Approval Date: January 25, 1988
Freedom of Information Summary
NADA 140-338
I. GENERAL INFORMATION:
NADA 140-338 Sponsor: The Upjohn Company
7000 Portage Road
Kalamazoo, Michigan 49001Generic Name: ceftiofur sodium sterile powder Trade Name: Naxcel Sterile Powder Marketing Status:
II. INDICATIONS FOR USE:
Naxcel Sterile Powder is indicated for treatment of bovine respiratory disease (shipping fever, pneumonia).III. DOSAGE FORM, ROUTE OF ADMINISTRATION AND RECOMMENDED DOSAGE:
Naxcel Sterile Powder is available in two package sizes: 1 gram and 4 gram vials.
Reconstituted product should be used within 12 hours if stored at controlled room temperature 15°-30°C (59°-86°F) or within 5 days if stored in a refrigerator.
1 gram vial
Reconstitute with 20 ml Sterile Water for Injection or with Bacteriostatic Water for Injection. Each ml of the resulting solution contains ceftiofur sodium equivalent to 50 mg ceftiofur.
4 gram vial
Reconstitute with 80 ml Sterile Water for Injection or with Bacteriostatic Water for Injection. Each ml of the resulting solution contains ceftiofur sodium equivalent to 50 mg ceftiofur.
Naxcel Sterile Solution should be administered by intramuscular injection to cattle at the dosage of 0.5 mg ceftiofur per pound of body weight (1 ml reconstituted sterile solution per 100 lb body weight). Treatment should be repeated every 24 hours for a total of three treatments. Additional treatments may be given on days four and five for animals which do not show a satisfactory response (not recovered) after the first three treatments.
IV. EFFECTIVENESS:
A. Pivotal Studies (4)
Summary
Four separate studies were completed to determine the efficacy of ceftiofur for treatment of bovine respiratory disease (BRD also referred to as shipping fever or bovine pneumonia). Each is considered a "pivotal" study. No "corroborative" efficacy studies were conducted.
The initial cattle study included 84 head with naturally occurring disease, 42 non-medicated controls and 42 ceftiofur treated animals. Drug treated animals each received ceftiofur at 1 mg/lb body weight intramuscularly for three consecutive days. Results showed that this drug, ceftiofur, was effective at 1 mg/lb body weight for treatment of BRD (see Table in section 1(d) for details).
In the first dose response study 220 cattle with spontaneously occurring disease were assigned to 44 blocks of five animals (treatments) per block. Treatments were 0, 0.5, 1.0, 1.5 or 2.0 mg/lb body weight given intramuscularly for three consecutive days. Results from this study showed all drug concentration treatment groups to be effective relative to no therapy and not different from each other (see Table in section 2(d) for details).
A second dose response study was conducted using 235 cattle blocked into 47 blocks of five animals (treatments) per block. Doses evaluated were 0, 0.125, 0.25, 0.50 and 1.0 mg/lb body weight given intramuscularly for three consecutive days. Results showed 0.5 and 1.0 mg/lb treatment groups to be similar and each superior to the non-medicated control group. Other treatment groups were not deemed different either from control or from the two higher dosed treatment groups.
Collectively considering data from the three previously described trials, a dose of 0.5 mg/lb body weight was the optimal dose of ceftiofur for treatment of BRD.
Finally, a seven location clinical trial was conducted. This included 629 cattle with spontaneously occurring disease and three treatment groups. These were 1) non-medicated control, 2) ceftiofur at 0.5 mg/lb and 3) ceftiofur at 1.0 mg/lb body weight. Placebo and ceftiofur treatments were administered by intramuscular injection once per day for three or five days. Results indicated that each of the two ceftiofur doses (0.5 and 1.0 mg/lb) was significantly better than placebo. The two ceftiofur dosed groups provided similar responses.
1. Initial Efficacy Study
(a) Type of Study
The first cattle study was an efficacy trial using calves with spontaneously occurring disease to determine whether or not ceftiofur would be effective for treatment of BRD when administered intramuscularly at 1.0 mg/lb body weight for three consecutive days.
(b) Investigator
G. G. Stocking, D.V.M.
The Upjohn Company
Kalamazoo, Michigan 49001(c) General Design
(d) Results Pasteurella hemolytica was present in 80 percent of the pre-treatment nasal swabs. None of the swabs showed growth of P. multocida or Hemophilus somnus . Mycoplasmas were isolated from 94 percent of the nasal swabs. Of the 20 percent of swabs without Pasteurella hemolytica , seven percent were in the ceftiofur treated group and 13 percent were in the control group. Of the six percent of swabs without Mycoplasma , all were in the ceftiofur treated group.
Purpose of this study was to determine whether ceftiofur would be effective for treatment of BRD in cattle with spontaneously occurring disease. Animals - Feeder cattle ranging in weight from 350 to 456 lbs
- approximate age was 6 mos.
- mixture of bulls and steers of Angus, Hereford and Angus/Hereford crossbreeding
- 42 head for each of two treatment groups.
Control was a non-drug sterile diluent administered intramuscularly at 8 ml/animal for three consecutive days. Diagnosis BRD was diagnosed by an elevated body temperature (104.5°F or greater) and the presence of at least two additional signs of the disease such as depressed physical appearance, increased or labored respiration, cough and anorexia. Nasal swabs were taken from each animal just prior to treatment to determine presence of BRD pathogens.
Dosage Form Dosage from was an aqueous solution containing 50 mg ceftiofur equivalents per ml.
Route of Administration: Intramuscular. Dose Doses tested were 0 and 1.0 mg ceftiofur per lb body weight administered intramuscularly three consecutive days.
Test Duration: This was a 28 day study. Pertinent Parameters Measured:
The primary parameter for determining drug effectiveness in this preliminary study was body temperature drop between days one and four of the study. Other measurements were:
gain or loss in body weight at days 4, 10, 28 of the study success (clinical) on days 4, 10, 28 of the study mortality at days 4, 10, 28 of the study. (Eds. note: The following table consists of 7 columns.)
Treatment Means ------------------------Day of Study------------------------ --------4-------- --------10------- --------28------- Variable Control Treated Control Treated Control Treated Body Temp.Drop 1.0 2.9* 1.0 1.9* -- -- Body Weight -4.2 4.1* -7.6 2.1* 9.2 17.1 Change (kg) Clinical Success 57 86 45 74 55 71 Mortality (%) 7.1 0 16.7 2.4 31.0 7.1 * Significant difference, at P ¾ 0.05, from control mean for same day of study.Cause of death for the 16 calves that died during the study was directly related to the severe bronchopneumonia that the cattle experienced during the course of their illness.
(e) Statistical Analysis
A randomized complete block design was used with each block consisting of two animals with similar body temperatures and body weights. The following analysis was used for body temperature drop and body weight changes:
(Eds. note: The following table consists of 3 columns.)
Source Degrees of Freedom Test Term Blocks 41 -- Treatment 1 Error Error 41 Differences between treatment and control groups were based on P ¾ 0.05 level of significance.(f) ConclusionsResults from this preliminary study showed that ceftiofur administered at an intramuscular dose of 1.0 mg/lb body weight was effective for treatment of BRD.
(g) Adverse Reactions
Except for an immediate and transient pain reaction to the local intramuscular injection of ceftiofur, there was no clinical evidence of adverse effect.
(h) Special Issues: None.
2. First Dose Response Trial
(a) Type of Study
Dose response study in cattle with spontaneously occurring BRD.
(b) Investigator
G. G. Stocking, D.V.M.
The Upjohn Company
Kalamazoo, Michigan 49001(c) General Design
(d) Results Pasteurella hemolytica was isolated from 77 percent of the cattle from the pre-treatment nasal swabs. Pasteurella multocida was found in three percent of the cattle. Mycoplasmas were present in the 92 percent of the cattle and there were only nine animals, of the 200, whose nasal swabs produced no organisms. These nine were distributed among the treatment groups as follows: 0.0 mg/lb-l, 0.5 mg/lb-2, 1.0 mg/lb-O, 1.5 mg/lb-4, 2.0 mg/lb-2.
Purpose of this study was to determine the optimum dose of ceftiofur for the treatment of BRD. Animals - 220 head of feedlot cattle ranging in initial weight from 330 to 486 lbs
- approximate age was 6 months
- mixture of Angus/Hereford pure and crossbred bulls and steers
- 44 head per treatment group.
Control was a non-drug sterile diluent administered intramuscularly at 8 ml/animal for three consecutive days. Diagnosis: BRD was diagnosed by an elevated body temperature (104.5°F or greater) and the presence of at least two additional signs of the disease such as depressed physical appearance, increased or labored respiration, cough and anorexia. Nasal swabs were taken from each animal just prior to treatment to determine presence of BRD pathogens.
Dosage Form: Dosage form was an aqueous solution containing 50 mg ceftiofur equivalent per ml.
Route of Administration: Intramuscular. Dose: Doses tested were 0, 0.5, 1.0, 1.5 and 2.0 mg/lb body weight administered intramuscularly three consecutive days.
Test Duration: This was a 28 day study. Pertinent Parameters Measured: - body temperature drop at day 4
- clinical success at day 10
- clinical success at day 28
- mortality by day 28
(Eds. note: The following table consists of 7 columns.)
