2. Title: Mutagenicity Test on AL-452 MS in the CHO/HGPRT Forward Mutation Assay

    Report Number: 9891-0-435

    Initiation Date: May 13, 1987

    Completion Date: August 4, 1987

    Study Director: Robert R. Young, M.S.

    Testing Laboratory:

    Hazelton Laboratories America, Inc.
    5516 Nicholson Lane
    Kensington, Maryland 20895

    Test Substance/Dosage Form: Colistimethate sodium powder, dissolved in water

    Species/Strain: in vitro study, not applicable

    Number of Animals: not applicable

    Drug Levels Tested and Duration of Dosing: 1.0, 2.0, 4.0, 6.0, 8.0 and 10.0 mg/mL plus controls (positive and negative)

    Route of Administration: not applicable

    Parameters Tested: Forward mutations at the HGPRT locus of the CHO-K1-BH 4 Chinese hamster ovary (CHO) cell line as assessed by colony growth in the presence of 6-thioguanine (TG).

    Significant Toxicities Observed: Concentrations up to 10 mg/mL did not exceed the historical maximum threshold mutant frequency.

    No-Observed-Effect Level: not applicable

    Statistical Analysis: Kastenbaum-Bowman Test

    Conclusions: The ability of the test material to induce forward mutations at the HGPRT locus in Chinese hamster ovary cells under both the S9 metabolic activation and nonactivation conditions of the assay remains unresolved.

  3. Title: Mutagenicity Test on AL-452 MS in an In Vitro Cytogenetic Assay Measuring Sister Chromatid Exchange and Chromosomal Aberration Frequencies in Chinese Hamster Ovary (CHO) Cells.

    Report Number: 9891-0-457

    Initiation Date: September 28, 1987

    Completion Date: November 20, 1987

    Study Director: Hemalatha Murli, Ph.D.

    Testing Laboratory:

    Hazelton Laboratories America, Inc.
    5516 Nicholson Lane
    Kensington, Maryland 20895

    Test Substance/Dosage Form: Colistimethate sodium powder, dissolved in McCoy's 5a culture medium

    Species/Strain: in vitro study, not applicable

    Number of Animals: not applicable

    Drug Levels Tested and Duration of Dosing: 16.7, 50.1, 167, and 501 µg/mL without S9, and 16.7, 50.1, 167, 501, 1670, and 5010 µg/mL with S9, plus positive and negative controls for the SCE test; 1250, 2510, 3760, and 5010 µg/mL with and without S9 for the 10-hour harvest, and 1420, 2850, 4270, and 5690 µg/mL with and without S9 for the 19.4-hour harvest, plus positive and negative controls for the chromosomal aberrations test.

    Route of Administration: not applicable

    Parameters Tested: An in vitro assay to evaluate the ability of the test material to induce sister chromatid exchange (SCE) and chromosomal aberrations in Chinese hamster ovary (CHO) cells with and without metabolic activation.

    Significant Toxicities Observed: Sister Chromatid Exchange Assay: There was a significant dose-related increase in SCE at 167 and 501 µg/mL under non-activation conditions. There was a significant dose-related increase in SCE at 501, 1670, and 5010 µg/mL under conditions of metabolic activation. Chromosomal Aberrations Assay: There was a significant dose-related increase in chromosomally aberrant cells at 2850, 4270, and 5690 µg/mL both with and without metabolic activation.

    No-Observed-Effect Level: not applicable

    Statistical Analysis: Fisher's Exact Test to compare the percentage of cells with aberrations in treated cells with pooled results from solvent and negative controls (Sokal and Rohlf, Biometry, 1981).

    Conclusions: The test article is considered positive for inducing sister chromatid exchanges and chromosomal aberrations in Chinese hamster ovary cells under both the metabolic activation and non-activation conditions of the assay.

