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U.S. Department of Health and Human Services

Animal & Veterinary

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NADA 141-077 ADSPEC™ Sterile Solution - original approval

Approval Date: January 28, 1998

I. GENERAL INFORMATION:

NADA141-077
Sponsor:Pharmacia & Upjohn Company
7000 Portage Road
Kalamazoo, Michigan 49001
Generic Name:spectinomycin sulfate tetrahydrate
Trade Name:ADSPEC™ Sterile Solution
Marketing Status:This is a prescription product and bears the caution statement as follows: Federal (USA) law restricts this drug to use by or on the order of a licensed veterinarian

 

II. INDICATIONS FOR USE IN CATTLE

ADSPEC™ Sterile Solution is indicated for the treatment of bovine respiratory disease (pneumonia) associated with Pasteurella haemolytica, Pasteurella multocida and Haemophilus somnus.

 

III. DOSAGE

A.DOSAGE FORMADSPEC™ Sterile Solution is available in three package sizes: 100, 250, and 500 mL vials.
B.ROUTE OF ADMINISTRATIONADSPEC™ Sterile Solution is to be administered to cattle by subcutaneous injection in the neck.
C.RECOMMENDED DOSAGES:ADSPEC™ Sterile Solution is to be administered to cattle at a daily dose of 10 to 15 mg spectinomycin per kg of body weight (4.5 to 6.8 mL per 100 lb body weight). Treatment should be administered at 24-hour intervals for 3 to 5 consecutive days. Selection of dose (10 to 15 mg/kg) and duration of treatment (3 to 5 days) should be based on an assessment of severity of disease, pathogen susceptibility, and clinical response.

 

IV. EFFECTIVENESS

Two separate pivotal clinical studies were conducted in cattle to evaluate the effectiveness of spectinomycin sulfate sterile solution as therapy for bovine respiratory disease (BRD).

A dose determination study evaluated the efficacy of spectinomycin sulfate sterile solution for treatment of the bacterial component of BRD in an induced challenge model in ruminating calves. Based on the results of this study, 10 and 15 mg spectinomycin/kg body weight were the doses selected for confirmation in a clinical field trial.

A multi-location field trial was conducted to evaluate the efficacy of spectinomycin sulfate sterile solution for the treatment of the bacterial component of spontaneously occurring BRD in feedlot cattle under natural conditions. Both doses (10 and 15 mg/kg) administered for either 3 or 5 days significantly (p<0.05) increased the percent clinical success on Day 14 of the study.

A. Pivotal Clinical Studies

1. Dose Determination Study

a. Type of Study: This dose determination study was conducted using an established model for inducing bovine pneumonic pasteurellosis.

b. Investigator:
Terry N. TerHune, D.V.M., Ph.D.
Health Management Services
Tulare, California 93275

c. General Design:

1) Purpose of Study: To determine a spectinomycin dose that was efficacious for the treatment of bovine respiratory disease.

2) Test Animals: Eighty-eight recently weaned male Holstein calves were acclimated to the test facilities for 5 to 9 days before challenge. Sixty eight animals met the inclusion criteria and were assigned to treatment groups according to a pre-arranged randomization scheme.

Table 4.1. Treatment groups, dose, number of treatments, and number of calves assigned to each treatment group in dose determination study

Treatment groupspectinomycin dose (mg/kg BW)Number of treatmentsNumber of calves
10314
25314
310314
415314
520312

3) Controls: The control group (1) received daily injections of sterile 0.9% sodium chloride for injection at 0.2 mL per kg body weight (equivalent to the high dose volume of spectinomycin sulfate sterile solution).

4) Challenge: To induce pneumonic pasteurellosis, the animals were challenged with a virulent strain of P. haemolytica administered by intratracheal injection.

5) Diagnosis: Following challenge, the animals were monitored daily by the study veterinarian for clinical signs of respiratory disease. Animals were assigned to treatment within the first 48 hours post-challenge if they met predetermined criteria regarding rectal temperature, respiratory rate, and depression.

6) Dosage Form: Spectinomycin Sulfate Sterile Solution containing 100 mg spectinomycin free base/mL

7) Route of Administration: The drug was administered by subcutaneous injection.

8) Doses: 0.2 mL Sterile 0.9% Sodium Chloride for Injection/kg body weight; 5, 10, 15, and 20 mg spectinomycin/kg body weight at 24-hour intervals for 3 consecutive days.

9) Test Duration: Animals were monitored by the study veterinarian for 10 days after initial treatment.

10) Pertinent Parameters Measured: The primary decision variables included rectal temperature, lung lesion score, and mortality.

d. Post-treatment Microbiology: At the time of necropsy, samples were obtained from lung lesions for identification of prevalent microorganisms.

e. Experimental Design: The study was designed as a randomized complete block design with five treatments and 14 blocks; two blocks were incomplete. The individual calf was the experimental unit. Calves were blocked on rectal temperature, body weight, and day of treatment initiation.

f. Statistical Analysis: Comparison of control to treated (a=0.05) for the decision variables: lung lesion scores and rectal temperature, was done using analysis of variance (SAS® proc mixed), and for mortality using the Cochran-Mantel-Haenszel mean scores statistic (SAS proc freq) controlling for blocks. To assist in determining the doses to be used in the pivotal dose confirmation study, the dose response for each decision variable was characterized through the use of linear-plateau regression modeling (SAS proc nlin).

g. Results: All dose levels of spectinomycin tested (5, 10, 15, and 20 mg/kg) prevented mortality; however, there was no statistically significant difference (p=0.08) in the mortality between the negative control group (21% mortality) and the spectinomycin groups (0% mortality). There was a trend toward lower lung lesion scores for animals treated with spectinomycin; however, the scores were highly variable. Mortality and lung lesion score did not meet all the criteria defined in the study protocol to allow their use in selecting a dose.

Rectal temperature at 1 day post-treatment met all the criteria as defined in the study protocol to be used to select a dose. At 1 day post-treatment, rectal temperatures of animals treated with 5, 10, 15, and 20 mg spectinomycin/kg were significantly (p£0.001) lower compared to non-medicated control group. The data for rectal temperature one day after last treatment were fit to a linear-horizontal regression model. The dose as predicted by the upper 95% confidence limit on the join point (beginning of dose response plateau) was 12.8 mg/kg.

Table 4.2. Mean daily rectal temperature (°F) by day post-treatment in cattle treated subcutaneously with spectinomycin sulfate for 3 consecutive days

Dose spectinomycin
Post-treatment day
(mg/kg body weight)1234567
0104.4104.4104.3103.7103.9103.6103.3
5103.1102.9103.0102.8102.9102.9102.8
10102.1102.2102.1102.6102.3101.8102.1
15101.9102.3102.1102.4102.2101.9102.1
20102.0101.9102.1102.4102.4102.0101.7

h. Conclusions:

Spectinomycin sulfate sterile solution administered subcutaneously was effective in reducing clinical signs of induced pneumonic pasteurellosis. Doses selected for testing in the dose confirmation study were 10 and 15 mg spectinomycin/kg body weight.

i. Adverse Reactions: None reported

2. Multi-Location Field Trial

a.Type of Study: Pivotal clinical field study designed to evaluate efficacy of spectinomycin sulfate sterile solution as therapy for naturally occurring BRD.

b.Investigators:

Terry N. TerHune, D.V.M., Ph.D.
Health Management Services
Tulare, California 93275
Janice Berg, D.V.M.
Lakeside Research Brooks,
Alberta Canada T1R 1B7
Edward G. Johnson. D.V.M.
Johnson Research
Parma, Idaho 83660
David T. Bechtol, D.V.M.
Agri Research Center, Inc.
Canyon, Texas 79015

c. General Design:

1) Purpose of Study: The objective of this study was to evaluate the efficacy of spectinomycin sulfate sterile solution at doses of 10 and 15 mg spectinomycin/kg body weight administered subcutaneously once daily for either 3 or 5 consecutive days as therapy for naturally occurring BRD (under field conditions) compared to a placebo control.

2) Test Animals: A total of 457 male cattle were used in this study. Of this total number, 120 were Holsteins, and 337 were crossbred beef. The age of cattle ranged from 4 to 10 months. The average weight of the Holsteins was 126 kg, and the average weight of the crossbred beef cattle was 227 kg. These cattle were randomly assigned to treatment groups as described in Table 4.3 below.

Table 4.3. Treatment groups, dosage, number of injections, and number of cattle assigned to each treatment group in multi-location field trial

Treatment groupdose of spectinomycin
(mg/kg body weight)
Number of injectionsNumber of cattle assigned
1a0577
210376
310578
415376
515576
6b0--c74

a Control group
b Treatment group 6 was administered an experimental compound
c Data not reported for the experimental compound

3) Controls: The control animals were injected once daily for 5 consecutive days with Sterile 0.9% Sodium Chloride for Injection equal in volume (0.15 mL/kg) to the dose volume of the highest spectinomycin sulfate sterile solution dose group.

4) Challenge: Naturally occurring BRD.

5) Diagnosis: Animals were randomly assigned (test Day 1) to treatment groups when they met the following inclusion criteria:

a) Rectal temperature >104.0 °F, AND
b) Respiratory index =1 (abnormal), AND
c)Depression index >1 (mildly depressed to severely depressed)

6) Dosage Form: Spectinomycin sulfate sterile solution contained 100 mg spectinomycin/mL and 9.47 mg benzyl alcohol/mL.

7) Route of Administration: Subcutaneous injection.

8) Doses: Animals were randomly assigned to receive either: 3 or 5 doses of 10 mg spectinomycin/kg, or 3 or 5 doses of 15 mg spectinomycin/kg, or 5 doses of 0.9% sodium chloride aqueous solution. Doses were administered once daily on consecutive days.

9) Test Duration: The combined treatment and observation period was 14 days.

10) Pertinent Parameters Measured: Animals were monitored daily for pertinent clinical signs of BRD. Those animals meeting the ancillary treatment criteria (same as inclusion criteria) during the post-treatment observation period were considered treatment failures and were treated with standard feedlot therapy.