Treatment Means Tempera- Tempera- Percent Percent ture Drop ture Clinical Clinical Percent Treatment Day 4 Day 4 Success/ Success/ Mortality mg/lb n (Degrees F) (Degrees F) Day 10 Day 28 Day 28 0.0 44 0.70 104.7 30 45 41 0.5 44 2.78 102.7 66 66 16 1.0 44 2.90 102.6 66 59 09 1.5 44 2.87 102.7 70 57 20 2.0 44 3.08 102.4 70 59 20 LSD* 0.36 0.34 17 17 14 * Least significant difference (P = .05) one-sided comparison of any dose with control.
Based on results of clinical, gross, histopathological and bacteriologic findings, the cause of death for the 47 calves that died during the 28 day period was consistent with BRD.
(e) Statistical Analysis
A randomized complete block design was used with five animals of similar body temperatures and initial body weights considered a block. The following analysis was conducted for each of the four critical variables:
(Eds. note: The following table consists of 3 columns.)
Source Degrees of Freedom Test Term Blocks 43 -- Treatments(a) 4 Error Error 172 (a) Partitioned into linear, quadratic and remainder for trend evaluation.(f) Conclusions This study demonstrated that ceftiofur at doses of 0.5, 1.0, 1.5 and 2.0 mg/lb body weight, by intramuscular injection for three consecutive days, is effective for the treatment of BRD.
Interpretations.
Body temperature drop: All drug treatment groups were significantly improved relative to control and not different from each other. Success rate at day 10: All drug treatment groups were significantly improved relative to control and not different from each other. Success rate at day 28: The 0.5 mg/lb treatment group was significantly improved relative to control. No other significant difference existed. Mortality: All drug treatment groups were significantly better (lower mortality) than control and these were not significantly different among drug treatment groups. Significance level used: P ¾ 0.05. (g) Adverse Reactions
Except for an immediate and transient local pain reaction to the intramuscular injection of ceftiofur, there was no evidence of any adverse effect.
(h) Special Issues: None.
3. Second Dose Response Trial
(a) Type of Study
Dose response study in cattle with spontaneously occurring BRD.
(b) Investigator
G. G. Stocking, D.V.M.
The Upjohn Company
Kalamazoo, Michigan 49001(c) General Design
(d) Results Pasteurella hemolytica was isolated from 128 (54%) of the 235 pre-treatment nasal swabs of cattle used in this study. Pasteurella multocida was found in two (0.8%) of the cattle. Although Hemophilus somnus was not detected from any of the nasal swabs, it was isolated from 18 (26%) of the lungs from cattle that died during the 28 day study, indicating that it was a part of the etiology of the disease in the study. Mycoplasmas were isolated from 199 (85%) of the nasal swabs and there were six animals whose nasal swabs produced no organisms. These were distributed among treatment groups as follows: 0, 3, 0, 1 and 2 at doses 0, 0.125, 0.25, 0.50 and 1.0 mg/lb body weight, respectively.
Purpose of this study was to determine the optimum dose of ceftiofur for the treatment of BRD. Animals - 235 head of feedlot cattle ranging in weight from 300-540 lbs.
- calves ranged in age from six to ten months,
- mixture of Angus/Hereford pure and crossbred bulls and steers
- 47 head per treatment group.
Control was a non-drug sterile diluent administered intramuscularly at 8 ml/animal for three consecutive days. Diagnosis BRD was diagnosed by an elevated body temperature (104.5°F or greater) and the presence of at least two additional signs of the disease such as depressed physical appearance, increased or labored respiration, cough and anorexia. Nasal swabs were taken from each animal just prior to treatment to determine presence of BRD pathogens.
Dosage Form: Dosage form was an aqueous solution containing 50 mg ceftiofur equivalents per ml.
Route of Administration: Intramuscular. Dose: Doses tested were 0, 0.125, 0.25, 0.5 and 1.0 mg ceftiofur per lb body weight administered intramuscularly three consecutive days.
Test Duration: This was a 28 day study. Pertinent Parameters Measured: - body temperature drop at day 4
- clinical success at day 10
- clinical success at day 28
- mortality by day 28
(Eds. note: The following table consists of 7 columns.)
Treatment Means Tempera- Tempera- Percent Percent ture Drop ture Clinical Clinical Percent Treatment Day 4 Day 4 Success/ Success/ Mortality mg/lb n (Degrees F) (Degrees F) Day 10 Day 28 Day 28 0.000 47 0.43 105.2* 31 39 38 0.125 47 1.86 103.8 45 51 30 0.250 47 1.89 103.7 36 36 32 0.500 47 2.18 103.4 55 51 23 1.000 47 2.41 103.2 43 51 23 LSD** 0.35 0.35 17 17 15 * N = 46 ** Least significant difference (P=.05) one sided comparison of any dose with control.Based on results of clinical, gross, histopathological and bacteriologic findings, the cause of death for the 69 calves that died during the 28 day period was consistent with BRD. (e) Statistical AnalysisA randomized complete block design was used with five animals of similar body temperatures and initial body weights considered a block. The following analysis was conducted for each of the four critical variables:
(Eds. note: The following table consists of 3 columns.)
Source Degrees of Freedom Test Term Blocks 46 -- Treatments(a) 4 Error Error 184 (a) Partitioned into linear, quadratic and remainder for trend evaluation.(f) Conclusions In this study, ceftiofur at doses of 0.5 and 1.0 mg/lb body weight, given intramuscularly for three days, was effective for treatment of BRD.
Interpretations
Body temperature drop: All drug treatment groups were significantly improved relative to non-medicated controls. Treatment group 0.5 was not significantly different from any other drug group but the 1.0 mg/lb group was significantly improved relative to the 0.125 and 0.25 mg/lb treatment groups. Success rate at day 10: The 0.5 mg/lb group was significantly better than non-medicated controls at day 10. No other differences among groups were significant. Success rate at day 28: No differences among groups were significant at day 28. Mortality: Treatment Groups 0.5 and 1.0 mg/lb were not different from each other and each was significantly different from controls. No other differences among treatments were significant. Significance level used for all comparisons: P ¾ .05. (g) Adverse Reactions
Except for an immediate and transient local pain to the intramuscular injection of ceftiofur, there was no evidence of any adverse effect.
(h) Special Issues: None.
4. Interpretation of Data from Two Multi Dose Trials and One Single Drug Dose Trial
(a) Type of Study
This summary is from the first three described under "Pivotal Studies".
(b) Results
(Eds. note: The following table consists of 6 columns.)
Treatment Means Percent Percent Cumulative Dose Number of Temp. Drop Clinical Clinical Percent mg/lb body Animals per Day 4 Success/ Success/ Mortality wt. Group (Degrees F) Day 10 Day 28 Day 28 0.0 133 0.68 35 46 37 0.125 47 1.86 45 51 30 0.25 47 1.89 36 36 32 0.5 91 2.47 60 58 20 1.0 133 2.72 60 60 14 1.5 44 2.87 70 57 20 2.0 44 3.08 70 59 20 LSD (0.05) .35 35 17 15The treatment means are unadjusted for differences that occurred between trials. The control group and the 1.0 mg/lb group were common to all three studies. The LSD values (0.05) are an over estimate since they are the values associated with sample sizes of 44 cattle per treatment group. The LSD values were nearly identical across the three studies, therefore, combined LSDs could only be smaller. Consequently, any difference significant with the above listed LSDs would be more significant with LSDs calculated from the combined data. (c) ConclusionsThe combined set of data from the three previously described studies support a conclusion that 0.5 mg/lb is an effective dose for the treatment of BRD.
5. Multi-Location Clinical Trial
(a) Type of Study
This study was a multi-location clinical trial in cattle with spontaneously occurring disease under typical feedlot conditions.
(b) Investigators
D. T. Bechtol, D.V.M.
Palo Duro Vet. Services
Route 1, Box 37
Canyon, Texas 79015Joseph G. Clark, D.V.M., M.S.
210 K Street
Brawley, California 92227Alvin J. Edwards, D.V.M., Ph.D.
Dept. of Surgery and Medicine
College of Vet. Medicine
Kansas State University
Manhattan, Kansas 66506Dallas P. Horton, D.V.M., Ph.D.
Horton Feedlot and Res. Ctr.
5100 E. County Rd. 70
Wellington, Colorado 80549Daryl G. Meyer, D.V.M.
Route 2, Box 23A
Gothenburg, Nebraska 69138Robert D. Glock, D.V.M., Ph.D.
Central Arizona Vet. Lab., Inc.
P.O. Box 1296
Casa Gande, Arizona 85222Leon J. Mills, D.V.M.
c/o Walter Koers
Koers Consulting Service
2000 Ridgeview
Salina, Kansas 67401(c) General Design
Purpose of this multi location study was to determine whether or not ceftiofur at 0.5 mg/lb body weight given intramuscularly for three to five days is effective for the treatment of BRD under typical feedlot conditions. Animals - 629 head of feedlot cattle ranging in weight from 281 to 562 lbs.
- calves ranged from six to ten months in age
- typical feedlot animals, mixed steers and bulls
- 209 head per treatment group.
Control was a non-drug sterile diluent administered intramuscularly at 8 ml/animal for three consecutive days. Diagnosis: BRD was diagnosed by an elevated body temperature (104.5° F or greater) and the presence of at least two additional signs of the disease such as depressed physical appearance, increased or labored respiration, cough and loss of appetite. Nasal swabs were taken from each animal just prior to treatment to determine presence of BRD pathogens.
Dosage Form: Dosage form was an aqueous solution containing 50 mg ceftiofur equivalents per ml.