  4. Title: Mutagenicity Test on AL-452MS in the In vivo Sister Chromatid Exchange Assay

    Report Number: 9891-0-458

    Initiation Date: October 5, 1988

    Completion Date: May 19, 1989

    Study Director: James L. Ivett, Ph.D.

    Testing Laboratory:

    Hazelton Laboratories America, Inc.
    5516 Nicholson Lane
    Kensington, Maryland 20895

    Test Substance/Dosage Form: Colistimethate sodium powder, dissolved in sterile, deionized water immediately before use.

    Species/Strain: adult mice, strain ICR

    Number of Animals: 5 males and 5 females per treatment group

    Drug Levels Tested and Duration of Dosing: 500, 2500 and 5000 mg/kg plus controls (positive and negative), given as a single dose

    Route of Administration: oral gavage Parameters Tested: sister chromatid exchanges in bone marrow cells

    Significant Toxicities Observed: No increase in SCE was observed at any treatment level, while the positive control (cyclophosphamide, 10 mg/kg) caused a significant increase in SCE.

    No-Observed-Effect Level: No level of the test article caused an effect in this study.

    Statistical Analysis: the data were analyzed by a parametric Analysis of Variance test (Sokal and Rohlf, 1981)

    Conclusions: The test article did not induce an increase in SCE in bone marrow cells under conditions of this assay and is considered negative in the in vivo mouse bone marrow SCE assay.

  5. Title: Mutagenicity Test on AL-452 MS in the Mouse Bone Marrow Cytogenic Assay

    Report Number: 9891-0-451

    Initiation Date: October 5, 1988

    Completion Date: May 15, 1989

    Study Director: James L. Ivett, Ph.D.

    Testing Laboratory:

    Hazelton Laboratories America, Inc.
    5516 Nicholson Lane
    Kensington, Maryland 20895

    Test Substance/Dosage Form: Colistimethate sodium powder, dissolved in deionized water immediately before use

    Species/Strain: adult mice, strain ICR

    Number of Animals: 5 males and 5 females per treatment group per harvest time

    Drug Levels Tested and Duration of Dosing: 500, 2500, and 5000 mg/kg plus controls (positive and negative), given as a single dose for the acute study; 500, 1000, and 2000 mg/kg plus control (negative), given on 5 consecutive days for the subacute study

    Route of Administration: oral gavage

    Parameters Tested: chromosomal aberrations in bone marrow cells harvested 6, 18, and 30 hours after dosing in the acute study or 6 hours after the final dose in the subacute study

    Significant Toxicities Observed: No increase in chromosomal aberrations was observed at any treatment level or harvest time in either the acute or subacute study, while the positive control (cyclophosphamide, 80 mg/kg) caused a significant increase in chromosomal aberrations.

    No-Observed-Effect Level: No level of the test article caused an effect in this study.

    Statistical Analysis: The data were analyzed using the Kruskal-Wallis test at the alpha = 0.05 level to determine whether any of the mean values of the treatment groups differed from the negative control.

    Conclusions: The test article was considered negative for inducing chromosomal aberrations in bone marrow cells of mice under the conditions of the test.

  6. Title: 13-Week Oral Subchronic Toxicity Study in Dogs

    Report Number: 2482-102

    Initiation Date: July 22, 1987

    Completion Date: October 19, 1988

    Study Director: David E. Bailey, Ph.D.

    Testing Laboratory:

    Hazelton Laboratories America, Inc.
    9200 Leesburg Turnpike
    Vienna, Virginia 22180

    Test Substance/Dosage Form: Colistin sulphomethate sodium USP; powder

    Species/Strain: beagle dogs

    Number Animals Per Sex Per Treatment Group: 4 males and 4 females

    Drug Levels Tested and Duration of Dosing: 0, 0.5, 1.6, and 5.0 percent of the diet for 13 weeks

    Route of Administration: oral

    Parameters Tested: survival, body weight, food intake, clinical signs, ophthalmoscopic and physical examinations, hematology, clinical chemistry, gross necropsy with organ weights, and histopathology

    Significant Toxicities Observed: All animals survived until the scheduled terminal sacrifice; body weights were somewhat decreased in the high-dose animals, but not significantly when compared to control values; no effect was observed on food intake; clinical signs were incidental and not treatment related; no treatment related ophthalmic lesions were noted; no treatment related effects were observed on clinical hematology or clinical chemistry values; gross pathology findings were scattered and considered incidental; the only histomorphic changes were described in the kidney as minimal cytoplasmic pallor of the distal tubules in both sexes of the high-dose group.