The decision variable for determining efficacy was overall treatment success on Day 14, and was defined as a live animal that did not require ancillary treatment for BRD at any time during the study, and that met the following criteria on test Day 14:

a)Rectal temperature <104.0 °F, AND
b)Respiratory index =0 (normal pattern and effort), AND
c)Depression index <1 (normal or mildly depressed), AND
d)Appetite score <1 (normal or mild anorexia)

11) Microbiology: Prior to treatment, nasal swabs were obtained from all animals assigned to the study and cultured for the major bacteria associated with BRD. Lung samples from animals that died during the 14 day study period were also cultured for BRD pathogens. The in vitro activity of spectinomycin against isolates from this study was determined using a broth microdilution method which conformed to the National Committee for Clinical Laboratory Standards (NCCLS) guideline, and included the appropriate quality control organisms.

d. Results: Treatment success: The number of successes compared to the number of animals on study for individual trials and the overall percent clinical success for each treatment group are shown in Table 4.4. The number of animals on study for treatment groups 1, 2, 3, 4, and 5 was reduced from 383 animals to 376 animals due to the removal of 7 animals for non-BRD reasons.

Table 4.4. Percent treatment success on Day 14 in cattle treated with spectinomycin sulfate at a dose of 10 to 15 mg/kg for 3 or 5 consecutive days

DoseDuration
Trial Location
(mg/kg)(days)CaliforniaTexasIdahoCanadaOveralla
0515 (3/20)19 (4/21)25 (5/20)54 (7/13)26 (19/74)
10347 (9/19)25 (5/20)62 (13/21)93 (14/15)55 (41/75)
10585 (17/20)32 (6/19)41 (9/22)93 (14/17)59 (46/78)
15370 (14/20)20 (4/20)70 (14/20)80 (12/15)59 (44/75)
15579 (15/19)50 (10/20)48 (10/21)86 (12/14)64 (47/74)

aOne-sided P-value for CONTROL vs TREATMENT group <0.001.

Microbiology Results: A total of 457 nasal swabs were collected immediately prior to initiation of treatment. Of this total, P. haemolytica was isolated from 234 (51%) swabs, P. multocida was isolated from 145 (32%) swabs, and H. somnus was isolated from 16 (4%) swabs. Lung samples from the 12 animals that died during the 14-day study period were cultured for BRD pathogens. Ten animals were positive for P. haemolytica; two animals were positive for P. multocida, and one animal was positive for H. somnus.

The in vitro activity of spectinomycin against 381 isolates from this study was determined. A summary of the spectinomycin minimum inhibitory concentration (MIC) data for P. haemolytica, P. multocida, and H. somnus isolated from nasal swabs obtained from cattle immediately prior to treatment is shown in Table 4.5. The MIC data for 13 isolates cultured from the lungs of animals that died during the study are shown in Table 4.6. Because of the low number of isolates cultured from the lungs, the MIC50 and MIC90 were not calculated; the MIC range for each isolate is provided.

Table 4.5. Minimum inhibitory concentration (MIC) of spectinomycin (µg/mL) against P. haemolytica, P. multocida, and H. somnus isolated from nasal swabs obtained from cattle with BRD

OrganismNumber of isolatesMIC50MIC90
P. haemolytica2231632
P. multocida1311632
H. somnus14216

Table 4.6. Minimum inhibitory concentration (MIC) of spectinomycin (µg/mL) against P. haemolytica, P. multocida, and H. somnus isolated from lungs of cattle with BRD

OrganismNumber of isolatesMIC Range
P. haemolytica1016 - >128
P. multocida216 - 32
H. somnus116

e. Experimental design: This study was designed as a multi-location study with randomized complete block designs run at each location. The blocking was done on rectal temperature and day assigned to study. If the number of animals to be assigned on a given day was not sufficient to fill a complete block those animals were maintained on study in an incomplete block. By design the treatments were assigned at random to those animals, thus treatments were approximately equally represented in the incomplete blocks.

f. Statistical Analysis: The statistical analysis included all treatment groups tested in this study (one control group, four spectinomycin groups, and one experimental treatment group); however, only results for the control and spectinomycin groups are reported herein.

To take into account the binary nature of the data, logistic regression was used to test the one-sided hypotheses that the mean success rate of each of the spectinomycin-treated groups was equal to the mean treatment success rate of the negative control group. SAS® proc genmod was used for analysis with a model including terms for TRIAL and TREATMENT and allowing for over-dispersion (i.e., treating the TRIAL x TREATMENT interaction as a random effect).

The number of animals determined to be clinical successes on Day 14 of the study period were significantly (p < 0.05) increased for all spectinomycin treatment groups as compared to a negative control.

g. Conclusions: Spectinomycin sulfate sterile solution at 10 or 15 mg/kg BW administered subcutaneously for either 3 or 5 consecutive days was shown to be an effective treatment against the bacterial component (P. haemolytica, P. multocida and H. somnus) of spontaneously-occurring BRD in feedlot cattle.

h. Adverse Reactions: In the trial in Texas, one animal died following the second treatment of 15 mg spectinomycin/kg. The cause of death was reported as anaphylactic reaction. No adverse effects were observed in the other trials.

B. In vitro Activity Studies

The following 4 studies were considered as supportive information regarding the in vitro susceptibility of bovine bacterial pathogens to spectinomycin. These studies demonstrate that spectinomycin has in vitro activity against Pasteurella haemolytica, Pasteurella multocida, Haemophilus somnus, Actinomyces pyogenes, and Mycoplasma bovis and Mycoplasma dispar.

1. A Four-Year Survey of Antimicrobial Susceptibility Trends for Isolates from Cattle with Bovine Respiratory Disease in North America (Journal of Clinical Microbiology, 1994; 32:725-731)

In a 4-year survey (1988-1992) of antimicrobial susceptibility trends for isolates from cattle with BRD in North America, a total of 880 isolates representing P. haemolytica, P. multocida and H. somnus were tested. The majority of the isolates were recovered from lungs of animals expiring from acute BRD. All MIC determinations with the Pasteurella spp. were performed using a broth microdilution method. All H. somnus isolates were tested using an agar dilution method. A summary of spectinomycin MIC data for P. haemolytica, P. multocida, and H. somnus isolated from cattle with BRD for all 4 years is shown in Table 4.7.

Table 4.7. Summary of spectinomycin MIC data for P. haemolytica, P. multocida, and H. somnus isolated from cattle with BRD from 1988 to 1992.

  
MIC
(µg /mL)
 
OrganismNumber of isolates50%90%ModeRangePercent Susceptiblea
P. haemolytica4613264320.5 > 12883.5
P. multocida31832>128160.13 > 12876.4
H. somnus1098328<0.13 > 12887.1

aBased on the NCCLS guideline which categorizes isolates with MIC of <32.0 µg/mL as susceptible.

2. Actinomyces pyogenes: Susceptibility of 103 Clinical Animal Isolates to 22 Antimicrobial Agents (Vet Res, 1993; 24:251-259)

Minimal inhibitory concentrations (MIC) of 22 antibiotics, including spectinomycin, for 103 strains of Actinomyces pyogenes of animal origin (97 strains from cattle) were determined by an agar dilution test. All strains were susceptible to spectinomycin as shown in Table 4.8. The MIC of spectinomycin for all 103 strains was <2 µg/mL.

Table 4.8. Minimum inhibitory concentrations (MIC) for 103 strains of Actinomyces pyogenes isolated from cattle, sheep, and goats

Inhibitory concentration (µg/mL)0.060.120.250.51.02.04.0
Number of A. pyogenes isolatesa for which growth was inhibited021735310

aOf the 103 strains isolated, 97 were from cattle, 4 were from sheep, and 2 were from goats

3. In Vitro Susceptibility of Veterinary Pathogenic Mycoplasma to Trospectomycin and Spectinomycin (Upjohn Technical Report 705-7922-86-005) and A Procedure for the in vitro Evaluation of Antimicrobial Agents Against Mycoplasma Pathogens of Veterinary Importance (Upjohn Technical Report 705-7922-86-004)

The in vitro effectiveness of spectinomycin against 8 mycoplasma strains from bovine were determined using an agar dilution procedure. The MIC was recorded as the lowest concentration of antibiotic that completely inhibited visible growth of the mycoplasma after 7 days. The acceptable reproducibility of the MIC test was within one 2-fold dilution of the actual endpoint. The MIC value for spectinomycin against each strain is shown in Table 4.9.

Table 4.9. Minimum inhibitory concentration (MIC) of spectinomycin against 8 mycoplasma strains isolated from cattle

Mycoplasma spp.Strain DesignationMIC (µg/mL)
M. canadenseATCC 294188
M. bovisDavis 1>128
M. bovisDavis 232
M. bovisp1132
M. bovisVLS 8202-10.25
M. bovisVLS 8202-34
M. bovisVLS 91784
M. bovisAmes IV16

4. Susceptibilities of Mycoplasma bovis, Mycoplasma dispar, and Ureaplasma diversum Strains to Antimicrobial Agents In Vitro (Antimicrobial Agents and Chemotherapy, 1993; 37(2):317-321)

The in vitro susceptibility of various strains of Mycoplasma bovis, Mycoplasma dispar, and Ureaplasma diversum, to selected antimicrobial agents including spectinomycin, were determined by a serial broth dilution method. The MICs for spectinomycin against the field isolates and type strains are shown in Table 4.10.

Table 4.10. Minimum inhibitory concentration (MIC)of spectinomycin against M. bovis, M dispar, and U. diversum isolates

  
MIC
(µg /mL)
OrganismNumber of field strains50%90%RangeType Strain
M. bovis16241 - 44
M. dispar19120.5 - 1.02
U. diversum178324 - 32-

 

V. ANIMAL SAFETY

Target animal studies were conducted in male and female crossbred beef cattle to address the tolerance to and safety/toxicity of spectinomycin sulfate tetrahydrate administered by subcutaneous injection. In the first study, a dose of 150 mg/kg/day (10X the maximum daily recommended therapeutic dose) was injected subcutaneously as spectinomycin sulfate sterile solution once daily for 5 days (the maximum label duration of administration). Treatment-related findings included increased relative kidney weights in both sexes, minimal microscopic findings of squamous and transitional epithelial cells in the urine of steers, and decreased urinary pH in steers. Minimal injection site reactions were also present at 1 day and 4 days post injection. In the second study, spectinomycin sulfate tetrahydrate was administered subcutaneously as spectinomycin sulfate sterile solution at doses of 15, 45, or 75 mg/kg/day (1X, 3X, or 5X the maximum daily recommended therapeutic dose) once daily for 15 days (3X the maximum label duration of administration). No animals died on study. Treatment-related findings included decreased urinary pH and mild swelling at the injection sites with minimal to moderate injection site reactions.