Route of Administration: Intramuscular. Dose: Control was the sterile vehicle (Bacteriostatic Water for Injection) given intramuscularly at 8 ml per animal. Ceftiofur was administered intramuscularly either at 0.5 or 0 mg/lb body weight three or five consecutive days. On day four of the trials, the investigators evaluated the condition of all surviving animals and determined whether or not they were recovered. If they were deemed "recovered", no further medication was given. If they were judged "not recovered" two more daily treatments were administered on days four and five. This procedure also included placebos.
Test Duration: This was a 28 day study. Pertinent Parameters Measured: - body temperature drop at day 4
- clinical success at day 10
- clinical success at day 28
- mortality by day 28(d) ResultsNasal swabs were taken for pre-treatment culture and organism identification from 618 cattle used in this study.
The most commonly isolated pathogen was Pasteurella hemolytica which was present in 385 (47%) of the specimens. Pasteurella multocida was found in 163 (20%) of the swabs. Hemophilus somnus was present in 41 (5%) and Mycoplasma spp. in 309 (37%). There were 210 (25%) cattle from whose nasal swabs no organisms could be detected.
(Eds. note: The following table consists of 7 columns.)
Treatment Means Tempera- Tempera- Percent Percent ture Drop ture Clinical Clinical Percent Treatment Day 4 Day 4 Success/ Success/ Mortality mg/lb no. (Degrees F) (Degrees F) Day 10 Day 28 Day 28 0.00 204 1.2 104.0 29 57 25 0.5 mg/lb 201 2.1 103.1 53 69 7 ceftiofur 1.0 mg/lb 201 2.2 103.0 61 69 3 ceftiofur LSD -1 sided .227 .226 7.4 7.4 5.1 -2 sided .270 .270 8.8 8.8 6.1Based on necropsy results, all but two of the 79 calves which died during the 28 day study were caused by typical BRD syndrome. Of the two, one was diagnosed as septicemia and the other anemia, dehydration and nutritional deficiency complicated by the stress of handling and shipping. (e) Statistical AnalysisA randomized complete block design was used with five animals of similar body temperatures and similar body weights being assigned per block. Treatments (3) were randomized within each block. All variables were analyzed according to the following Analysis of Variance:
(Eds. note: The following table consists of 4 columns.)
Source DF Test Term Decision Location 6 -- -- Block 180 -- -- Doses 2 -- -- 0 vs 0.5 mg/lb (1) error + significance at one sided.05 infers 0.5 dose is efficacious 0 vs 1.0 mg/lb (1) error + significance at one sided .05 infers 1.0 dose is efficacious 0.5 vs 1.0 mg/lb (1) error + significance at one sided .05 infers 1.0 dose is more effective than 0.5 dose Pooled Error 417* dose* location (12) pure error significance at .25 warrants use of dose*location as test term for contrasts pure error 405** + error = dose*location if P < .25 for test of dose* location * df = 400 for variables temperature reduction, and temperature on day 4 ** df = 388 for variables temperature reduction, and temperature on day 4Significance level used P ¾ 0.05.(f) Conclusions
Interpretations
Body temperature drop - Both ceftiofur treatment groups were significantly improved relative to non-medicated control, not different from each other. Success rate at day 10 - Ceftiofur treatment groups were each significantly better than non-medicated control, and not significantly different from each other. Success rate at day 28 Ceftiofur treatment groups were each significantly better than non-medicated controls and not different from each other. Mortality at day 28 Ceftiofur treatment groups were each significantly better than non-medicated control and not different from each other. Results from this study demonstrated that ceftiofur at doses of either 0.5 or 1.0 mg/lb was effective for treatment of BRD under typical feedlot conditions.
(g) Adverse Reactions
Except for an immediate and transient local pain reaction to the intramuscular injection of ceftiofur, there was no evidence of any adverse effect.
(h) Special Issues: None.
V. ANIMAL SAFETY:
Summary
Three specifically designed Target Animal (cattle) Safety Studies were conducted to address the tolerance to and safety of ceftiofur. Additionally, four clinical efficacy trials were conducted wherein target animal safety was also determined.
Based on results of these seven trials, ceftiofur is safe when administered intramuscularly at doses up to 1.0 mg. ceftiofur per lb body weight daily for five days. Local pain reaction to the injection is deemed transient and not detrimental to safety of an already sick animal.
A. Pivotal Studies
1. Safety/Toxicity Study
(a) Type of Study
This was a five day study in cattle where drug was administered at daily doses of 0, 1 mg/lb, 3 mg/lb, or 5 mg/lb body weight to determine the pharmacologic and toxicologic effects of ceftiofur following the intramuscular injection.
(b) Investigator
Study Director:
A. D. Hall, D.V.M.
The Upjohn Company
Kalamazoo, Ml 49001(c) General Design
(d) Results
Purpose of this study was to determine the pharmacologic and toxicologic effects of ceftiofur following the intramuscular injection in the bovine. Potential target organs and tissues were to be identified through clinical observations, histologic examination, hematology and serum chemistries. Red blood cell counts, hemoglobin, packed cell volume, serum creatinine and blood urea nitrogen values were also parameters of interest. Finally, injection site irritation was evaluated by use of histopathology and serum creatinine phosphokinase (CPK). Animals - Crossbred beef type steers
- body weight range 370 to 450 lb
- 20 animals for four treatment groups.
Control was a non-drug containing sterile diluent administered intramuscularly daily at 5 ml/100 lb body weight. Dosage Form: Sterile aqueous solution containing 100 mg drug/ml. Dosage Used: 0, 1, 3 or 5 mg/lb body (e.g., 1, 3, or 5 ml sterile solution containing 100 mg/ml per 100 lbs. Controls received 5 ml per 100 lb body weight.
Route of Administration: Intramuscular Test Duration: 7 days Pertinent Parameters Measured: Clinical observations, hematologic and serum chemistry measurements, body weight changes, organ weights and gross and microscopic evaluations were used to assess the potential toxicity in the steers.
(Eds. note: The following table consists of 6 columns.)
Clinical Observations: No clinical signs of toxicity or illness were observed during this study. Occasionally a steer would react to the injection of the compound by vocalizing and/or exhibiting a limp on the injected rear leg for the first few strides out of the squeeze-chute. The limp was always transient and was no longer noticeable by the time the steer returned to its pen.
Hematology: Statistical analysis of the data identified a slight but significant decrease in hematocrit (HCT) and hemoglobin (HGB) values when the high dose animals were compared to controls. However, the pretest hematology data contained a comparable statistically significant difference in hematocrit values between the control and high dose group. It should also be noted that the hematocrit and hemoglobin values decreased similarly in all groups, including the untreated controls, over the duration of the study. All mean hematologic values were within the normal biologic range for the bovine, and since there was no significant decrease in red blood cell numbers, the hematologic differences noted were not attributed to treatment.
Serum Chemistries: Significantly elevated CPK values (P ¾ .05) were observed in the high dose animals when compared to the controls. The CPK values also increased in the 3 mg/lb group but were not statistically different from the control value. In contrast, the untreated control and 1 mg/lb group CPK values were similar. The elevated CPK values correlated with the muscle damage at the injection site which was observed histopathologically. There were no other treatment related differences between treated and controls regarding clinical chemistry measurements.
Body Weights: The five day treatment regimen with U-64,279 had no significant effect on body weight in any of the groups.
Organ Weights: There were no significant differences between control and any of the dose groups for any of the five organs weighed.
Gross and Histologic Observations: The only gross change associated with the use of this drug was the focal areas of hemorrhage and/or muscle discoloration at the injection sites. Occasional, focal lung lesions were observed, but the incidence was uniformly distributed among the treatment groups. Drug related histopathologic changes were confined to the skeletal muscle at the injection sites. There appeared to be greater muscle fiber degeneration and localized inflammation at the highest dose. Localized edema, hemorrhage, mononuclear inflammatory cell infiltration and myofibril degeneration and mineralization were observed at the injection sites, leading to the morphologic diagnosis of mild to severe subacute necrotizing myositis. However, this study was not designed to correlate sequential histopathologic changes in the intramuscular injection sites with time.
Two steers in the 10X group had evidence of a mild abomasitis. One lesion was composed of a mild infiltrate of eosinophils, plasma cells and lymphocytes and may have been a response to a parasite or a piece of plant material that might have become lodged in that area of the abomasum. The other lesion was a focal lymphoid reaction that was probably triggered by a local irritation. The findings of abomasitis appeared to be unrelated tissue changes with no evidence of being an effect of treatment.
Other histopathologic findings were seen with similar frequencies in all dose groups and were not attributable to treatment.
Frequencies of Various Findings Organ and Diagnosis Sex -----Groups----- 1 2 3 4 Skeletal Muscle: Examined: M 5 5 5 5 Myositis Chronic Localized Severe M 0 0 1 0 Myositis Subacute Localized Mild M 4 0 0 0 Myositis Subacute Localized Moderate M 1 2 1 0 Myositis Subacute Localized Severe M 0 3 3 5 Group 1 Vehicle Control Group 2 U-64,279, 1 mg/lb/day Group 3 U-64,279, 3 mg/lb/day Group 4 U-64,279, 5 mg/lb/day(e) Statistical Analysis A randomized complete block design was used with a pen of four similar animals representing each block. There were five blocks.(Eds. note: The following table consists of 3 columns.)