    No-Observed-Effect Level: 1.6% of colistin sulphomethate sodium USP in the diet

    Statistical Analysis: The body weight change, total food consumption, clinical pathology and organ weight data were analyzed for homogeneity of variances using Levene's test; if variances were found to be heterogeneous, data were transformed using a variety of transformations until homogeneity could be demonstrated; homogeneous data were analyzed by Analysis of Variance; if significant in the ANOVA, Dunnett's was used to compare treatments to control.

    Conclusions: The NOEL for colistin sulphomethate sodium in beagle dogs is 1.6% in the diet, which is equivalent to 457 mg/kg body weight/day.

  7. Title: 13-Week Subchronic Oral Toxicity Study in Rats

    Report Number: 2482-101

    Initiation Date: September 28, 1987

    Completion Date: October 14, 1988

    Study Director: David E. Bailey, Ph.D.

    Testing Laboratory:

    Hazelton Laboratories America, Inc.
    9200 Leesburg Turnpike
    Vienna, Virginia 22180

    Test Substance/Dosage Form: Colistin sulphomethate sodium USP; powder

    Species/Strain: Sprague-Dawley Crl:CD7BR rats

    Number Animals Per Sex Per Treatment Group: 20 males and 20 females

    Drug Levels Tested and Duration of Dosing: 0, 0.5, 1.6, and 5.0 percent of the diet for 13 weeks

    Route of Administration: oral

    Parameters Tested: survival, body weight, food intake, clinical signs, ophthalmoscopic and physical examinations, hematology, clinical chemistry, gross necropsy with organ weights, and histopathology

    Significant Toxicities Observed: All male animals survived until the scheduled terminal sacrifice, while 2 high-dose females died prior to their scheduled terminal sacrifice; no effect was observed in food intake; clinical signs were incidental and not treatment related; no treatment-related ophthalmic lesions were noted.

    The toxic effects observed were: statistically significant reductions in mean body weight in females of the intermediate-dose group and in males and females of the high-dose group; significant increases in segmented neutrophils in males and females of the high-dose group; statistically significant increases in blood urea nitrogen and alanine aminotransferase in males of the high-dose group; mucosal erosions of the glandular stomach in males and females of the high-dose group. These erosions were characterized by focal areas of ischemic necrosis with occasional bacteria on the luminal surface of the foci.

    No-Observed-Effect Level: 0.5% of colistin sulphomethate sodium USP in the diet

    Statistical Analysis: The body weight, total body weight change, total food consumption, mean clinical pathology and organ weight data were analyzed for homogeneity of variances using Levene's Test; if variances were found to be heterogenous, data were transformed using a variety of transformations until homogeneity could be demonstrated; homogeneous data were analyzed by Analysis of Variance (ANOVA); if significant in the ANOVA, Dunnett's Test was used to compare treatments to control.

    Conclusions: The NOEL for colistin sulphomethate sodium USP in rats is 0.5% in the diet, which is equivalent to 387 mg/kg body weight/day.

  8. Title: Two-Generation Reproduction Study in Rats with a Teratology Phase

    Report Number: 2482-103

    Initiation Date: July 22, 1987

    Completion Date: August 1, 1989

    Study Director: David E. Bailey, Ph.D.