A. Pivotal Studies

1. Drug Tolerance Test: Upjohn Technical Report 7219-95-038, issued Sept. 5, 1995

  • a. Study Director:
    R.K. Frank, D.V.M., Ph.D.
    The Upjohn Company
    Kalamazoo, MI
  • b. General Design: 1) Purpose of study: To characterize the pharmacotoxic effects of formulated drug in the target species at 10X the maximum daily recommended therapeutic dose.

    2) Test Animals: Eight 6- to 12-month-old crossbred beef-type cattle weighing from 251.5 to 308.5 kg were used. Two animals per sex (steers and heifers) were used as controls and 2 animals per sex were administered spectinomycin at a dose of 150 mg/kg/day.

    3) Control: The control was 0.9% sodium chloride injection, USP (saline) administered subcutaneously at 1.5 mL/kg body weight.

    4) Test article: The test article was the proposed dosage form and formulation of spectinomycin sulfate solution (ADSPEC™ Sterile Solution).

    5) Dose Used: Daily doses of 150 mg/kg were administered subcutaneously in a sterile solution which contained 100 mg/mL (free base equivalents).

    6) Route. Subcutaneous injection at 8 to 10 sites. No site was injected with more than 50 mL.

    7) Test Period: Animals were treated for 5 days, then euthanized and necropsied on Study Day 6.

    8) Pertinent Parameters Measured: Toxicological evaluation was based on clinical observations, injection site irritation, food consumption, physical examinations, body weight changes, hematology and serum chemistry parameters, fecal examinations, urinalysis, gross necropsy observations, absolute and relative organ weights, and histologic evaluation of tissues.

e. Statistical Evaluation: None.

f. Results: No deaths occurred prior to study termination. Treatment-related increases in relative kidney weights were observed in both steers and heifers. Treated steers also had more acidic urine and more transitional or squamous epithelial cells in the urine relative to controls. Injection sites had dark red/tan areas in the subcutis 1 day after injection, but this lesion was observed in only 1 of 4 animals 4 days after injection.

g. Conclusions: Spectinomycin sulfate tetrahydrate injected subcutaneously at a dose of 150 mg/kg/day once daily for 5 days in male and female crossbred beef cattle was well tolerated. The urinary system is the target organ system for spectinomycin sulfate tetrahydrate toxicity. Treatment-related findings included increased relative kidney weights, increased urine acidity, microscopic observation of transitional or squamous epithelial cells in urine. In addition, gross lesions were observed at all labeled injection sites at 1 day post injection of 50 mL spectinomycin and at 1 of 4 labeled injection sites at 4 days post injection of 50 mL spectinomycin.

2. Toxicity Test: Upjohn Technical Report 7219-95-024, issued September 1, 1995

  • a. Study Director:
    R.K. Frank, DVM, PhD
    The Upjohn Company
    Kalamazoo, MI
  • d. General Design: 1) Purpose of study: To characterize the margin of safety of formulated drug in the target species following subcutaneous administration once daily for 15 days (3X the maximum label duration of administration) in male and female crossbred beef cattle at 1X, 3X, and 5X the maximum daily recommended therapeutic dose (15 mg/kg/day).

    2) Test Animals: Thirty-two 6- to 12-month-old crossbred beef-type cattle weighing from 271.0 to 376.0 kg were used. Four animals per sex (steers and heifers) were used as controls and 4 animals per sex were administered spectinomycin at a doses of 15, 45, and 75 mg/kg/day.

    3) Control: The control was 0.9% sodium chloride injection, USP (saline) administered subcutaneously at 0.75 mL/kg body weight.

    4) Dosage Form: The test article was the proposed formulation of spectinomycin sulfate solution (ADSPEC™ Sterile Solution).

    5) Doses Used: Daily doses of 15, 45, and 75 mg/kg body weight were given subcutaneously in a sterile solution which contained 100 mg/mL (free base equivalents).

    6) Route: Subcutaneous injection were administered at 2 to 10 sites. No site was injected with more than 30 mL.

    7) Test period: Animals were treated for 15 days and euthanized and necropsied on Study Day 16.

    8) Pertinent Parameters Measured: Toxicological evaluation was based on clinical observations, injection site irritation (30 mL per labeled injection site on study days 1, 8, and 15), food consumption, physical examinations, body weight changes, hematology and serum chemistry parameters, fecal examinations, urinalysis, gross necropsy observations, absolute and relative organ weights, and histologic evaluation of tissues and organs.

e. Statistical Evaluation: Body weights, food consumption, and hematology and serum chemistry parameters were analyzed using a repeated measures analysis of variance, using the average of the pretest values as covariates. If the TREATMENT x TIME interaction was not significant, then treatment groups were compared to the control group using the overall means. If the interaction was significant (p<0.10), the data were analyzed at each time point. Statistical calculations were made using PROC GLM and PROC MIXED of SAS, and group comparisons were made at the p=0.10 level. Urinalysis endpoints and organ weights were analyzed using a one-way analysis of variance. If the overall test for treatment effect was significant, the treatment-to-control comparisons were made using the Least Significant Difference (LSD) method.

f. Results: No deaths occurred prior to study termination. Treatment-related observations included decreased urinary pH and mild swelling at labeled injection sites. The decrease in urinary pH was statistically significant for heifers at the 5X (75 mg/kg/day) dose level only. At necropsy, injection sites of cattle treated with test article on Study Days 8 and 15 had dark red, tan, brown, and/or dark brown areas, often with some expansion (thickening) of the subcutis; only mild discoloration remained at 15 days after injection.

g. Conclusions: Spectinomycin sulfate tetrahydrate solution (100 mg/mL free base equivalents) is safe when injected subcutaneously according to label directions (up to 15 mg/kg/day once daily for up to 5 days). Since gross injection site lesions were observed to persist beyond the established withdrawal period, a statement is included in labeling regarding potential trim loss of edible tissue at slaughter.

B. Corroborative Studies: None.

 

VI. HUMAN SAFETY

A. Toxicity Studies

Repeated-dose toxicity studies with orally administered spectinomycin sulfate tetrahydrate were conducted in rats and dogs. In pivotal GLP toxicity studies, spectinomycin sulfate tetrahydrate was administered at dose levels up to 3000 mg/kg/day (free base equivalents) to rats and dogs for 90 days. Typical antibiotic effects on the intestinal microflora (i.e., soft formless feces/diarrhea) were observed for both rats and dogs following systemic exposure to spectinomycin. However, spectinomycin sulfate tetrahydrate was well tolerated in both species at dose levels up to 3000 mg/kg/day.

Toxicokinetic evaluation indicated that spectinomycin sulfate tetrahydrate is poorly absorbed from the intestinal tract. In the pivotal 90-day rat study, the high-dose (3000 mg/kg/day) group had an average serum concentration of 10.2 µg/mL during a four-hour period following dose administration, while the 400 mg/kg/day and 50 mg/kg/day dose groups had values that were near, or below, the limit of assay quantitation (5 µg/mL). In the pivotal 90-day dog study, toxicokinetic assays of serum samples showed that spectinomycin sulfate tetrahydrate was poorly absorbed at all dose levels and that serum levels increased, but not proportionally, as the dose increased.

In conclusion, the no-observed-effect level (NOEL) for spectinomycin sulfate tetrahydrate in the rat following 90 days of oral administration was 400 mg/kg/day. The no-adverse-effect level for dogs was 50 mg/kg/day following 90 days oral administration of spectinomycin sulfate tetrahydrate. To assess the effects of spectinomycin sulfate tetrahydrate on fertility and general reproductive performance, a pivotal GLP two-generation reproduction study was conducted in rats. In addition, a pivotal GLP Segment II teratology study was conducted in rats to assess the teratogenic and embryotoxic potential of spectinomycin sulfate tetrahydrate. The drug was administered orally in both studies.

Oral treatment of rats with spectinomycin sulfate tetrahydrate at single daily doses of 0, 400, 1000, or 2000 mg/kg/day throughout two generations resulted in pharmacologic effects of soft stools in F0 and F1 males in the 1000 and 2000 mg/kg/day groups, soft stools in the 2000 mg/kg/day F1 females and diarrhea in the 2000 mg/kg/day F1 males. A slight, but statistically significant increase in postpartum body weights of the 1000 and 2000 mg/kg/day F1 females appeared to be treatment-related; however, the body weight change was not considered to be biologically relevant. No treatment-related effects were seen for F0 or F1 copulation and fertility indices, estrous cycle length, gestation length, numbers of live, dead, or abnormal pups, or post-implantational loss.

The results of this study indicated that the NOEL (no-observed-effect level) was 400 mg/kg/day.

In the Segment II teratology study, spectinomycin sulfate tetrahydrate was administered orally at dose levels of 0, 1000, 2000, or 4000 mg/kg/day to rats beginning on day 6 of gestation and continuing through day 17 of gestation. Rats in the 2000 and 4000 mg/kg/day dose groups experienced maternal toxicity which may have led to mild fetal toxicity in the 4000 mg/kg/day dose group, as evidenced by delayed skeletal development. However, spectinomycin sulfate tetrahydrate was not teratogenic at any dose tested in this study. The results of the study indicated the NOAEL in the rat for maternal toxicity was 1000 mg/kg/day, while the NOAEL for developmental toxicity was 2000 mg/kg/day.

Spectinomycin sulfate tetrahydrate was not mutagenic or genotoxic in four in vitro assays: the Ames assay using Salmonella typhimurium strains TA-97a, TA-98, TA-100, TA-102, and TA-1535, the AS52/XPRT mammalian cell forward gene mutation assay, the chromosome aberration assay in Chinese hamster ovary (CHO) cells, and the unscheduled DNA synthesis assay, or in the in vivo micronucleus test in the mouse. The following table summarizes the genetic toxicology profile for spectinomycin sulfate tetrahydrate. Table 6.1: Summary of Mutagenic Potential - Spectinomycin Sulfate Tetrahydrate

Type of StudyResultsInterpretation
Gene Mutation (bacterial)
Ames/Salmonella
NegativeNo evidence of bacterial mutagenicity with or without S9 metabolic activation at doses up to 2000 µg/plate.
Gene Mutation (mammalian)
AS52 Chinese Hamster Ovary Cells (XPRT Locus)
NegativeNo evidence of increase in mutant frequency with or without S9 metabolic activation at dose levels of 313 to 10,000 µg/mL.
Primary DNA Damage
Rat Hepatocyte Primary Culture/Unscheduled DNA Synthesis (UDS)
NegativeNo evidence of increase in UDS over negative controls at dose levels of 10 to 3000 µg/mL.
In Vitro Cytogenetics Chinese Hamster Ovary (CHO) Cells/Chromosome AberrationsNegativeNo evidence of induction of chromosome aberrations with or without S9 metabolic activation at dose levels of 2113 to 5000 µg/mL.
In Vivo Cytogenetics Mouse MicronucleusNegativeNo evidence of induction of micronuclei in polychromatic erythrocytes of mouse bone marrow at dose levels of 625 to 2500 mg/kg.