Source Degrees of Freedom Test Term Treatment 3 Error Blocks 4 -- Error 12The hypothesis of no treatment effect associated with the measurements of most interest, RBC count, Hgb, PCV, BUN and serum creatinine, was tested at the .10 level of significance. Planned contrasts of dose groups with control were one-sided tests at the .10 level of significance, since only increases in BUN and serum creatinine and decreases in RBC count, Hgb, and PCV were of concern. The hypothesis of no treatment effect associated with the remainder of the variables was tested at the .05 level. Planned contrasts of dose groups with control were two-sided at the .05 level. Organ weights were also analyzed using least squares analysis of variance.The histopathologic data were analyzed using Fisher's Exact test at the .10 level of significance.
(f) Conclusions
No systemic drug related effects were detected following intramuscular administration of aqueous U-64,279 at dosages of 1, 3 and 5 mg/lb of body weight for five days, as was evidenced by the various parameters that were monitored during this study.
Gross and microscopic tissue changes were confined to localized areas of muscle damage at the injection site. This lesion was most pronounced at the highest dose tested (5 mg/lb) which correlated well with the only significantly elevated (P ¾ .05) CPK value.
2. Five Day Tolerance Study In Feeder Calves
(a) Type of Study
This was a five day tolerance study in feeder calves. Formulated drug was administered Intramuscularly up to 50X the highest proposed labeled dose.
(b) Investigator
Study Director
T. J. Kakuk
Path and Tox Research
The Upjohn Company
Kalamazoo, Ml 49001(c) General Design
Purpose of this tolerance study was to determine the pharmacotoxic effects of formulated freeze dried ceftiofur following intramuscular injection at an exaggerated daily dosage of 50X the highest labeled dose (0.5 mg/lb body weight) for five consecutive days. Animals
- crossbred beef type animals (steers)
- starting weight ranged from 182-316 kg and age ranged from 6-10 months
- eight animals total, four used as controls and four given 50X drug treatment.Control was bacteriostatic water for injection (vehicle used as diluent for making sterile solution from the ceftiofur sterile powder) 50 ml per 100 lb body weight.
Control and Treatment each administered intramuscularly.
Dose of 25 mg/lb body weight given in a 50 mg/ml formulation or 50 ml per 100 lb body weight.
Test period was 15 days, first five as daily treatments of either 25 mg/lb or vehicle.
Pertinent Parameters Measured:
Toxicological evaluation was predicated on clinical signs including body weight changes, six hematological and 17 clinical chemistry measurements, and gross pathologic observations. Tests of statistical significance were made at the P < 0.05 level.
(d) Results
All of the calves survived. Formulated ceftiofur did not cause any overt signs of toxicity during the five day dosing period or during the 10 day post treatment observation interval. The only changes were the significant elevations in aspartate transaminase (AST) and in creatinine phosphokinase (CPK) in both vehicle and drug treated animals on study day five relative to pretreatment baseline values. These AST and CPK values returned to baseline by day 15 of the study. These transient elevations depict local muscle irritation associated with the formulated drug and the vehicle per se .
(e) Statistical Analyses
The hematologic and clinical chemistry parameters were analyzed by the BMDP repeated measure routine to test the hypothesis that there is no difference between the means of dose groups. The adjustments were made for the initial values of the parameters (covariate). Tests of statistical significance were made at the 0.05 level.
A time by time t-test supported general findings of the repeated measures analysis.
(f) Conclusions
In conclusion, formulated ceftiofur diluted in Bacteriostatic Water for Injection, USP with Benzyl Alcohol 0.945% w/v was well tolerated in feeder calves at 50 times the highest recommended dosage of 1.0 mg/lb/day (25 mg/lb/day) for 5 consecutive days. Results showed that formulated ceftiofur administered intramuscularly had no systemic toxic effects. Local effects of muscle irritation were detected after the last dose (five consecutive daily doses) as evidenced by significant elevated AST and CPK values. However, these transient elevated values returned to baseline values 9 days post treatment.
3. 15 Day Intramuscular Safety/Toxicity Study
(a) Description
In this study cattle were administered formulated ceftiofur at daily doses up to 20X the highest proposed doses for 15 days, which is 3X the longest proposed duration of use. Toxicological assessment was based on clinical signs, hematological and clinical chemistry measurements, urinalysis measurements and gross and microscopic evaluations. The study included 5 M and 5 F, each at daily doses 0, 1, 3, 5 or 10 mg/lb body weight.
(b) Study Director
T. J. Kakuk
The Upjohn Company
Kalamazoo, MI 49001(c) General Design
(d) Results There were no systemic effects attributable to formulated ceftiofur sodium based on clinical signs, hematological, clinical chemistry and urinalysis parameters and on gross and microscopic determinations.
Purposes of this study were to determine the pharmacotoxic effects of formulated freeze dried ceftiofur sodium following intramuscular injection at the recommended and exaggerated daily doses given for an exaggerated duration and to measure the potential local muscle irritancy properties of the formulated product. Animals - 5 M and 5 F per treatment group
- crossbred beef type
- initial weights ranged from 180-290 kg
- age at start of trial ranged from 6-12 months.
Dose Form: The product was a reconstituted sterile solution injected intramuscularly. A freeze dried sterile powder was reconstituted with Bacteriostatic Water for injection, USD to make the resulting solution.
Dose Used: Daily doses of 1, 3, 5 or 10 mg drug/lb body weight was given intramuscularly using a sterile solution which contained 50 mg/drug/ml. Controls were administered 30 ml vehicle per head/day (based on volume the mg/lb treatment group would receive).
Route: Intramuscular injection (no injection site was injected with more than 10 ml; when >10 ml was indicated multiple sites of the same anatomical area were used).
Test Duration: 15 days; animals were sacrificed on day 16 and necropsied. Pertinent Parameters Measured: Toxicological evaluation was based on clinical signs including body weight changes, hematological and clinical chemistry measurements, urinalysis measurements and gross and microscopic evaluation. All tests of statistical significance were made at the P < 0.05 level.
Local effects of muscle irritation were evidenced by histopathologic evaluation. Lesions were microscopic in size and difficult to find even with the aid of injection site markers.
(Eds. note: The following table consists of 5 columns.)
Percent Calves (of 10) with Findings Treatment Groups Day Post Injection Findings Vehicle 1.0 mg/lb 3.0 mg/lb None 10 30 30 1 Acute Myositis 60 40 40 Subacute Myositis 30 30 30 Chronic Myositis -- -- -- None 0 30 40 3 Acute Myositis 0 20 0 Subacute Myositis 100 40 60 Chronic Myositis -- 10 -- 7 None 0 40 30 Chronic Myositis 100 60 70 14 None 100 80 70 Chronic Myositis 0 20 30(e) Statistical Evaluation Data for each sex were analyzed separately. The body weight was analyzed by BMDP repeated measures routine to test the following null hypothesis: (i) there is no difference between the means of dose groups, (ii) the dose groups behave the same over time. The organ weight, ratio organ weight to body weight, blood chemistry and hematology variables were analyzed by one way analysis of variance. Whenever the analysis indicated a dose difference, a Dunnett's t test was used to compare each active group to the control group. All tests of statistical significance were made at P<0.05 level.(f) Conclusions
Formulated ceftiofur is safe when injected intramuscularly into cattle. The formulation is deemed to be a slight muscle irritant.
4. Four Clinical Trials
The specific design including routes of administration, doses tested, numbers of animals, duration of tests, variables measured, results, statistical procedures and conclusions are each addressed in Section IV, EFFECTIVENESS, of this FOI Summary. Please refer to those sections for additional comments regarding Animal Safety.
VI. HUMAN SAFETY STUDIES
A. Toxicity Studies
1. Acute Oral Single Dose Study in Rats
Significant Toxicity Observed -
Technical Report No. 7263-85-002, June 3, 1985 Starting Date - August 2, 1984 Termination Date - August 16, 1984 Study Director - T. J. Kakuk Location of Study - The Upjohn Company, Kalamazoo MI Identification of Substance and Dosage Form - Ceftiofur sodium oral Species and Strain - Sprague Dawley Rats Number Animals Per Sex Per Treatment Group - 10 M and 10 F Drug Levels Tested and Duration of Dosing - 0, 996, 2120, 4276 and 7760 mg/kg body weight one day dosing followed by 15 day observation interval. Route of Drug Administration - oral Parameters Tested
Clinical daily observations were made to determine onset, severity and duration of behavioral changes and to note any evidence of toxicity. Body weights on days 1 and 15 of study. Gross necropsy on each animal and all organs and tissues were examined in situ . No deaths occurred at any dose level, nor were there any significant findings at the terminal necropsy. At 24 hours post treatment diarrhea was observed for 25% and 50% of the animals at 4276 and 7760 mg/kg, respectively. No such effects were observed at 996 and 2120 mg/kg. By day two all animals appeared clinically normal. A few animals in the two highest dose groups only had transient diarrhea on days seven and eight, returning to a normal clinical state thereafter. Body weight gains were similar for all animals independent of dose.
No Observed Effect Level - the oral single lethal dose value is >7760 mg/kg body weight.
Statistical Analysis - none
Conclusions - The No Observed Effect Level based on the oral single lethal dose in rats is >7760 mg/kg body weight.
2. 30 Day Oral Toxicity Study in Rats
Technical Report No. 7263-85-071, December 12, 1985
Starting Date - August 20, 1984
Termination Date - September 20, 1984
Study Director - T. J. Kakuk
Location of Study - The Upjohn Company, Kalamazoo, MI
Identification of Substance and Dosage Form - Ceftiofur sodium in solution
Species and Strain - Sprague Dawley Rats
Number Animals Per Sex Per Treatment Group - 15 M and 15 F per group
Drug Levels Tested and Duration of Dosing - 0, 1500, 3000 and 6000 mg/kg body weight administered once daily for 30 or 31 days.