    Testing Laboratory: Hazelton Laboratories America, Inc.
    9200 Leesburg Turnpike
    Vienna, Virginia 22180

    Test Substance/Dosage Form: Colistin sulphomethate sodium UPS; powder

    Species/Strain: Sprague-Dawley Crl:CD7BR rats

    Number Animals Per Sex Per Treatment Group: 27 males and 27 females per generation; 2 litters were allowed in each generation

    Drug Levels Tested and Duration of Dosing: 0, 0.5, 1.6, and 5.0 percent of the diet; first generation males were treated for at least 10 weeks prior to mating until termination, while first generation females were treated for at least 2 weeks prior to mating and through mating; treatment of second generation males and females began while they were nursing and continued until termination.

    Route of Administration: oral

    Parameters Tested: The following general parameters were recorded in the parental generations: survival, body weight (in females, on Days 0, 7, 14, and 20 of gestation and 0, 4, 7, 14, and 21 of lactation), food consumption, clinical signs, physical examinations, and gross necropsy. The reproductive parameters recorded were males and females mated, pregnancy rates, number of pups, pup survival with periodic pup weights (until Day 21 postpartum), and gross necropsy findings. The following parameters were recorded in the teratology phase: number of males and females mated, pregnancy rate at cesarean section, numbers of corpora lutea, number of implantation sites, resorptions, live or dead fetuses, fetal weights, and gross fetal observations followed by soft tissue and skeletal examinations.

    Significant Toxicities Observed: First generation, general parameters: two female animals from the high-dose group were found dead prior to the scheduled terminal sacrifice; clinical observations were sparse and considered incidental; food intake values were generally higher for mid- and high-dose males and high-dose females; significant reductions in body weight were observed in both males and females of the high-dose group. Reproductive parameters: the reproductive performance of this generation was excellent for all treatment groups; no females aborted or delivered early; indices for male and female fertility, gestation, pup viability, weaning and sex ratio were comparable for all treatment groups; the number of pups was slightly reduced in the high-dose group; mean pup weight was reduced in the mid- and high-dose groups; at necropsy, there was a significant increase in the incidence of no milk in the stomach in the high-dose group; gross pathology findings for parental animals and pups were comparable in all treatment groups.

    Second generation, first litter: general parameters: two male animals from the high-dose group were found dead prior to the scheduled terminal sacrifice; clinical observations were sparse and considered incidental; food intake values were generally higher for mid- and high-dose males and high-dose females, but during the first week food intake values were lower for the high-dose males and females; significant reductions in body weight were observed in both males and females of the high-dose group and in females of the mid-dose group. Reproductive parameters: reproductive performance of this generation was excellent for all treatment groups; all pregnant females delivered live litters and all litters survived to weaning; none of the females aborted or delivered early; indices for male and female fertility were somewhat reduced in the mid- and high-dose groups, probably as a consequence of the reduced body weight at time of breeding: indices of gestation, pup viability, weaning and sex ratio were comparable for all treatment groups.

    Second generation, second litter: at cesarean section, the mean numbers of corpora lutea, implantation sites, resorptions, live fetuses, fetal weight, and pup sex ratio were comparable for all groups. Statistically significant reductions in mean maternal net body weight changes from Day 0 of pregnancy were seen in the mid- and high-dose groups; decreased gravid uterine weights were also seen in the mid- and high-dose groups. No external fetal variations were noted; external fetal malformations, as well as visceral variations and malformations, were considered incidental; no skeletal malformations were noted; skeletal variations included varying degrees of incomplete ossification of the sternebrae and ribs in the mid- and high-dose groups.

    No-Observed-Effect Level: 0.5% of colistin sulphomethate sodium USP in the diet

    Statistical Analysis: The body weight, total body weight change, total food consumption, duration of gestation, total number of pups delivered, number of live pups data were analyzed for homogeneity of variances using Levene's test; if variances were found to be heterogenous, data were transformed using a rank transformation; data were then analyzed by Analysis of Variance (ANOVA); if significant in the ANOVA, Dunnett's Test was used to compare treatments to control.