1. Thirteen-Week Oral Toxicity Study in Rats

  • a. Upjohn Technical Report 7226-95-020, issued November 29, 1995
  • b. Date of First Dose: June 1, 1994
  • c. Date of Last Dose: September 1, 1994
  • d. Study Director: P.K. Brown, B.S.
  • e. Location of Study: The Upjohn Company, Kalamazoo, MI
  • f. Identification of Substance and Dosage Form: Solution (5 or 40 mg/mL [free base equivalents]) or suspension (300 mg/mL [free base equivalents]) of spectinomycin sulfate tetrahydrate in vehicle (purified water, carboxymethylcellulose and benzyl alcohol).
  • g. Species and Strain: Rat, Crl:CD®BR (Sprague-Dawley)
  • h. Number of Animals Per Sex Per Treatment Group: Twenty males and 20 females per dose group were included in Segment A. Ten males and 10 females per dose group were included in Segment B. The first 5 rats/sex/group in Segment B were used for the determination of serum levels of drug. The last 5 rats/sex/group in Segment B were used for interim hematology and serum chemistry determinations.
  • i. Drug Levels Tested and Duration of Dosing: 0, 50, 400, or 3000 mg/kg/day (free base equivalents) administered once daily for 13 weeks. This study was conducted in and inspected for compliance with Good Laboratory Practices Regulations.
  • j. Route of Drug Administration: Drug was administered orally via gastric intubation.
  • k. Parameters Tested: The parameters tested included daily clinical observations, weekly body weight and food consumption, ophthalmologic examinations, hematology and serum chemistry, gross necropsy observations, absolute and relative organ weights, and microscopic evaluation of tissues and organs. Serum drug levels were evaluated in separate groups (Segment B) of identically dosed animals.
  • l. Significant Toxicity Observed: One control female and one 50 mg/kg/day male died (or was sacrificed) before the scheduled necropsy. These early deaths were not considered treatment-related. The only in-life change that was considered to be treatment-related was a change in stool consistency from normal to soft and formless. Soft formless feces were noted for many high-dose (3000 mg/kg/day) male and female rats. Statistically significant increases in cecum weight were observed in all spectinomycin sulfate tetrahydrate-treated groups (male and female) relative to controls. Clinical pathology determinations made on day 28 were unremarkable. Statistically significant decreases in total serum protein and total serum globulin were observed in the high-dose male and female rats following 13 weeks of oral exposure to spectinomycin sulfate tetrahydrate; however, values were still in the low-normal range. The drug was poorly absorbed from the gastrointestinal tract. The high-dose group had an average spectinomycin serum concentration value of 10.2 µg/mL during the four-hour period following dose administration, while the mid-dose group and the low-dose group had values that were below, or near, the limit of assay quantitation (5 µg/mL).
     

    Treatment-related changes were observed microscopically in the stomach and rectum. Vacuolation of the glandular epithelium was seen in the fundic and pyloric regions of the stomach in the high-dose group males and females. The toxicologic significance of the vacuolar epithelial change is not known. The nonglandular stomach and the rectum had treatment-related changes that were considered of doubtful toxicologic importance. These included lymphocytic inflammation of the submucosa of the nonglandular stomach, which was present in one control rat and three high-dose rats, and congestion of the submucosal lymphatics in the rectum, for which the incidence was increased in all treated groups relative to controls.

  • m. No-Observed-Effect Level: The NOEL for this study is 400 mg/kg/day.
  • n. Statistical Analysis: Body weight, food consumption, hematology, serum chemistry and organ weight data from Segment A were analyzed statistically. Treatment group differences were analyzed using analysis of variance on raw data in conjunction with analysis of variance on ranks of data. All statistical calculations were made using PROC GLM of SAS. Statistical analysis was also conducted on hematology and serum chemistry data collected from Segment B rats.
  • o. Conclusions: Once-daily oral administration of spectinomycin sulfate tetrahydrate for 13 weeks to male and female Sprague-Dawley rats (Crl:CD®BR) was well tolerated at doses of 0 (vehicle control), 50, 400, or 3000 mg/kg/day (free base equivalents). Increased cecum weight, soft formless feces and low-normal values for serum protein and serum globulin were seen, but they were considered secondary changes (an antibiotic effect on the intestinal microflora) rather than direct toxic effects resulting from systemic exposure to spectinomycin sulfate tetrahydrate. Spectinomycin was poorly absorbed from the gastrointestinal tract following oral administration and detectable levels of drug were consistently present in only the high-dose group. Vacuolar changes in the epithelium of the glandular stomach (fundic and pyloric regions) were detected microscopically in male and female rats administered 3000 mg/kg/day and the significance of the change is not known. Microscopically detected changes in the nonglandular stomach and the rectum were considered of doubtful toxicologic importance. Based on the results of this study, the no-observed-effect level (NOEL) for 13-week oral administration of spectinomycin sulfate tetrahydrate was 400 mg/kg/day in rats.

2. Ninety-Day Oral Toxicity Study in Dogs

  • a. Upjohn Technical Report 7226-95-013, issued September 26, 1995
  • b. Date of First Dose: July 12, 1994
  • c. Date of Last Dose: October 12, 1994
  • d. Study Director: A.D. Hall, D.V.M., Ph.D.
  • e. Location of Study: The Upjohn Company, Kalamazoo, MI
  • f. Identification of Substance and Dosage Form: Spectinomycin sulfate tetrahydrate was a solution at the low and mid doses and a suspension at the high dose. Test article was formulated in vehicle (0.19% carboxymethylcellulose sodium in an aqueous vehicle containing 0.945% benzyl alcohol) at 5, 40, or 300 mg/mL (free base equivalents).
  • g. Species and Breed: Dog, Beagle
  • h. Number of Animals Per Sex Per Treatment Group: 4/sex/group for a total of 32 dogs
  • i. Drug Levels Tested and Duration of Dosing: 0, 50, 400, or 3000 mg/kg/day (free base equivalents) once daily for 92 (males) or 93 (females) days. This study was conducted in and inspected for compliance with Good Laboratory Practices Regulations.
  • j. Route of Drug Administration: Drug was administered orally via gastric intubation.
  • k. Parameters Tested: Dogs were observed daily for the presence of clinical signs; body weight data were recorded weekly from day -28 to the end of the study. Food consumption data were collected for 4 consecutive days per week and summarized weekly for weeks -3 through 12, except weeks 5 and 9. Ophthalmic examinations were conducted on study days -15 and 84. Blood and urine samples were collected at pretest (study days -26 and -1) and on study days 30, 58, and 86 for hematology, serum chemistry, and urinalysis. Blood samples for evaluation of spectinomycin serum levels prior to dose initiation were collected from all dogs on study day -1; additional samples were collected from the dogs in the 50, 400, and 3000 mg/kg/day groups at 1.5 and 3 hours post dose on study day 1 and at 0 (prior to dosing [nadir]), 1.5 and 3 hours post dose on study days 30, 58, and 86. All dogs from all groups were euthanized and necropsied at the end of the dosing phase. At necropsy, preselected organs were weighed, gross observations were recorded, and protocol tissues were collected and processed for histologic evaluation.
  • l. Significant Toxicity Observed: Excessive salivation at dosing, emesis at dosing, and diarrhea were the clinical signs associated with treatment. The incidence of each sign as well as the frequency of occurrence of diarrhea increased as the dose increased to 400 and 3000 mg/kg/day; however, the frequency of occurrence of salivation and emesis was only notably increased at 3000 mg/kg/day. There were no biologically significant treatment effects on body weight, food consumption, hematology, serum chemistry, or urinalysis. No unscheduled mortality occurred during the study. There were no treatment-related gross lesions, organ weight changes, or histopathologic observations. Despite poor absorption, detectable serum levels of spectinomycin were seen at all three doses (50, 400, and 3000 mg/kg/day). Peak serum levels of spectinomycin were observed at 1.5 hours post dose in all three treatment groups. Although serum levels of spectinomycin increased as the dose increased, the serum level increases were not proportional to the dose increases. There was no evidence of spectinomycin serum accumulation in any of the treatment groups over the course of the 3-month dosing period.
  • m. No-Adverse-Effect Level: 50 mg/kg/day for both male and female dogs
  • n. Statistical Analysis: Body weights, hematology, serum chemistry, urinalysis, and organ weights (absolute and relative to necropsy body weight and relative to brain weight) were analyzed statistically. Food consumption was not analyzed due to the large number of censored data points (animals that ate all their food). Data were analyzed using a two-way analysis of variance which tested for the effects of TREATMENT, SEX, and TREATMENT X SEX interaction. If the interaction was not significant (p>0.10), then the treated groups were compared to the control group for sexes combined using the protected Least Significant Difference method. If the interaction was significant, these comparisons were made separately for male and female dogs. All comparisons were made at the p=0.05 level, except for interaction, which was tested at the p=0.10 level. All statistical calculations were made with PROC GLM in SAS.
  • o. Conclusions: The oral administration of spectinomycin sulfate tetrahydrate as a solution at 0 (vehicle control), 50, or 400 mg/kg/day (free base equivalents) and as a suspension at 3000 mg/kg/day (free base equivalents) to beagle dogs via gastric intubation once daily for 3 months resulted in an increased incidence of salivation, emesis, and diarrhea at 400 and 3000 mg/kg/day as well as an increased frequency of occurrence for these signs at 3000 mg/kg/day; diarrhea was the only clinical sign to show an increased frequency at 400 mg/kg/day. No major treatment-related adverse effects were observed in body weights, clinical pathology, organ weights, or gross and microscopic pathology at doses up through 3000 mg/kg/day during this study. Toxicokinetic assays of serum samples revealed that spectinomycin was poorly absorbed at all doses and that peak serum levels increased with, but not proportional to, dose increases. Nadir (0 hour) serum samples taken on study day 86 indicated that no significant accumulation occurred during the 3-month dosing period. Based on clinical signs, the no-adverse-effect level for the 3-month oral administration of spectinomycin sulfate tetrahydrate was 50 mg/kg/day for both male and female dogs.