Route of Drug Administration - oral intragastric intubation
Parameters Tested - clinical signs, weekly body weight and food consumption, seven hematologic, 15 clinical chemistry and eight urinalysis measurements, selected organ weight, and gross and microscopic evaluation.
Significant Toxicity Observed - Clinical signs of toxicity observed included diarrhea at 1500 mg/kg or greater, and distended abdomen and hardened stomach contents at 3000 mg/kg or greater. These signs were most prevalent in the 6000 mg/kg group with an onset of four to five days after ceftiofur dosing was initiated. The incidence of clinical signs in the other treated groups are presented in table form.
(Eds. note: The following table consists of 5 columns.)
Percent Incidence of Various Signs of Toxicity Group Number 1 2 3 4 Diarrhea 0 10 33 97 Distended abdomen 0 0 10 89 Stomach hard upon palpation 0 0 20 92 Groups 1 0 (Control) 2 1,500 mg/kg 3 3,000 mg/kg 4 6,000 mg/kgData for each sex were analyzed separately. Treatment group differences were analyzed using analysis of variance on raw data in conjunction with analysis of variance on ranks of data. For statistically significant variables, treatment groups were compared to the vehicle control using the Least Significant Difference method. Tests of statistical significance were made at the 0.05 level.
No Observed Effect Level - <1,500 mg/kg Statistical Analysis The following parameters were examined for significant differences between treated and control groups:
Body Weight Urine Measurements Hemogram Measurements Organ Weights Clinical Chemistry Measurements Statistical analysis was conducted on the histopathological findings using the following method:
Data for each sex were analyzed separately. The program used contains methods for the analysis of unadjusted proportions as well as the life table related techniques of Tarone (1975), Cox (1972) and Breslow (1970) for analysis of lesion incidence.
For the unadjusted analysis, an exact test for trend was used to test for an increase (or decrease) in lesion incidence as the dosage administered increased. Additionally, Fisher's exact test was used to compare the proportion exhibiting a particular lesion in each treated group to the proportion exhibiting that same lesion in the vehicle control group. Both tests were two-tailed, using the P value obtained by doubling the one-tailed probability.
Similarly, for the adjusted analysis, tests for trend and treatment groups to vehicle control group comparisons were conducted on the time adjusted proportions.
Conclusions
Ceftiofur sodium, administered orally to rats for 30 days, was markedly toxic at 6,000 mg/kg, moderately toxic at 3,000 mg/kg and minimally toxic at 1,500 mg/kg. The target organ was the gastrointestinal tract as evidenced by the clinical signs of diarrhea, distended abdomen, and hardened stomach contents, and grossly by the formation of stomach concretions of the drug food mixture at 3,000 mg/kg or greater.
3. 90 Day Oral Toxicity Study in Rats
Technical Report No. 7263-85-075, December 17, 1985
Starting Date - December 18, 1984
Termination Date - March 20, 1985
Study Director - T. J. Kakuk
Location of Study - The Upjohn Company, Kalamazoo MI
Identification of Substance and Dosage Form - Ceftiofur sodium oral solution
Species and Strain - Sprague Dawley Rats
Number Animals Per Sex Per Treatment Group - 20 M and 20 F
Drug Levels Tested and Duration of Dosing - 0, 30, 100, 300, 1,000 and 3,000 mg/kg body weight for 90 or 91 days.
Route of Drug Administration - Oral intragastric intubation
Parameters Tested
Parameters utilized to assess toxicity included clinical signs, weekly body weight and food consumption, body weight gains, hematologic, clinical chemistry and urinalysis measurements, selected organ weights, and gross and microscopic evaluation. Tests of statistical significance on the parameters denoting toxicity per sex per group were made at the .05 level.
Significant Toxicity Observed
The target organ was the gastrointestinal tract as evidenced by the clinical signs of diarrhea and hardened stomach contents and grossly by a dose-related increase in stomach content weights at 300 mg/kg/day or greater. In males and females at 3,000 mg/kg/day, the incidence of diarrhea was -5 to 83% and the incidence of hardened stomach contents was 86 to 100%. At 1,000 mg/kg/day diarrhea occurred at an incidence of 6 to 42% in males and females and there was a 5% incidence of hardened stomach contents for the males. Only transient diarrhea (incidence of 5 to 11%) occurred in males at 300 mg/kg/day and in females at 30 and 100 mg/kg/day. This transient diarrhea did not occur in females at 300 mg/kg/day.
At 3,000 mg/kg/day there was formation of gastric concretions of the drug food mixture at an incidence of 83%. These concretions in the stomach could not pass through the pylorus ("mechanical toxicity"), which led to poor or impaired nutritional status and secondary effects of suppressed body weight gain (-36%) in the males only, gastric erosions and/or ulcers, thickening of the gastric ridge (males only), and death of 10 percent (4/40) of the rats after 9 to 91 days of treatment. The impaired or poor nutritional condition at 3,000 mg/kg/day was also associated with decreased serum glucose values, electrolyte imbalance, sparseness of fat deposition in the abdominal cavity, and, microscopically, by the lack of hepatocellular glycogen deposition and atrophy of the germinal centers of the spleen, lymph nodes, and thymus. The impaired nutrition and lowered glucose values at 3,000 mg/kg/day, along with a significant increase in food consumption and significant changes in urinary ketones (increase) and pH (decrease) at both 1,000 and 3,000 mg/kg/day, suggest a deficiency in carbohydrate metabolism at 1,000 and 3,000 mg/kg/day. In addition, the lowered urinary pH at 100 mg/kg/day or greater probably reflected the excretion of the compound or its metabolites, including furic acid. Hence, the lowered pH at 100 mg/kg/day, without any other significant changes, suggested that the compound was being metabolized and excreted as acidic metabolites.
Additionally, based on microscopic findings, the compound caused colitis at an incidence of 25% for males at 1,000 mg/kg/day or greater and in females at an incidence of 10%, 30% and 15% at 300, 1,000 and 3,000 mg/kg/day, respectively. Also, cecitis and/or ileitis occurred at an incidence of 10% (females) to 20% (males) at 3,000 mg/kg/day. Colitis, cecitis, and ileitis were probably associated with the changes in bacterial flora in the gut produced by the antibiotic properties of ceftiofur sodium. Males at 300 mg/kg/day or greater had an inflammatory response (mononuclear cells) located in the renal corticomedullary junction. This change may have been related to the compound favoring the formation of a possible enterotoxin in the gut of these animals. The acid urine (lowered pH) associated with the metabolism of ceftiofur apparently resulted in a significant increase in nephrocalcinosis for females only given ceftiofur sodium at 3,000 mg/kg/day.
No Observed Effect Level - 30 mg/kg
Statistical Analysis:
Statistical Evaluation
The following parameters were examined for significant differences between treated and control groups
Body Weight Urine Measurements Food Consumption Organ Weights Hemogram Measurements Histopathologic Findings Clinical Chemistry Measurements
Statistical Method - Data for each sex were analyzed separately. Treatment group differences were analyzed using analysis of variance on raw data in conjunction with analysis of variance on ranks of data (Conover and Iman, 1981). For statistically significant variables, treatment groups were compared to the vehicle control using the Least Significant Difference method. Tests of statistical significance were made at the 0.05 level.
Statistical analysis was conducted on the histopathological findings using the following method -
The computer program of Thomas, Breslow and Gart (1977) was used to analyze the data. This program contains methods for the analysis of unadjusted proportions as well as the life table related techniques of Tarone (1975), Cox (1972), and Breslow (1970) for analysis of lesion incidence.
For the unadjusted analysis, an exact test for trend was used to test for an increase in lesion incidence as the dosage administered increased. Additionally, Fisher's exact test was used to compare the proportion exhibiting a particular lesion in each treated group to the proportion exhibiting that same lesion in the vehicle control group.
Similarly, for the adjusted analysis, tests for trend and treatment groups to vehicle control group comparisons were conducted on the time adjusted proportions.
Conclusions
Based on the results, the NOEL was 30 mg ceftiofur sodium per kg of body weight for 90 days. Also, the results indicated that ceftiofur administered orally to the rats for 90 days was markedly toxic at 3,000 mg/kg/day, moderately toxic at 1,000 mg/kg/day and minimally toxic at 300 mg/kg/day.
4. Oral Two Generation Fertility and Reproductive Study in Rats - Performance of F0 Generation
Technical Report No. 7263-85-082, December 17, 1985
Starting Date - March 27, 1985
Termination Date - August 21, 1985
Study Director - C. J. Price
Location of Study - Research Triangle Institute, Research Triangle, North Carolina 27709
Identification of Substance and Dosage Form - ceftiofur sodium dosing solution.
Species and Strain - Sprague Dawley Rats
Number Animals Per Sex Per Treatment Group - 0 M and 0 F per group
Drug Levels Tested and Duration of Dosing - 0, 100, 300 or 1000 mg/kg/day. Males dosed 70 days and females 14 days prior to the breeding stage.