    Conclusions: Colistimethate sodium did not interfere with the reproductive performance of male and female rats through two generations and did not produce teratogenic effects.

B. Calculation of a Safe Concentration (SC) for Colistin Base:

  1.  No Observed Effect Level (NOEL)

    The most sensitive species to this product in the toxicity studies was the rat. The lowest NOEL was obtained with 0.5% (387 mg/kg/day) of colistin sulphomethate sodium USP in the diet. This NOEL was observed in the 13-week oral toxicity study and in the 2-generation reproduction study with a teratology phase. The NOEL from the 13-week oral toxicity study in the rat and a safety factor of 1000 will be used to calculate the safe concentration.

    Colistin sulphomethate sodium will be used to calculate the safe concentration of residues since it is the active molecular entity being tested. For the calculations, a molecular weight of 1168 for the colistin base and 1620 for the colistin sulphomethate sodium will be used. The latter molecular weight assumes an average of three sodium methyl sulfonate and two sodium methylol groups per molecule of colistin base.

  2. Safe Concentration (SC) Calculations

    The NOEL of 0.5% CMS USP (387 mg/kg/day), obtained in the 13-week oral toxicity study in the rat, corresponds to 297 mg/kg/day of colistin sulphomethate sodium. The latter is calculated from the colistin base value of 214 mg/kg/day and a ratio of 72.1% (base to colistin sulphomethate sodium). If calculated from the 387 mg/kg/day CMS USP, which has a colistin sulphomethate sodium content of 75.1%, the value would be 291 mg/kg/day of colistin sulphomethate sodium. Using either of the calculated values for colistin sulphomethate sodium would give approximately the same Safe Concentration. Using the 297 mg/kg/day value as the NOEL, along with a safety factor of 1000, the safe concentration is:

    SC = 297x60/1000x0.3 = 59 ppm

    The Safe Concentration in muscle is limited to 5.0 ppm of colistin sulphomethate sodium (as the molecular entity with a molecular weight of 1620) because no chronic toxicity testing has been performed with the product. This Safe Concentration is solely limited to the colistin sulphomethate sodium and not to the base or its potency. For microbiologically-active residues above 5 ppm, testing would need to be done to demonstrate that the intestinal microflora in humans is not altered. The Safe Concentrations for poultry tissues are shown in Table 6.1.

    Table 6.1. Safe Concentrations (SC) for total residues of colistin sulphomethate sodium in edible chicken tissues using the revised food consumption factors

    Edible TissueAmount Consumed/DaySafe Concentration (SC)
    Muscle300 g5 ppm
    Liver100 g15 ppm
    Fat/Skin50 g30 ppm
    This compound is a category A compound as derived from the Threshold Assessment considerations. Based on Structural Activity Assessment, it was assigned to Category C (non-carcinogen). Subsequent to this, the 90-day feeding studies allowed it to be classified as Category A. Because of the therapeutic use and the low dose it is considered a Low Use Drug. Accordingly, chronic studies were not required, and, based on 90-day studies, a Safe Factor of 1000 is used in the Safe Concentration calculations.

C. Total Residue Depletion Study:

Title: 14C-Colistimethate Sodium Total Residues in Chickens

Report No.: APA1/960195

Initiation Date: November 1, 1995

Completion Date: June 14, 1996

Investigator:

Dr. L. F. Elsom Huntingdon
Life Sciences Ltd.
P.O. Box 2
Huntingdon, Cambridgeshire
PE18 6ES England

Description of Animals Used: Domestic chicks (Gallus gallus domesticus, Cobb Broiler, fast growing broiler strain)

Route of Administration: subcutaneous injection in neck

Time and Duration of Dosing: Each bird received a single dose of 0.2 mg colistin potency at one day of age

Radioisotope Used: 14C-Colistimethate sodium

Sampling Times: 14, 21, and 28 days post dose

Number of Animals/Sampling time: 12 males, 12 females: tissues pooled in group of 4 by sex

Inspection of the data in Tables 6.2 and 6.3 confirms that there is no difference in residue concentration in males or females.