3. Two-Generation Oral Rat Reproduction Study

  • a. Upjohn Technical Report 7227-95-034, issued December 6, 1995
  • b. Date of First Dose: January 17, 1994
  • c. Date of Last Dose: September 4, 1994
  • d. Study Director: T.A. Marks, Ph.D. and D. F. Morris, B.S.
  • e. Location of Study: The Upjohn Company, Kalamazoo, MI
  • f. Identification of Substance and Dosage Form: Solution of spectinomycin sulfate tetrahydrate in vehicle (9.45 mg/mL benzyl alcohol and 0.2% carboxymethylcellulose in water) at 20, 50 or 100 mg/mL
  • g. Species and Strain: Rat, Crl:CD(SD)BR (Sprague-Dawley)
  • h. Number of Animals Per Sex Per Treatment Group: 30 males and 30 females per group per generation in the F0 and F1 generations
  • i. Drug Levels Tested and Duration of Dosing: 0 (vehicle control), 400, 1000, or 2000 mg/kg/day. In the first (F0) generation, male rats were dosed for 99 days beginning 70 days prior to cohabitation (mating). F0 females were dosed once daily for 57 to 69 days beginning 14 days prior to cohabitation (mating). Dosing for F1 rats began at 22 days of age (the day after weaning). Dosing continued in F1 males for 72 to 85 and dosing continued in F1 females for 93 to 116 days. This study was conducted in and inspected for compliance with Good Laboratory Practices Regulations.
  • j. Route of Drug Administration: Drug was administered orally by gastric intubation.
  • k. Parameters Tested: F0 and F1 rats were observed daily during treatment for pharmacologic or toxic signs. Body weights were recorded once weekly for males and once weekly prior to confirmed mating for females. Body weights of bred females were recorded on gestation days 0 and 6 and postpartum days 0, 7, 14, and 21. F1 and F2 preweaning pups were counted, sexed and weighed on postpartum days 0, 1, 4, and 21. Estrous cycles were recorded for 7 days prior to dose initiation continuing through the first 14 days of treatment for F0 females and for 14 days prior to cohabitation for F1 females. The number of implantation sites (scars) was recorded at necropsy for gravid F0 and F1 females. Gross lesions seen at necropsy in adults and pups, tissues (heart; lungs; liver; spleen; kidneys; adrenals; and ovaries, vagina, and uterus or testes, epididymides, prostate, and seminal vesicles) from dosed rats that died on study, and reproductive organs (testes, epididymides, prostate, and seminal vesicles) from males whose assigned females were nongravid were saved and examined for histopathologic findings.
  • l. Significant Toxicity Observed: Soft stools were observed in F0 and F1 males in the 1000 and 2000 mg/kg/day groups and in 2000 mg/kg/day F1 females. Diarrhea was noted in 2000 mg/kg/day F1 males. In general, these pharmacologic effects were more pronounced in the males of each generation and the severity increased in the F1 generation. Since other indications of toxicity were not seen, the soft stools and diarrhea, common pharmacologic effects when antibiotics are given at high doses, were not considered to be toxicologic findings. Three rats were found dead, but the deaths were not treatment-related. A slight, but statistically significant increase in postpartum body weights was observed in 1000 and 2000 mg/kg/day F1 females but was not considered to be biologically relevant. No treatment-related effects were seen for F0 and F1 copulation and fertility indices, estrous cycle length, gestation length, numbers of live, dead or abnormal pups, or postimplantational loss. Spectinomycin sulfate tetrahydrate did not affect pup body weights or survival (viability) during lactation in either generation. No treatment-related effects were seen grossly at necropsy or histopathologically.
  • m. No-Observed-Adverse-Effect Level: 2000 mg/kg/day No-Observed-Effect Level: 400 mg/kg/day
  • n. Statistical Analysis: The Modified Cochran-Armitage Chi-square statistic was used to test whether there was a significant dose response in the fertility index. Pairwise comparisons of group proportions were made using a two-sided Fisher's exact test. Postimplantation losses were analyzed using the Modified Jonckheere Ordered Alternatives Test to test for a significant dose-response trend. Jackknife methodology was used to estimate group proportions and make pairwise comparisons among them. Body weights and body weight change, estrous cycle lengths, gestation lengths, and numbers of implants, live pups and dead pups were analyzed using analysis of variance on raw data in conjunction with analysis of variance on ranks of the data. Pup body weights were analyzed using a weighted analysis of variance with the litter as the experimental unit. Pup body weights were analyzed by sex, and also, if appropriate, combined over sexes. Tests of statistical significance were made at the p=0.05 level.
  • o. Conclusions: Spectinomycin sulfate tetrahydrate was administered to male and female rats through two generations at single daily oral doses of 0 (vehicle control), 400, 1000, or 2000 mg/kg/day. The results of this study indicated that the NOAEL (no-observed-adverse-effect level) for maternal and paternal toxicity, spermatogenesis, fertility, length of gestation, parturition, lactation, and offspring survival and growth was 2000 mg/kg/day and the NOEL (no-observed-effect level) was 400 mg/kg/day.

4. Segment II Teratology Study (Oral) in Rats

  • a. Upjohn Technical Report 7227-94-018, issued November 9, 1994
  • b. Date of First Dose: February 7, 1994
  • c. Date of Last Dose: February 21, 1994
  • d. Study Director: D.L. Black, B.S.
  • e. Location of Study: The Upjohn Company, Kalamazoo, MI
  • f. Identification of Substance and Dosage Form: Solution (100 mg/mL) or suspension (200 or 400 mg/mL) of spectinomycin sulfate tetrahydrate in 9.45 mg/mL benzyl alcohol and 0.2% carboxymethylcellulose in water
  • g. Species and Strain: Rat, Crl:CD[BR] (Sprague-Dawley)
  • h. Number of Animals Per Sex Per Treatment Group: 24 females per dose group
  • i. Drug Levels Tested and Duration of Dosing: 0 (vehicle control), 1000, 2000, or 4000 mg/kg/day. Treatments were administered once daily beginning on day 6 of gestation (day 0 was the day of insemination) and continuing through day 17 of gestation. This study was conducted in and inspected for compliance with Good Laboratory Practices Regulations.
  • j. Route of Drug Administration: Drug was administerd orally via gastric intubation.
  • k. Parameters Tested: Maternal body weights were measured on gestation days 0, 6, 9, 12, 15, 18, and 20. Cesarean section data (gestation day 20) included the numbers of corpora lutea, resorptions, and live and dead fetuses, and the sex ratio, body weights and crown-to-rump lengths of live fetuses. The condition of the dam's major abdominal and thoracic organs was evaluated. External, visceral and skeletal examinations were conducted on the live fetuses.
  • l. Significant Toxicity Observed: Soft stools, diarrhea, and blood in the stools were observed in rats treated with 4000 mg/kg/day. Blood in the stool was also observed in 1 female treated at 2000 mg/kg/day. Although the pharmacologic signs soft stools or diarrhea or both were seen in all high-dose rats and in 67% of the mid-dose rats, these findings are common in rats treated with an antibiotic at high-dose levels and are not, by themselves, considered an indication of toxicity. The maternal toxicity experienced by the high-dose dams was not severe enough to affect maternal body weight gain, survival, or fertility. Additionally, embryonic implantation and pre-implantational/post-implantational survival, as well as fetal body weight, crown-to-rump length and sex ratio, were not adversely affected in any group. The maternal toxicity experienced by the high-dose dams may have led to mild fetal toxicity, as evidenced by delayed skeletal development.
  • m. No-Observed-Adverse-Effect Level Maternal toxicity: 1000 mg/kg/day Developmental toxicity: 2000 mg/kg/day
  • n. Statistical Analysis: Body weights and body weight gains, corpora lutea, live fetuses, resorptions and implants were analyzed using analysis of variance on raw data in conjunction with analysis of variance on ranks of the data. Fetal body weights and lengths were analyzed using a weighted analysis of variance with the litter as the experimental unit. Pre- and postimplantational losses were analyzed using the Modified Jonckheere Ordered Alternatives Test for dose response. In the case of a significant trend, Jackknife methodology was used to estimate group proportions and make pairwise comparisons among them. Tests of statistical significance were made at the p=0.05 level.
     

    Statistical analyses were conducted on litter proportions of affected fetuses (number affected/number examined) exhibiting gross malformation, visceral malformations, skeletal malformations, total malformations, gross variations, visceral variations, skeletal variations and total variations. Specific malformations and variations observed were also analyzed. Litter proportions of affected fetuses were analyzed using the Modified Jonckheere Ordered Alternatives Test to test for a significant dose-response trend. Jackknife methodology was used to estimate group proportions and make pairwise comparisons among them. Probabilities associated with the pairwise comparisons are for two-sided t-tests and do not reflect experiment-wise error rates. Tests of statistical significance were made at the 0.05 level.

  • o. Conclusions: Spectinomycin sulfate tetrahydrate was given orally by gastric intubation at dose levels of 0 (vehicle control), 1000, 2000, or 4000 mg/kg/day to groups of 24 female Crl:CD[BR] (Sprague-Dawley) rats once daily beginning on day 6 of gestation (day 0 was the day of insemination) and continuing through day 17 of gestation. Rats in the 4000 mg/kg/day group experienced maternal toxicity which may have led to mild fetal toxicity; however, spectinomycin sulfate tetrahydrate was not teratogenic at any dose. Spectinomycin sulfate tetrahydrate may have been mildly toxic to dams in the 2000 mg/kg/day group; however, no adverse maternal effects were observed in the 1000 mg/kg/day group. Thus, based on the results of this study, the no-observed-adverse-effect level (NOAEL) in the rat for maternal toxicity was 1000 mg/kg/day, while the NOAEL for developmental toxicity was 2000 mg/kg/day.