Route of Drug Administration - via gavage
Parameters Tested
Males (4-47 days of age) were dosed daily via gavage for 70 days and females (5-59 days of age) were dosed daily via gavage for 14 days prior to the cohabitation of F0 breeding pairs (1 male and 1 female per cage); daily dosing of males and females was continued throughout the study. Females were singly housed on the day sperm were found in the vaginal lavage or at the end of the 21 day cohabitation period. On the day a litter was cast (pnd 0), the following reproductive parameters were recorded: (1) day of delivery and length of gestation for each dam, (2) the number of live or dead pups in each litter, and (3) the sex and clinical condition of each live pup. The body weight of each live pup was recorded on pnd 0, 4, and 21. On pnd 4, live litters were randomly culled to a maximum size of 10 pups per litter. Postnatal viability of Fl pups was evaluated on pnd 0, during the perinatal period (days 0-4) and during the remainder of lactation (days 4-21). On pnd 21 (day of weaning), 30 male and 30 female pups per group were randomly selected, orally dosed at the prescribed levels and designated for subsequent use of F1 breeders. All remaining F1 pups were sacrificed and necropsied and observations were recorded. Following weaning of F1 litters, F0 rats were sacrificed (79th day of exposure for females; 136th day of exposure for males); each F0 rat was evaluated at necropsy and reproductive organs, gross lesions and/or masses from the control and high dose groups were evaluated histopathologically. In addition, histopathologic evaluation was performed on reproductive organs, heart, lungs, spleen, kidneys, adrenal or gross lesions and/or masses of animals that died or were moribund during the course of the study.
Significant Toxicity Observed
Body weight gain (g) for males was significantly suppressed in a dose-related manner throughout the study (weeks 0-2, 2-6, 6-10, 10-14, 14-18 and 18-necropsy). The effect of ceftiofur upon male body weight gain varied in severity during different phases of the study, suggesting a possible interaction of treatment with length of exposure and/or growth rate during development. Weight gain during the first 2 weeks of exposure was suppressed in the mid and high dose groups. All ceftiofur treated groups were below controls for body weight gain (g) and percent body weight gain for the treatment period as a whole (weeks 0-necropsy). Thus, potential effects of ceftiofur on male reproductive capacity were evaluated within a dose range which produced a statistically significant suppression in male body weight gain.
Female body weight gain was not affected during the first two weeks of exposure to ceftiofur (i.e., prior to cohabitation). For confirmed mated females with litters, body weight gain was significantly suppressed for all ceftiofur exposed groups during gestational days (gd) 16-20, and weight gain during gestation (days 0-20) was significantly suppressed at the high dose. Body weight gain for females with litters was significantly increased during the first 2 weeks of lactation at the mid and high doses, but was not affected during the third and final week of lactation or for the lactation period as a whole. Thus, female body weight gain was not affected prior to breeding, but dose related changes in body weight gain were observed during gestation and lactation.
No dose-dependent adverse effects on fertility or reproductive performance were associated with exposure to ceftiofur. Ceftiofur treated groups were similar to controls for the following endpoints: (1) the proportion of cohabited females with confirmed mating, (2) the proportion of mated females with confirmed pregnancies, (3) the proportion of confirmed pregnancies producing live litters, (4) the length of gestation in confirmed pregnant females, (5) the number of live pups per litter on pnd 0, 4 or 21, and (6) the proportion of live male pups on pnd 0 or 4. Postnatal viability of pups in ceftiofur treated groups was equivalent to controls on pnd 0, and for pnd 4 to 21. During the perinatal period (pnd 0 to 4), 100% of pups in the high dose group survived, thus contributing to a statistically significant trend toward increased survival; this statistical effect appears to lack biological relevance since survival in all groups was high (avg. % viable pups per litter ranging from 97.57-100%), and since the relevant statistical tests are not optimized when zero variance is observed in any dose group. Live pup body weight per litter was not affected by ceftiofur exposure on pnd 0 or 4; on pnd 21, the low dose group exhibited a significant suppression in body weight relative to the control group (-7.3%), but the absence of an effect at higher exposure levels suggests that this was not a treatment related effect.
Grossly, enlargement of the cecum occurred at an incidence of 59 to 97% in the treated groups compared to 0% in the control. In survivors, no compound related microscopic lesions were observed in the reproductive organs of either sex at the high dose (1000 mg/kg/day) compared to the controls. Moreover, there were no compound related changes in the reproductive organs, heart, lungs, spleen, kidneys or adrenal of animals that died during the course of the study. Documented histopathological findings were considered to be secondary to gavage accidents, unspecified etiologies or background lesions common to rats of this strain and age.
No Observed Effect Level - 1000 mg/kg for reproductive performance.
Statistical Analysis
Endpoints were statistically evaluated using SAS® software. ANOVA including linear trend analysis and Dunnett's tests for comparison of treatments with controls was used for most variables. Where appropriate non parametric procedures such as Chi Square and Fishers exact test were used.
Conclusions
Daily oral administration of ceftiofur sodium at dosages of 0, 100, 300 and 1000 mg/kg/day in Sprague Dawley rats failed to produce any adverse effects upon fertility or reproductive performance, or any histopathological alterations in the reproductive organs of either sex in the F0 generation. Hence, the no observable effect level (NOEL) for reproductive performance and fertility of the F0 generation in rats was 1000 mg ceftiofur sodium per kg of body weight.
5. Oral Two-Generation Fertility Reproductive Study in Rats - Performance of F1 Generation
Technical Report No. 7263-86-031, May 13, 1986
Starting Date - July 31, 1985
Termination Date - January 7, 1986
Study Director - C. J. Price
Location of Study - Research Triangle Institute, Research Triangle, North Carolina 27709
Identification of Substance and Dosage Form - Ceftiofur sodium dosing solution
Species and Strain - Sprague Dawley Rats
Number of Animals Per Sex Per Treatment Group - 30 males and 30 females of the F1 generation
Drug Levels Tested and Duration of Dosing
0, 100, 300 or 1,000 mg/kg/day. Animals (F1) were dosed once daily beginning on the day of weaning (postnatal 21) and continuing until the day before scheduled sacrifice (females for an average of 153 days and males an average of 152 days). At 12-15 weeks of age the F1 males and females were cohabited for 21 days. Resulting F2 pups were sacrificed at time of weaning (day 21 of age).
Route of Administration - via gavage
Variables Tested
In this study, F1 males and females from each respective group were dosed daily via gavage beginning on pnd 21 (weaning) and continuing until the day before scheduled sacrifice. At 12-15 weeks of age, the F1 males and females were cohabited in nonsibling breeding pairs for a maximum of 21 days. On the day sperm were found in the vaginal lavage or at the end of the 21 day cohabitation period, females were singly housed and checked at least twice daily thereafter for the presence of pups in the cage. On the day a litter was found (pnd 0), the following reproductive parameters were recorded: (1) day of delivery and length of gestation for each dam, (2) the number of live and dead pups in each litter, and (3) the sex and clinical condition of each live pup. The number of live pups of each sex and the average pup body weight for live pups of each sex per litter were recorded on pnd 0, 4, and 21. On pnd 4, live litters were randomly culled to a maximum size of ten pups per litter. Postnatal viability of F2 pups was evaluated on pnd 0, during the perinatal period (days 0 to 0) and during the remainder of lactation (days 4-21). On pnd 21 (day of weaning), all F2 pups were sacrificed, necropsied, and observations were recorded. Following sacrifice of F2 litters, F1 adult animals were sacrificed (146th-160th day of exposure for females; 145th-159th day of exposure for males). Each F1 animal was evaluated at scheduled sacrifice and the gastrointestinal tract, gross lesions and masses from all dose groups were evaluated histopathologically. The reproductive organs of both sexes were evaluated histopathologically for all control and high dose animals. In the absence of any compound related changes in the reproductive organs at the high dose relative to the controls, comparable tissues from the low and mid dose groups were not microscopically examined, as per study protocol. For animals that died or were moribund during the course of the study histopathologic evaluation was performed on reproductive organs, heart, lungs, spleen, kidneys, adrenal, or gross lesions and masses.
Significant Toxicity Observed
Except for mechanical effects due to dosing, clinical signs were not related to treatment. Males and females in all ceftiofur sodium groups had an increased tendency to struggle during dosing. Thirty three (33) rats (18 males and 15 females) died or were moribund over the course of the study. When these data from males and females were combined, the incidence of dead or moribund animals increased in a dose-related manner (0/60, 1/60, 11/60, and 21/60 at 0, 100, 300, and 1,000 mg/kg/day, respectively). Ninety-four percent (17/18) of these occurrences in males and 87% (13/15) in females were associated with evidence of accidental causes. For the remaining three (3) (e.g., one male at 1,000 mg/kg/day, one female at 300 mg/kg/day, and one female at 1,000 mg/kg/day), the cause of death could not be determined.
The potential effects of ceftiofur sodium on F1 male reproductive capacity were evaluated within a dose range which produced a statistically significant increase in male body weight gain (weaning necropsy) at the low and mid doses, but did not affect weight gain at the high dose. Body weight gain for females was not affected during the first twelve weeks of exposure to ceftiofur sodium (i.e., prior to cohabitation) or during gestation, but was significantly increased at the high dose throughout lactation.
Fertility and reproductive performance of the F1 generation and development of F2 litters through weaning (pnd 21) were not adversely affected by daily ceftiofur sodium exposure. Nor were there any treatment related gross lesions observed in the F2 pups at scheduled sacrifice. Thus, ceftiofur sodium had no adverse effects on fertility and reproductive performance of the F1 adults or the resulting F2 pups.