Table 6.2. Concentrations of radioactivity in male chickens after a single subcutaneous dose of 14C-colistimethate sodium at a dose level of 0.2 mg potency/bird at 14, 21, and 28 days post-dose

 Mean PPM
Tissue14 days21 days28 days
liver0.510.170.07
muscle0.510.230.12
skin0.290.120.07
injection site0.510.210.12

Table 6.3. Concentrations of radioactivity in female chickens after a single subcutaneous dose of 14C-colistimethate sodium at a dose level of 0.2 mg potency/bird at 14, 21, and 28 days post-dose

 Mean PPM
Tissue14 days21 days28 days
liver0.520.180.08
muscle0.550.240.14
skin0.320.120.07
injection site0.650.220.12

The droppings from all of the birds were collected twice a day for Days 1 and 2, and then daily through Day 28. The droppings were homogenized, sampled and assayed for total radioactivity. A total of 32.6% of the dose was excreted through Day 28. The first excreta sample, 0 to 12 hours, showed microbiological activity equivalent to 0.074% of the total dose and was confirmed as colistin base via HPLC. All subsequent excreta samples contained no microbiological activity.

The nature of the tissue residue is one of absorption and incorporation of the radioactivity into the tissue. This statement is supported by the excretion profile and the tissue residue pattern in muscle. The excretion profile with the bulk of the radioactivity excreted by Day 7 (25.6% C equivalent to 78.5% of the total excretion) and a steady state reached after Day 9 post-dose indicate tissue incorporation with the low level of excretion at 0.3%/day equivalent to tissue turnover.

The tissue pattern for muscle is shown in Table 6.4 as the largest tissue by weight with the highest radioactivity. When the muscle tissue residue value is multiplied by the total body weight, a constant value is obtained, indicating that the decrease in tissue residue values is due to body weight gain and not excretion, which is in agreement with the total residue excretion values.

Table 6.4. Muscle total tissue residue (TTR) value times body weight (BW)

Chicken SexWithdrawal DayTTRBWTTR x BW
Males14
21
28
0.51
0.23
0.12
367
809
1320
187
186
158
Females14
21
28
0.55
0.24
0.14
333
722
1150
183
174
161

In Table 6.5 the measured 28-day total residue values for each tissue are compared to the applicable safe concentration.

Table 6.5. Comparison of safe concentrations (SC) to the total residue in chicken tissues measured at 28 days post-dose

TissueSexSC (ppm)28 Day (ppm)SC Tissue Residue
livermale
female
15
15
0.07
0.08
214
188
musclemale
female
5
5
0.12
0.14
42
36
skinmale
female
30
30
0.07
0.07
429
429
injection sitemale
female
5
5
0.12
0.12
42
42

The nature of the radioactive residues in muscle samples from Day 21 and 28 sacrifices were investigated since they represent the highest total tissue residues at each of those time points. The muscle samples were extracted with chloroform:methanol and 0.5% sulfuric acid with the results listed in Table 6.6.

Table 6.6. Chicken muscle fractionation

DaySexTRCHCI3:MeOH Extractable (%)0.5% H2SO4 Extractable (%)Bound Residue Non-Extractable (%)
21M
F
0.23
0.24
15.9
15.6
0.8
0.9
83.3
83.5
28M
F
0.12
0.14
8.0
7.4
<0.1
<0.1
92.0
92.6

The bound residue fractions were incubated with an established bacterial protease that is a standard for tissue digestion at pH 5, then followed by pH 10. The results of these incubations are listed in Table 6.7, below.