5. Ames Assay

  • a. Upjohn Technical Report 7228-90-095, issued December 4, 1990
  • b. Beginning of Laboratory Phase: October 31, 1990
  • c. End of Laboratory Phase: November 5, 1990
  • d. Study Director: C. S. Aaron, Ph.D.
  • e. Location of Study: The Upjohn Company, Kalamazoo, MI
  • f. Identification of Substance and Dosage Form: Spectinomycin sulfate tetrahydrate dissolved in dimethylsulfoxide
  • g. Species and Strain: In vitro assay in Salmonella typhimurium strains TA-97a, TA-98, TA-100, TA-102 and TA-1535
  • h. Number of Animals Per Sex Per Treatment Group: Not applicable.
  • i. Drug Levels Tested and Duration of Dosing: 250, 500, 1000 or 2000 µg/plate with and without S9 (rat liver homogenate) metabolic activation.

    Positive controls: with S9: 2-aminoanthracene; without S9: 2-nitrofluorene, sodium azide, cumene hydroperoxide and 4-nitro-o-phenylenediamine.

    Negative control: solvent (dimethylsulfoxide).

    Treatment period: 2 days.

  • j. Route of Drug Administration: Not applicable.
  • k. Parameters Tested: Spectinomycin sulfate tetrahydrate was evaluated for its ability to induce reversion mutations in Salmonella typhimurium strains TA-97a, TA-98, TA-100, TA-102, and TA-1535. The assay measures increases in mutant colonies in histidine-deficient medium and was conducted in and inspected for compliance with Good Laboratory Practices Regulations.
  • l. Significant Toxicity Observed: The data showed no evidence of bacterial mutagenicity at any dose, whether or not exogenous in vitro liver homogenate (S9) was used to supply metabolic activation. Strain-specific positive controls and the solvent (dimethylsulfoxide) control performed within the expected pattern; therefore, the results of these experiments failed to reveal any tendency of spectinomycin sulfate tetrahydrate to produce mutations under the conditions of the test.
  • m. No-Observed-Effect Level: Not applicable.
  • n. Statistical Analysis: Not applicable.
  • o. Conclusions: Based on these results, spectinomycin sulfate tetrahydrate was judged to be nonmutagenic in the Salmonella/microsome test (Ames Assay) at doses of 250 to 2000 µg/plate with and without S9 metabolic activation.

6. AS52/XPRT Mammalian Cell Forward Gene Mutation Assay

  • a. Upjohn Technical Report 7228-94-030, issued July 26, 1994
  • b. Beginning of Laboratory Phase: October 19, 1993
  • c. End of Laboratory Phase: January 18, 1994
  • d. Study Director: L. F. Stankowski, Jr., Ph.D.
  • e. Location of Study: Pharmakon Research International, Inc., Waverly, PA
  • f. Identification of Substance and Dosage Form: Spectinomycin sulfate tetrahydrate suspended or dissolved in dimethylsulfoxide
  • g. Species and Strain: Not applicable (in vitro assay in AS52 [Chinese hamster ovary] cells).
  • h. Number of Animals Per Sex Per Treatment Group: Not applicable.
  • i. Drug Levels Tested and Duration of Dosing: 313, 625, 1250, 2500, 5000, or 10,000 µg/mL with and without S9 (rat liver homogenate) metabolic activation.

    Positive controls: dimethylnitrosamine with S9, ethyl methanesulfonate without S9.

    Negative controls: solvent (dimethylsulfoxide), untreated culture medium.

    Treatment period: 5 hours.

  • j. Route of Drug Administration: Not applicable.
  • k. Parameters Tested: Spectinomycin sulfate tetrahydrate was evaluated for its potential to induce forward gene mutations at the xanthine-guanine phosphoribosyl transferase (XPRT) locus in AS52 cells. The assay measures the production of XPRT-deficient mutants resistant to 6-thioguanine and was conducted in and inspected for compliance with Good Laboratory Practices Regulations.
  • l. Significant Toxicity Observed: Data from the original mutation assay show a statistically significant increase in average mutant frequency for cultures treated with 5000 µg/mL in the presence of S9. This finding was not dose-dependent, did not reach a net increase of 30 mutants/106clonable cells and was not repeated in the confirmatory assay. Thus, the increase observed at 5000 µg/mL in the presence of S9 in the original assay was considered to be not biologically significant. All positive and negative control values in both assays were within acceptable ranges.
  • m. No-Observed-Effect Level: Not applicable.
  • n. Statistical Analysis: The mutant frequency observed at each concentration of test article was compared to that for the pooled concurrent negative controls. A test article was considered positive if it exhibited a dose-dependent increase in average mutant frequencies (least-squares method of linear regression analysis; p<0.05), with at least one concentration producing a statistically significant increase in average mutant frequency that was greater than or equal to two times the average mutant frequency of the pooled concurrent negative controls and represented a net increase of 30 mutants/106 clonable cells. A test article which exhibited only one of these two responses was considered to be equivocal.
  • o. Conclusions: At doses of 313 to 10,000 µg/mL with and without S9 metabolic activation, spectinomycin sulfate tetrahydrate was considered to be negative in the AS52/XPRT mammalian forward cell gene mutation assay under the conditions of the assay.

7. In Vitro Chromosome Aberration Assay

  • a. Upjohn Technical Report 7228-91-007, issued January 28, 1991 b. Beginning of Laboratory Phase: November 7, 1990 c. End of Laboratory Phase: December 4, 1990 d. Study Director: R. Marshall, Ph.D. e. Location of Study: Hazleton Microtest, York, England f. Identification of Substance and Dosage Form: Spectinomycin sulfate tetrahydrate dissolved in culture medium g. Species and Strain: Not applicable (in vitro assay in Chinese hamster ovary [CHO] cells) h. Number of Animals Per Sex Per Treatment Group: Not applicable. i. Drug Levels Tested and Duration of Dosing: Table 6.2: Drug levels tested and duration of dosing in the in vitro chromosome aberration assay
Drug Levels AnalyzedaTreatment Period
Experiment 1: 2113, 3250, or 5000 µg/mL2 hours with S9c with 18-hour recovery; 20 hours without S9
Experiment 2: 2450, 3500, or 5000 µg/mL2 hours with S9 with 18- or 42b- hour recovery; 20 or 44b hours without S9

aSelection of doses for analysis based on effect on mitotic index. Drug levels initially tested but not analyzed include the following:
Experiment 1: 43.75, 67.31, 103.6, 159.3, 245.1, 377.1, 580.1, 892.5, and 1373 µg/mL.
Experiment 2: 201.8, 288.2, 411.8, 588.2, 840.4, 1201, and 1715 µg/mL.

bAnalyzed at 5000 µg/mL only

cS9 (rat liver homogenate) metabolic activation

Positive controls: cyclophosphamide with S9, methyl methanesulfonate without S9

Negative control: solvent (culture medium)

  • j. Route of Drug Administration: Not applicable.
  • k. Parameters Tested: This assay evaluated the clastogenic potential of spectinomycin sulfate tetrahydrate by examining its ability to induce chromosome aberrations in CHO cells. The assay measures increases in the proportions of aberrant cells and was conducted in and inspected for compliance with Good Laboratory Practices Regulations.
  • l. Significant Toxicity Observed: There was no evidence of any induction of chromosome aberrations by spectinomycin sulfate tetrahydrate under the conditions of this in vitro test for chromosomal damage using CHO cells either with or without S9. The frequencies of cells with aberrations were not significantly different from concurrent negative controls and the proportions of cells with aberrations were within historical negative control ranges. The positive and negative controls performed as expected.
  • m. No-Observed-Effect Level: Not applicable.
  • n. Statistical Analysis: The proportion of cells with structural aberrations excluding gaps for each test group was compared with the proportion for the negative controls by Fisher's exact test. Probability values of p<0.05 were accepted as significant.
  • o. Conclusions: Spectinomycin sulfate tetrahydrate was unable to induce chromosome aberrations in CHO cells when tested at doses of 2113 to 5000 µg/mL with and without S9 metabolic activation. Therefore, spectinomycin sulfate tetrahydrate was judged to be nongenotoxic under the conditions of the assay.

8. Unscheduled DNA Synthesis Assay

Drug Levels TestedaTreatment Period
Experiments 1 and 2: 10, 30, 100, 300, 1000, or 3000 µg/mL18-20 hours in both experiments

aDoses were selected according to the standard operating procedure of the test facility, i.e., up to 3000 µg/mL if there is no limitation due to precipitation or toxicity.
Positive control: 2-acetylaminofluorene
Negative control: solvent (culture medium)

  • a. Upjohn Technical Report 7228-90-094, issued December 17, 1990
  • b. Beginning of Laboratory Phase: October 11, 1990
  • c. End of Laboratory Phase: November 8, 1990
  • d. Study Director: C.S. Aaron, Ph.D.
  • e. Location of Study: The Upjohn Company, Kalamazoo, MI
  • f. Identification of Substance and Dosage Form: Spectinomycin sulfate tetrahydrate dissolved in culture medium
  • g. Species and Strain: Not applicable (in vitro assay in rat primary hepatocytes)
  • h. Number of Animals Per Sex Per Treatment Group: Not applicable.
  • i. Drug Levels Tested and Duration of Dosing: Table 6.3: Drug levels tested and duration of dosing in the unscheduled DNA synthesis assay
    Drug Levels TestedaTreatment Period
    Experiments 1 and 2: 10, 30, 100, 300, 1000, or 3000 µg/mL18-20 hours in both experiments

    aDoses were selected according to the standard operating procedure of the test facility, i.e., up to 3000 µg/mL if there is no limitation due to precipitation or toxicity.

    Positive control: 2-acetylaminofluorene

    Negative control: solvent (culture medium)

  • j. Route of Drug Administration: Not applicable.
  • k. Parameters Tested: Spectinomycin sulfate tetrahydrate was evaluated for its potential to induce repairable DNA damage without inhibiting that repair in rat primary hepatocytes. The assay measures increases in net grains/nucleus (NG) and the proportion of cells in repair and was conducted in and inspected for compliance with Good Laboratory Practices Regulations.
  • l. Significant Toxicity Observed: The NG counts were negative for the solvent control and for all doses of spectinomycin sulfate tetrahydrate tested in both experiments. A positive response was demonstrated by the positive control.
  • m. No-Observed-Effect Level: Not applicable.
  • n. Statistical Analysis: Regression analysis was used to test for a significant dose response. Since non-normality and heterogeneity of variance in the data were possible, regression analysis was performed on the ranked NG means as well as the NG means. A Dunnett's t-test was used to compare means of NG data (as well as means of ranked NG data) of each test article treatment group with those of the concurrent solvent control group. The proportions of cells in repair (cells with >5 NG) for the test article treatment groups were tested for a positive dose response using a modified Jonckheere test.
  • o. Conclusions: Under the conditions of this in vitro rat hepatocyte DNA repair assay, the results with spectinomycin sulfate tetrahydrate, at doses of 10 to 3000 µg/mL, were negative.