Evaluation of F2 animals at scheduled sacrifice revealed an increased incidence of enlargement of the cecum at 300 and 1,000 mg/kg/day, but not at 100 mg/kg/day when compared to the controls. In addition, males in the 300 and 1,000 mg/kg/day groups and females in all ceftiofur sodium dose groups exhibited increased weight of the gastrointestinal tract (i.e., with or without stomach contents) relative to the vehicle control group. At 300 and 1,000 mg/kg/day, there was a greater incidence of degenerative changes in the non glandular stomach (six and 92%, respectively) and hypersecretion of mucus in the neck cells of the glandular stomach (12 and 79%, respectively) as compared to the control and 100 mg/kg/day groups.
For F1 animals which survived to scheduled sacrifice, no compound related microscopic findings were observed in the reproductive organs of either sex at the high dose (1,000 mg/kg/day) compared to the vehicle controls. Microscopic changes in the reproductive organs, heart, lungs, spleen, kidneys, or adrenal of animals that died or were moribund during the course of the study were not compound related.
No Observed Effect Level - 1,000 mg/kg for reproductive performance and fertility.
Statistical Analysis
Endpoints were statistically evaluated using SAS® software. ANOVA including linear trend analysis and Dunnett's tests for comparison of treatments with controls was used for most variables. Where appropriate non parametric procedures such as Chi Square and Fishers exact test were used.
Conclusions
Daily oral administration of ceftiofur sodium at dosages of 0, 100, 300, or 1,000 mg/kg/day to Sprague Dawley rats produced gross enlargement of the cecum and microscopic changes in the stomach at 300 and 1,000 mg/kg/day as compared to the controls. These effects were not observed at 100 mg/kg/day. Ceftiofur sodium failed to produce any adverse effects upon fertility or reproductive performance of the F1 generation.
Likewise, no adverse effects were observed with regard to the growth and viability of F2 litters through weaning (pnd 21). Hence, the no observable effect level (NOEL) for reproductive performance and fertility of the F1 generation in rats was 1,000 mg ceftiofur sodium per kg of body weight.
6. Segment II Teratology Study in Rats
Technical Report No. 7259-85-011, December 12, 1985
Starting Date - February 12, 1985
Termination Date - March 14, 1985
Study Director - T. A. Marks
Location of Study - The Upjohn Company, Kalamazoo MI
Identification of Substance and Dosage Form - ceftiofur sodium powder formulated with sterile filtered deionized water to form a solution.
Species and Strain - Sprague Dawley Rats
Number Animals Per Sex Per Treatment Group - 24 bred rats per group
Drug Levels Tested and Duration of Dosing - 0, 800, 1,600 and 3,200 mg/kg/day on days 6-15 of gestation
Route of Drug Administration - Oral by gastric intubation
Parameters Tested
All dams were weighed on the day of insemination (day 0 of gestation), throughout the dosing period, and on day 20, the day cesarean sections were performed. Sex, weight, number and location of each dead and reabsorbed fetus. Live fetuses were evaluated for gross, visceral and skeletal anomalies.
Significant Toxicity Observed
Ceftiofur administration led to dose-related maternal toxicity (soft stools, porphyrin staining of the eye and nares) especially in the high dose group (diarrhea, blood in stools). However, in spite of administration at such high dosages, this cephalosporin did not appear to adversely affect the reproductive capacity of the dams. Although there was a statistically significant (P < 0.01) decrease in the proportion of dams in the high dose group that conceived, the fact that the drug was not given until day 6 of gestation, as well as the lack of significant effects on early embryo development (early implant sites and resorption), suggested that this effect occurred by chance.
The only statistically significant adverse effect on the embryos was a dose-related decrease in mean fetal body weights. However, the actual decreases did not exceed 7% and no other indication of toxicity toward the offspring was found. Thus, it was concluded that ceftiofur was not embryotoxic. Since there were no biologically significant increases in the incidence of variations or malformations, it was also concluded that this drug was not teratogenic to the rat.
No Observed Effect Level - 3,200 mg/kg body weight for teratogenic effects.
Statistical Analysis
Several different procedures were used, depending on the end point, ranging from non parametric procedures to Analysis of Variance with LSD Method for pair wise comparisons.
Conclusions - Ceftiofur sodium is not teratogenic in the rat at doses up to and including 3,200 mg/kg body weight.
7. 51 Day Oral Toxicity Study in Dogs
Technical Report No. 7263-85-077, December 12, 1985
Starting Date - September 4, 1984
Termination Date - October 26, 1984
Study Director - T. A. Jackson
Location of Study - The Upjohn Company, Kalamazoo MI
Identification of Substance and Dosage Form - Ceftiofur sodium in solution
Species and Strain - Purebred Beagle Dogs
Number Animals Per Sex Per Treatment Group - 4 M and 4 F per group
Drug Levels Tested and Duration of Dosing - 0, 300, 1,000 and 3,000 mg ceftiofur/kg body weight administered daily for 51 or 52 days
Route of Drug Administration - Oral intragastric intubation
Parameters Tested
Dogs were evaluated prior to study initiation by physical examination which included ophthalmologic examination and analysis of clinical pathology data. During the study, food consumption data were collected 5 days per week and clinical observations were made daily; body weights were documented on a weekly basis. Blood chemistry and urinalysis data were collected during week 4 and at study termination; ophthalmologic examinations were done at the same period. Hematology data was collected from all dogs at approximately weekly intervals, beginning at week 4. All dogs were necropsied; selected organs were weighed and protocol tissues were fixed in formalin, processed, and evaluated histopathologically.
Significant Toxicity Observed
Administration of ceftiofur to dogs at levels of 300 mg/kg/day, 1,000 mg/kg/day, or 3,000 mg/kg/day for a period of up to 51 consecutive days resulted in anemia and thrombocytopenia in both male and female beagles. The two higher dose levels were also associated with the development of elevated neutrophil counts and depression. Two females given 1,000 mg/kg/day and 2 males and 2 females given 3,000 mg/kg/day died prior to the termination of the study due to the clinical associated with exposure to this compound.
No Observed Effect Level - not determined
Statistical Analysis
Body weights, food consumption, organ weights, hematology, blood chemistry, urinalysis, and histopathology were evaluated statistically.
Conclusions
Under conditions of this study, ceftiofur produced multiple adverse effects on the hematopoietic system of dogs at all dose levels tested.
8. 90 Day Oral Toxicity Study in Dogs
Technical Report No. 7263-85-079, December 11, 1985
Starting Date - December 20, 1984
Termination Date - March 22, 1985
Study Director - T. A. Jackson
Location of Study - The Upjohn Company, Kalamazoo MI
Identification of Substance and Dosage Form - Ceftiofur sodium in solution
Species and Strain - Purebred Beagle Dogs
Number Animals Per Sex Per Treatment Group - 5 M and 5 F per group
Drug Levels Tested and Duration of Dosing - 0, 10, 30, 100 and 300 mg/kg body weight given daily for 90 days
Route of Drug Administration - Oral intragastric intubation
Parameters Tested
Dogs were evaluated prior to study initiation by physical examination. During the study, food consumption data were collected 7 days per week and clinical observations were made daily; body weights were documented on a weekly basis. Blood chemistry and urinalysis data were collected prior to study initiation, during week 6, and before study termination; ophthalmologic examinations were done prior to study initiation and at termination of the study. Hematology data were collected from all dogs prior to study initiation and 6 times during the study (including termination) at approximately 2-week intervals. Coombs' tests were performed in blood from all dogs given 300 mg/kg/day and 3 dogs per sex given vehicle only at day 41; at day 83 blood from all dogs was tested. Examinations of blood smears and differential leukocyte counts were done for all dogs prior to study initiation, at the 6-week bleeding, and at the last bleeding; smears were analyzed from every bleeding for dogs which became anemic and/or Coomb's test positive. A complete necropsy was done on all dogs; selected organs were weighed and protocol tissues were fixed in formalin, processed, and evaluated histopathologically.
Significant Toxicity Observed
Administration of 100 mg/kg/day or more of ceftiofur was associated with a non-progressive thrombocytopenia in at least 4 dogs. Multiple dogs treated with 300 mg/kg/day became positive for the Coombs' test indicating the presence of immunoglobulin on the surface of erythrocytes. Two dogs treated at this level became severely anemic and there was no apparent regenerative response by bone marrow until administration of compound ceased (one dog). Major clinical signs were associated with anemia and included pale mucous membranes and depression.
Necropsy observations and major histopathologic findings were related to the process of anemia. A minor inflammatory lesion was noted in the pelvis of kidneys from dogs given 100 mg/kg/day or more of compound; its significance is unknown.
No Observed Effect Level - 30 mg/kg
Statistical Analysis
Body weights, food consumption, organ weights, hematology, blood chemistry, urinalysis, and his- topathology were evaluated statistically.
Conclusions
Ceftiofur has obvious effects on the hematopoietic system, causing thrombocytopenia in dogs given 100 mg/kg/day or 300 mg/kg/day and severe anemia in dogs given 300 mg/kg/day. The no observed effect level (NOEL) for ceftiofur in dogs was 30 mg/kg/day in this study.
9. Genotoxicity Study - Ames Assay - Ceftiofur
Technical Report No. 7268-83-019, October 31, 1983
Starting Date - May 4, 1983
Termination Date - May 9, 1983
Study Director - D. H. Swenson
Location of Study - The Upjohn Company, Kalamazoo Ml
Identification of Substance and Dosage Form - ceftiofur sodium pulverized powder
Species and Strain - in vitro study ceftiofur dissolved in dimethylsulfoxide at 0.01 mg/ml immediately before use.