Table 6.7. Chicken muscle bound residue digestion/extraction

DaySexBound Residue (% of total)pH 5 Protease (% extracted)pH 10 Protease (% extracted)
21M
F
83.3
83.5
1.9
1.2
65.0
61.5
28M
F
92.0
92.6
1.2
1.3
67.0
66.9

The pH 10 protease samples were freeze dried, resuspended in pH 6 buffer, centrifuged and assayed microbiologically. No microbiological activity was noted in any of the pH 10 protease incubation extracts.

In a separate study the effect of the primary digestive enzymes (pepsin at pH 2 and pancreatin at pH 7.5) and a plant protease (papain at pH 6.2) on the chicken muscle total residues was evaluated. Control and 21-day treated chicken muscle samples were incubated with the three separate enzyme systems for 48 hours, followed by centrifugation. The supernatant of each was removed, volume measured and assayed for radioactivity. The total residue percent solubilized value for each enzyme treatment is listed in Table 6.8.

Table 6.8. Enzyme digestions of 21 day post-dose chicken muscle

Enzyme Treatment
(48 hrs at 37 °C)
Total Residue
(% Solubilized)
Total Residue
(% Non-Solubilized)
Pepsin at pH 237.654.3
Pancreatin at pH 7.573.026.9
Papain at pH 6.279.620.0

The digestion supernatant for each enzyme was freeze dried, resuspended in buffer, centrifuged, and processed for microbiological assay. There were no microbiological activity differences between the control muscle tissue and treated muscle tissue enzyme incubation supernatants.

D. Tolerance for the Marker Residue: Total residues at 28 days (the earliest time FDA considers broilers to be marketable) following the injection of colistimethate sodium in chicks are a minimum of 36 times lower than the calculated Safe Concentration. Therefore, a tolerance is not required.

E. Withdrawal Time: At 28 days post dose, total residues in edible tissues of chickens are a minimum of 36 times below the established safe concentrations. This finding supports the assignment of a zero-day withdrawal period. No additional residue depletion studies are required.

F. Regulatory Method: A regulatory method is not required for this use because the toxicology and residue data support a zero-day withdrawal for this use of colistimethate sodium.

G. User Safety Concerns: The labeling for this product warns users that topical exposures to skin and mucous membranes could cause irritation or elicit allergic reactions in individuals sensitized to antibiotics of the polymyxin class. Persons with known hypersensitivity to antibiotics in the polymyxin class should avoid any exposure to this product.

 

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 colistimethate sodium is safe and effective when administered subcutaneously in 1- to 3-day-old chickens at 0.2 mg colistin activity per chick for the control of early mortality associated with Escherichia coli susceptible to colistin.

Based on a battery of toxicology tests, the Safe Concentrations for total residues of colistimethate sodium in edible chicken tissues are 5 ppm in muscle, 15 ppm in liver, and 30 ppm in fat/skin. Total residues at 28 days following the injection of colistimethate sodium in chicks are a minimum of 36 times lower than the calculated safe concentration. Husbandry practices for broilers are such that they will only be handled at one to three days of age for injection with colistimethate sodium; they will not enter the human food chain until at least 28 days of age in the case of Cornish hens, or, more likely, not until 42 or more days of age. Therefore, a withdrawal period will not be required for this use of colistimethate sodium in one- to three-day-old chickens, and a target tissue, marker residue, and tolerance have not been assigned. An official regulatory method is not required because the residue and toxicology data support a zero-day withdrawal.

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 early chick mortality associated with E. coli 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 colistimethate sodium-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 excretion of colistimethate sodium-resistant zoonotic pathogens into the environment.

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 Dockets Management Branch (HFA-305), Park Building (Room 1-23), 12420 Parklawn Dr., Rockville, Maryland 20857.

Under section 512(c)(2)(F)(i) of the FFDCA, this approval qualifies for FIVE years of marketing exclusivity beginning on the date of approval because no active ingredient (including any ester or salt of the active ingredient) has been approved in any other application.

 

VIII. LABELING (Attached)

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

Freedom of Information Office
Center for Veterinary Medicine, FDA
7500 Standish Place
Rockville, MD 20855