9. Micronucleus Test

  • a. Upjohn Technical Report 7228-91-010, issued January 28, 1991
  • b. Beginning of Laboratory Phase: November 5, 1990
  • c. End of Laboratory Phase: December 12, 1990
  • d. Study Director: R. Marshall, Ph.D.
  • e. Location of Study: Hazleton Microtest, York, England
  • f. Identification of Substance and Dosage Form: Spectinomycin sulfate tetrahydrate dissolved in 0.9% saline
  • g. Species and Strain: CD-1 mouse
  • h. Number of Animals Per Sex Per Treatment Group: 5/sex/group/sacrifice time (24, 48 and 72 hours after dosing)
  • i. Drug Levels Tested and Duration of Dosing: 625, 1250, or 2500 mg/kg

    Selection of doses was based on the results of a range-finding study which evaluated doses of 126 to 4500 mg/kg in groups of 3 male and 3 female mice. Deaths occurred in 3/3 males and 3/3 females at 4500 mg/kg and the estimated LD50 was 3486 mg/kg. The high dose for the micronucleus test, 2500 mg/kg, was approximately 70% of the estimated LD50.

    Positive control: cyclophosphamide (40 mg/kg; 5/sex sacrificed 24 hours after dosing)

    Negative control: vehicle (0.9% saline)

    Treatment Period: Single dose

  • j. Route of Drug Administration: Intraperitoneal
  • k. Parameters Tested: Spectinomycin sulfate tetrahydrate was evaluated for its ability to induce micronuclei in the polychromatic erythrocytes (PCE) of mouse bone marrow. The test measures increases in the frequencies of micronucleated PCE and was conducted in and inspected for compliance with Good Laboratory Practices Regulations.
  • l. Significant Toxicity Observed: No statistically significant differences in the frequencies of micronucleated polychromatic erythrocytes were observed in any group treated with spectinomycin sulfate tetrahydrate compared to the negative control. The ratios of polychromatic to normochromatic erythrocytes in the treated groups were similar to the negative control at all sampling times. Both the positive and negative controls performed as expected.
  • m. No-Observed-Effect Level: Not applicable.
  • n. Statistical Analysis: Bone marrow slides from all groups sacrificed at 24 hours and from the high-dose and negative control groups sacrificed at 48 and 72 hours were scored. The numbers of micronucleated PCE in each treated group (males and females, separately and combined) were compared with the numbers in the negative control groups by using a 2 x 2 contingency table to determine c². Probability values of p<0.05 were accepted as significant. A linear trend test was used to evaluate possible dose-response relationships.
  • o. Conclusions: Spectinomycin sulfate tetrahydrate was unable to induce micronuclei in polychromatic erythrocytes at doses of 625 to 2500 mg/kg in the mouse bone marrow micronucleus test. Therefore, spectinomycin sulfate tetrahydrate was judged to be nongenotoxic under the conditions of the test.

B. Safe Concentration of Total Residues

1. No-Observed-Effect Level (NOEL):

The safe concentration of total residue was determined from the lowest NOEL in the most sensitive species from the various toxicology studies conducted. The lowest NOEL from the 90-day oral feeding studies was 50 mg/kg body weight (bw)/day in dogs.

2. Calculation of the Acceptable Daily Intake (ADI):

In the revised guideline General Principles for Evaluating The Safety of Compounds Used in Food-Producing Animals (FDA/CVM, revised July 1994), it is indicated that without chronic toxicity data, an ADI limit of 0.025 mg/kg bw/day will be applied. Therefore, an ADI of 0.025 mg/kg bw/day was used to calculate the safe concentration of total residue.

3. Calculations of Safe Concentrations (SC):

For calculations of the SC, the average human weight is approximated at 60 kg. The daily consumption values of edible tissues of cattle are approximated as 300 g muscle, 100 g liver, 50 g kidney, and 50 g fat. Because spectinomycin sulfate sterile solution will not have a label claim for use in lactating dairy cattle, none of the ADI is allocated to milk.

SC = ADI x 60kg /Consumption factor

SC (muscle) = 0.025 mg/kg bw/day x 60kg/0.3kg = 5 ppm

SC (liver) = 0.025 mg/kg bw/day x 60kg/0.1kg = 15 ppm

SC (kidney) = 0.025 mg/kg bw/day x 60kg/0.05kg = 30 ppm

SC (fat) = 0.025 mg/kg bw/day x 60kg/0.05kg = 30 ppm

Table 6.4 shows the safe concentrations for total residues.

Table 6.4. Safe Concentrations (SC) for total residues of spectinomycin sulfate in edible tissues of cattle using the revised food consumption factors

Edible TissueAmount Consumed/DaySafe Concentration (SC)
Muscle300 g5 ppm
Liver100 g15 ppm
Kidney50 g30 ppm
Fat50 g30 ppm

4. Threshold Assessment

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 on specific animals, it is considered a Low Use Drug. Accordingly, chronic studies were not required and based on the 90-day studies, a Safety Factor of 1000 was used in the Safe Concentration calculations.

C. Total Residue Depletion and Metabolism Studies

A total residue and metabolism study was conducted by R. E. Hornish, Ph.D. at the Animal Health Drug Metabolism, Pharmacia & Upjohn, Inc., Kalamazoo, Michigan and K. L. Davison, Ph.D. at USDA/ARS Biosciences Research Laboratory, Fargo, North Dakota. The purpose of the study was to determine the disposition and metabolism of 3H-spectinomycin sulfate that was subcutaneously injected into cattle.

Eighteen beef cattle (9 steers, 9 heifers), weighing 157-187 kg were used. Sixteen animals were injected subcutaneously with 15 mg 3H-spectinomycin/kg body weight daily for 5 consecutive days. The radiolabeled test article was labeled with 3H on the 6'-methyl. Two control cattle (1 steer, 1 heifer) received a placebo vehicle by subcutaneous (SC) injection one time. Blood, urine, and feces were collected at 24-hour intervals during treatment and after dosing until slaughter. The control cattle were slaughtered one day following the placebo treatment. The treated cattle were slaughtered at 1, 5, 10, and 15 days after the fifth dose. Intestinal contents, liver, kidney, muscle, fat, fifth injection site, lungs and heart were collected. Tissue and fluids were analyzed for total radioactive residues and metabolic profiles.

The drug was eliminated mostly by renal excretion; 70% of the dose for heifers and 83% of the dose for steers was recovered in the urine. Feces accounted for 10.8% of the dose for heifers but only 3% for the steers. Greater than 90% of the excreted dose was eliminated within 24 hours after the last dose. The terminal elimination phase of the total residue plasma pharmacokinetics had a half-life of 8 days. The mean disposition as a percent of dose is shown in Table 6.5.

Table 6.5 Summary of dose accountability

Withdrawal TimeMean (n=4) Percent of Dose ± StdD
(Days)UrineFecesTissuesTotal
169.35 ± 10.047.67 ± 7.183.30 ± 0.4280.31 ± 3.29
584.48 ± 7.515.32 ± 2.691.42 ± 0.1091.23 ± 4.91
1077.07 ± 8.746.26 ± 3.940.89 ± 0.1384.21 ± 5.18
1577.17 ± 8.348.35 ± 6.460.71 ± 0.0986.22 ± 5.70

Tritiated water accounted for less than 4% of the dose and confirmed the utility of 6'[3H]-methyl as a viable radiolabel to study the in vivo disposition and metabolism of spectinomycin. Eight metabolites were found in urine by HPLC radiotracer techniques. Parent spectinomycin accounted for approximately 62-64% of the on-treatment urinary residue. All other metabolites were minor components. The only significant residue in kidney was parent spectinomycin. In contrast, the only significant residue in liver was dihydrospectinomycin. Kidney is an appropriate target tissue for residue monitoring, and parent spectinomycin is the appropriate marker residue. The total residue and parent spectinomycin concentration in kidney and liver are shown in Table 6.6.

Table 6.6 Summary of total residue and of parent spectinomycin in kidney and liver

Withdrawal Time
(Days)
Mean (n=4) of Total Residue and of Parent Spectinomycin
(µg/g ± StdD)
 KidneyLiver
 Total ResidueParentTotal ResidueParent
159.6 ± 8.69.12 ± 1.2432.5 ± 5.81.36 ± 0.18
514.2 ± 3.61.74 ± 0.7518.8 ± 4.10.58 ± 0.12
104.5 ± 0.180.42 ± 0.027.55 ± 2.40.20 ± 0.09
152.66 ± 1.840.20 ± 0.214.56 ± 1.660.14 ± 0.06

A comparative metabolism study in rats was conducted by R. E. Hornish, Ph.D. at Pharmacia & Upjohn, Inc., Animal Health Drug Metabolism, Kalamazoo, Michigan. The purpose of the study was to determine the disposition and metabolism of 3H-spectinomycin sulfate in rats.

Eight Sprague-Dawley rats (3 males, 3 females), weighing 217-260 grams were used. Six rats were administered 7 consecutive daily oral gavage doses of 3H-spectinomycin at a constant dose of 25 mg/day (96-110 mg of spectinomycin free base equivalents per kg of body weight). The radiolabeled test article was labeled with 3H on the 6'-methyl. Two control rats (1 male, 1 female) were administered placebo for 6 days. Blood and urine were collected daily. The animals were slaughtered 4 hours after the final dose. Blood, kidney, liver, muscle, and abdominal fat were collected. Total residues and metabolic profiles were determined.

The percentage of tritiated water in urine varied from 1.4% to 17.3% with the mean of 7.5%. The percentage of tritiated water in the feces varied from 0.4% to 5.7% with mean of 1.0%. Tritiated water accounted for nearly all of the residue in the muscle, liver, kidney, and plasma. Overall tritiated water accounted for 1% of the dose. The urine, tissues, and feces were analyzed for metabolite profiles and identification. Several metabolites were found in the urine and feces by HPLC with a radioactivity detector. HPLC/APCI/MS produced tentative identification for some of these. Parent spectinomycin was the major residue in the feces, accounting for 62%. All other metabolites in the feces were minor. In the urine, parent spectinomycin was a minor component of the residue. HPLC/APCI/MS analysis produced tentative identification for some of these metabolites and are proposed to be dihydroxyspectinomycin, acetylated spectinomycin, and dihydrospectinomycin. These same metabolites were observed in the urine from cattle treated subcutaneously with spectinomycin.