Species and Strain - in vitro
Number Animals Per Sex Per Treatment Group - not applicable
Drug Levels Tested and Duration of Dosing - 0.125, 0.25, 0.5 and 1.0 µg/plate plus controls (positive and negative).
Route of Drug Administration - not applicable
Parameters Tested
The procedures for the Ames assay are the standard ones developed in the laboratory of Dr. B. N. Ames. Briefly, histidine auxotrophs of Salmonella typhimurium are mixed with the test compound in 0.1 ml dimethylsulfoxide, the 9,000 x g supernatant of liver homogenate (or saline) in molten (45°C) agar. The molten agar mix is poured onto a Petri plate containing a histidine deficient base agar. Revertants to histidine prototropy are scored as colonies, after incubation at 37°C for 22 days. A single plate is used for each dose level and the experiment is repeated. Vehicle controls are run in triplicate for each strain in each experiment and reported as an average of the three values.
Significant Toxicity Observed
At no dose level was an appreciable increase in colonies per plate observed, while the positive controls 2 acetyl amino fluorene (TA98, TA100, and TA1538), cyclophosphamide (TA1535) and 9 amino acridine (TA1537) showed positive mutagenicity.
No Observed Effect Level - Not applicable
Statistical Analysis - Not applicable
Conclusions
The results of this study show that ceftiofur was not a mutagen for S. typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 with or without metabolic activation.
10. Genotoxicity Study - The V79 Mammalian Cell Mutation Assay - Ceftiofur
Technical Report No. 7268-83-030, November 29, 1983
Starting Date - June 14, 1983
Termination Date - August 2, 1983
Study Director - D. H. Swenson
Location of Study - The Upjohn Company, Kalamazoo MI
Identification of Substance and Dosage Form - ceftiofur in solution
Species and Strain - Not Applicable
Number Animals Per Sex Per Treatment Group - Not Applicable
Drug Levels Tested and Duration of Dosing - 1, 2 and 4 mg/ml plus control (positive and negative)
Route of Drug Administration - Not Applicable - in vitro Test
Parameters Tested
The mammalian cell mutagenesis assay is an accepted in vitro short term testing procedure for detecting potential mutagens and carcinogens. This assay uses V79 (Chinese hamster) cells and measures the production of mutants resistant to 6-thioguanine (TG) as a result of exposure to the test compound. Mutant cells without HGPRT activity cannot metabolize TG, and thus escape its lethal effect.
Some compounds are negative in this assay unless they are metabolized to reactive forms. Because the V79 cell is incapable of such metabolic activation, an exogenous activation system is required. The rat liver S9 system is a widely used activation system for bacterial and mammalian cell mutagenesis. Thus, ceftiofur was tested with and without an S9 activation system.
Significant Toxicity Observed
There were no significant differences between the mutation frequencies of cultures treated with ceftiofur and control cultures. All the treatment/control ratios were very close to 1.0, and there was no significant linear dose response. Thus, ceftiofur with or without S9 activation, was not mutagenic in this study.
No Observed Effect Level - not applicable
Statistical Analysis
The raw data was entered into a computer program which calculated the mutant frequencies and generated summary tables and a statistical analysis. The mutation frequencies of the cultures were transformed to (1 + mutants/10e6)raised to 0.15 power). The mean response at each dose was compared to the mean control (no treatment) response using the one sided t-test. The mean square error from a two way analysis of variance (AOV) was used for a variance estimate. In addition, a one sided t-test was used to test for an increasing linear relationship between log dose and the transformed response. The mean square error from a covariance model with assays as the grouping variable was used for the variance estimate. To increase the sensitivity of the latter test, the control vehicle responses were incorporated into the analysis by assigning them dose values equal to R times the smallest dose where R is the ratio of two successive doses.
The alpha levels of the statistical tests were adjusted so that the experimental alpha level was not greater than .1. Thus, the alpha levels were set at .025 for the four-dose assays.
Conclusions - Ceftiofur was not mutagenic in this study.
11. Genotoxicity Study - Unscheduled DNA Synthesis (UD) Assay - Ceftiofur
Technical Report No. 7268-84-018, May 24, 1984
Starting Date - May 24, 1983
Termination Date - May 31, 1983
Study Director - D. H. Swenson
Location of Study - The Upjohn Company, Kalamazoo MI
Identification of Substance and Dosage Form - A solution of ceftiofur at 10 mg/ml in dimethylsulfoxide (DMSO)
Species and Strain - Not applicable
Number Animals Per Sex Per Treatment Group - Not applicable
Drug Levels Tested and Duration of Dosing - 0.03, 0.1, 0.3 and 1 mg ceftiofur/ml with a solvent control and UV light as a positive control.
Parameters Tested
Genotoxins are agents that can damage DNA or alter chromosome structure and with very few exceptions are generally mutagenic or carcinogenic. As a class of compounds, genotoxins have wide structural diversity. Some are active per se but most require metabolic activation before they are capable of exerting their biological effects. Not all types of DNA damage lead to mutagenic or carcinogenic endpoints. Some types of DNA damage are repaired by the cellular repair system before the deleterious consequences are manifest. DNA repair, therefore, implies DNA damage and as such can be used as an endpoint for assaying genotoxic action. Cells vary greatly in their ability to repair genotoxic lesions and depending on the type of lesion formed can be repaired in a variety of ways. A brief description of DNA excision repair serves to illustrate the repair process. Briefly, a genotoxic lesion is recognized and excised from the DNA strand by repair endonucleases, along with several nucleotides on either side of the lesion. The resulting gap is patched with nucleotides, using the opposite strand as a template. This repair is known as unscheduled DNA synthesis or UDS and can be measured by a variety of methods. The most common method used to monitor unscheduled DNA synthesis is by observing increased incorporation of [3H] thymidine into the repair patches by autoradiography.
Primary rat hepatocytes have three characteristics that make them useful for detecting UDS. First, hepatocytes are nondividing, therefore, a UDS response is not confused with cells undergoing semi-conservative replication (scheduled DNA synthesis). Second, hepatocytes have good capabilities for metabolic activation of genotoxins. Third, hepatocytes have excellent DNA repair capabilities.
For this test an increased frequency of nuclear grains indicates DNA repair and implies that the agent responsible for the increase may be carcinogenic and mutagenic.
Significant Toxicity Observed: UV light (1.5 joules/sq m) was used as a positive control and resulted in a 4-fold increase of nuclear grains as compared to controls.
Ceftiofur did not cause a dose-related increase of UDS nor did it cause a doubling of UDS relative to untreated control cultures that received equivalent volumes of vehicle (dimethylsulfoxide).
No Observed Effect Level - Not applicable
Conclusions - Under the conditions employed for this study, ceftiofur did not act as a DNA damaging agent.
12. Genotoxicity Study - The Micronucleus Test - Ceftiofur
Technical Report No. 7268-84-011, March 16, 1984
Starting Date - June 1, 1983
Termination Date - June 3, 1983
Study Director - D. H. Swenson
Location of Study - The Upjohn Company, Kalamazoo MI
Identification of Substance and Dosage Form - Ceftiofur dissolved in sterile water to make 100 mg drug/ml solution.
Species and Strain - Male Sprague Dawley Rats
Number Animals Per Sex Per Treatment Group - 10 M per group
Drug Levels Tested and Duration of Dosing
Ceftiofur at 250, 500 and 1,000 mg/kg body weight given in two equal doses 24h apart intraperitoneal (ip), water controls and cyclophosphamide as a positive control at 1 ml/kg.
Route of Administration - intraperitoneal
Parameters Tested
The micronucleus test is an in vivo procedure used for the detection of compounds that cause chromosome damage or nondisjunction of chromosomes (as induced by spindle poisons).
Micronuclei are chromosome fragments (as a result of clastogenic events) or whole chromosomes (a result of nondisjunctional events) that are left behind after expulsion of the nucleus during maturation of erythroblast to erythrocytes in the bone marrow of mammals. For this test an increased frequency of micronucleated polychromatophilic erythrocytes indicates chromosome damage, nondisjunction, or chromosomal deletions from the genome.
At 30 and 48 hours after the first dose (i.e., 6 and 24 hours after the second dose), the rats were sacrificed by cervical dislocation. The long bones of the hind quarters were exposed and the epiphyseal ends were snipped off to expose the marrow. A 20-gauge needle, on a 1 ml syringe containing 0.2-0.3 ml fetal calf serum, was inserted into the marrow cavity while simultaneously providing aspiration with the syringe plunger. The marrow plugs of the long bones of the hind quarters were dispersed in a disposable test tube, after which the cell suspension was placed on a microscope slip and spun at maximum speed for ten seconds in a Perkin Elmer slide spinner. The slides were air dried overnight at room temperature.
Significant Toxicity Observed
Cyclophosphamide, a mutagen that requires metabolic activation, was used as a positive control and significantly increased the incidence of micronucleated PCEs. None of the ceftiofur treatment groups exhibited a statistically significant increase in the rate of micronucleated PCEs over that of the control group, nor was the test for a positive dose response relationship statistically significant. These results indicate that ceftiofur did not have clastogenic or nondisjunctional effects on the chromosomes of rat bone marrow cells.
No Observed Effect Level - Not applicable
Statistical Analysis
Summary statistics were calculated