D. Tolerance for the Marker Residue

The above studies show that parent spectinomycin was the only major residue found in kidney, and therefore, is an appropriate target tissue for residue monitoring programs. Data from the radiolabel residue decline study were used to determine the relationship of parent spectinomycin to total residue and to establish the tolerance concentration of parent spectinomycin (Rm) relative to the safe concentration of total residue in kidney. Based on the relationship of parent spectinomycin to total residue and a safe concentration of 30 ppm total residue, a tolerance (Rm) of 4 ppm was established for the marker residue, parent spectinomycin, in cattle kidney.

E. Tolerance for Parent Spectinomycin in Muscle

Data from the radiolabel residue decline study and tissue residue depletion study were used to determine the relationship of parent spectinomycin to total residue and to establish the tolerance concentration of parent spectinomycin relative to the safe concentration of total residue in muscle. Based on the relationship of parent spectinomycin to total residue and a safe concentration of 5 ppm total residue, a tolerance of 0.25 ppm was established for parent spectinomycin in cattle muscle.

F. Study Establishing Withdrawal Period

A residue depletion study was conducted by R. E. Hornish, Ph.D. at Pharmacia & Upjohn, Inc., Animal Health Drug Metabolism, Kalamazoo, Michigan. The purpose of the study was to determine the depletion of spectinomycin in cattle following administration of subcutaneous injections of 15 mg spectinomycin/kg body weight every 24 hours for 5 consecutive days.

Twenty-four beef cattle (12 steers, 12 heifers), weighing 217-284 kg were used. The animals were injected subcutaneously with 15 mg spectinomycin/kg body weight daily for 5 consecutive days. Groups of 6 animals (3 males, 3 females) were slaughtered at 5, 10, 15, and 20 days following the fifth dose. Kidneys, liver, the fifth injection site, muscle, and fat were collected and assayed for parent spectinomycin using the determinative method described below. Liver was assayed for parent spectinomycin by a reverse phase chromatographic procedure (Weist RP method). Residues depleted most slowly from kidney.

Table 6.7 Parent spectinomycin in tissues of cattle injected subcutaneously with 15 mg spectinomycin/kg body weight daily for 5 consecutive days

Withdrawl TimeMean Concentration (n=6), µg/g ± StdD
(Days)Kidney*Liver**
53.97 ± 1.280.28 ± 0.04
100.97 ± 0.560.12 ± 0.01
150.27 ± 0.09<0.07
200.16 ± 0.07<0.07

*Limit of Quantitation = 0.10µg/g
**Limit of Quantitation = 0.07µg/g

The statistical method used to calculate the withdrawal time was that described in the Guideline for Establishing a Withdrawal Period, guideline VI found in General Principles for Evaluating The Safety of Compounds Used in Food-Producing Animals (FDA/CVM, July 1994). Using a statistical tolerance limit for the 99th percentile of the population with 95% confidence, a withdrawal time of eleven days was assigned for cattle administered subcutaneous injections of 10 to 15 mg spectinomycin/kg body weight every 24 hours for 3 to 5 consecutive days.

G. Regulatory Methods

1. Determinative Method

a. Type: The method utilizes an HPLC ion exchange separation with post column oxidation and derivatization to enable fluorescence detection of the analyte.

b. Scope: This method describes a high-performance liquid chromatographic (HPLC) quantitative analysis of parent spectinomycin residue in bovine kidney over a range of 1 µg/g to 10 µg/g.

c. Applicability: The high performance attainable with this method requires strict attention to system suitability parameters by sufficiently experienced laboratory analysts. The method is not intended to be used in a occasional manner, but is best applied in sustained residue analytical programs.

d. Principle: The isolation of parent spectinomycin residue from kidney is accomplished in a two-step procedure involving solvent extraction and solid phase extraction (SPE) clean-up followed by HPLC analysis. The spectinomycin residue is extracted from the kidney with a mixture of citrate buffer, trichloroacetic acid, and dichloromethane. The mixture is shaken then centrifuged to separate the layers. The supernatant is decanted into a clean tube, the pH adjusted to 6.8, then passed through a carboxylic acid SPE cartridge to trap the parent spectinomycin residue. Parent spectinomycin is liberated from the SPE cartridge with a citrate buffer. An aliquot of the volume-adjusted SPE eluant is analyzed by the HPLC procedure. A set of 12 samples can be processed in 6-8 hours.

e. Limit of Quantitation: 0.10 µg/g

f. Results of Method Validation: The Methods Trial of the determinative method for parent spectinomycin in bovine kidney was successfully completed by FDA and non-FDA laboratories. The method has been validated to measure parent spectinomycin residues at the tolerance of 0.4 micrograms/gram.

g. Method Availability: The validated regulatory analytical determinative method for parent spectinomycin in bovine kidney is on display in Dockets Management Branch (HFA-305), FDA/CVM, 12420 Parklawn Dr., Rockville, MD 20857.

2. Confirmatory Method

a. Type: The method utilizes a high performance liquid chromatograph (HPLC) interfaced to a triple-quadrupole mass spectrometer (MS/MS) with an atmospheric pressure chemical ionization (APCI) interface. Identification is accomplished by a collision induced dissociation (CID) mass spectrometric (MS/MS) technique, commonly referred to as HPLC/MS/MS.

b. Scope: This method describes a confirmatory procedure for the identification of parent spectinomycin residue in bovine kidney over a range of 1 µg/g to 10 µg/g. Although the method is described for the identification of parent spectinomycin in bovine kidney, the procedure is equally applicable to other tissues as well - muscle, liver, and fat.

c. Applicability: The high performance attainable with this method requires strict attention to system suitability parameters by sufficiently experienced laboratory analysts. The method is not intended to be used in an occasional manner, but is best applied in sustained residue analytical programs.

d. Principle: The isolation of parent spectinomycin residue from kidney is accomplished in a two-step procedure involving solvent extraction and solid phase extraction (SPE) clean-up followed by HPLC separation prior to APCI/MS/MS. The spectinomycin residue is extracted from the kidney with a mixture of citrate buffer, trichloroacetic acid and dichloromethane. The mixture is shaken then centrifuged to separate the layers. The supernatant is decanted into a clean tube, the pH adjusted to 6.8, then passed through a carboxylic acid SPE cartridge to trap the parent spectinomycin residue. Parent spectinomycin is liberated from the SPE cartridge with a citrate buffer. An aliquot of the volume-adjusted SPE eluant is analyzed by the HPLC/APCI/MS/MS procedure. The protonated molecular ion of spectinomycin, which is generated in the APCI source, is selectively filtered through quadrupole 1 into quadrupole 2 where it undergoes collisionally induced dissociation with energized argon atoms into various characteristic fragment ions (m/z 189, 158, 116, and 98) which are then analyzed in quadrupole 3. A set of 12 samples can be processed in 6-8 hours. Each HPLC/MS/MS run takes about 5 minutes per sample.

e. Limit of Confirmation: 0.10 µg/g

f. Results of Method Validation: The Methods Trial of the confirmatory method for parent spectinomycin in bovine kidney was successfully completed by an FDA laboratory.

g. Method availability: The validated regulatory analytical confirmatory method for parent spectinomycin in bovine kidney is on display in the Dockets Management Branch (HFA-305), Park Building (Room 1-23), 12420 Parklawn Drive, Rockville, MD 20857.

H. User Safety

User safety concerns regarding potential sensitization and/or allergic reaction resulting from accidental exposure have been satisfactorily addressed by establishing label warnings. In addition, a toll-free telephone number will be available on the label to inform users of where they can obtain additional information concerning user safety relative to the MSDS and to report adverse events.

 

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 Title 21, Part 514 of the implementing Code of Federal Regulations (21 CFR 514). The data demonstrate that ADSPECTM Sterile Solution, an aminocyclitol antibiotic, when administered subcutaneously to cattle as a daily injection at a dose of 10 to 15 mg/kg body weight for 3 to 5 days, is safe and effective for the treatment of bovine respiratory disease (pneumonia) associated with Pasteurella haemolytica, Pasteurella multocida, and Haemophilus somnus.

Based on a battery of toxicology tests, an acceptable daily intake of 25 mcg/kg body weight/day was calculated, which further yielded safe concentrations for total spectinomycin-related residues of 5 ppm in muscle, 15 ppm in liver, 30 ppm in kidney, and 30 ppm in fat. A tolerance of 4 ppm for parent spectinomycin (the marker residue) in kidney (the target tissue) is established for the subcutaneous treatment of cattle with spectinomycin. Based on the relationship of parent spectinomycin to total residue and a safe concentration of 5 ppm total residue, a tolerance of 0.25 ppm was established for parent spectinomycin in cattle muscle. The tolerance refers to the residue measured by the regulatory method described herein.

A pre-slaughter withdrawal period of 11 days was assigned for cattle administered subcutaneous injections of 10 to 15 mg spectinomycin/kg body weight once daily for 3 to 5 consecutive days. The withdrawal period was based on a statistical analysis of the depletion data, using an upper tolerance limit containing 99 percent of the population with a 95 percent confidence limit.

Labeling restricts this drug to use by or on order of a licensed veterinarian. This decision was based on the following factors: a) adequate directions cannot be written to enable lay persons to appropriately diagnose and subsequently use this product to treat bovine respiratory disease (BRD) associated with Pasteurella haemolytica, Pasteurella multocida, and Haemophilus somnus, (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 spectinomycin-resistant organisms may be reduced by the involvement of veterinarians in product use.

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)(ii) of the Federal Food, Drug, and Cosmetic Act, this approval for food producing animals qualifies for THREE years of marketing exclusivity beginning on the date of approval because the application contains substantial evidence of the effectiveness of the drug involved, any studies of animal safety, or, in the case of food producing animals, human food safety studies (other than bioequivalence or residue studies) required for the approval of the application and conducted or sponsored by the applicant. ADSPECTM Sterile Solution is not under any unexpired U.S. patents.

 

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