A. Toxicity Tests:
Toxicity and teratogenicity studies
were presented in the original NADA 128-620 and were conducted in Hoechst Research
Laboratories in Frankfurt, Germany and in the United States. Fenbendazole was
determined to be safe to human health when food derived from treated animals
is ingested (48 FR 42809, September 20, 1983).
B. Safe Concentrations and Tolerances
Safe concentrations for fenbendazole
total residues in cattle tissues were established with the original NADA 128-620
and are listed below along with the tissue consumption factors that were used.
Tissue Safe Concentration
muscle 5 ppm
liver 10 ppm (factor of 2)
kidney l5 ppm (factor of 3)
fat 20 ppm (factor of 4)
The tolerance and marker residue for
fenbendazole in cattle also were assigned with the original NADA 128-620. The
tolerance in cattle liver (the target tissue) is 0.8 ppm parent fenbendazole (the
marker residue) as measured by the regulatory assay.
Newly established with this supplement
to NADA 132-872 are a safe concentration and a tolerance for residues of fenbendazole
in milk. The safe concentration for fenbendazole total residues in milk is set
at 1.67 ppm (1/3 of the 5 ppm safe concentration in muscle tissue). The 1.67
ppm value was determined using FDA's approach to assigning safe concentrations
based on food factors (44 FR 17070, March 20, 1979).
As explained in Part C below, the
marker residue for fenbendazole in milk is the sulfoxide of parent fenbendazole.
The tolerance is assigned at 0.6 ppm, although the marker residue never reaches
that level in the milk from cattle treated at the approved dosing rate of 5
mg/kg body weight. The tolerance value was calculated from the marker residue
to total residue percentage when total fenbendazole residues are at a maximum
in milk. That maximum occurs in the range of 24 to 36 hours following dosing,
and at that time, the sulfoxide represents approximately 35% of the total residue
present. Accordingly, the tolerance for the fenbendazole sulfoxide is set at
0.6 ppm (35% of the 1.67 ppm safe concentration).
C. Total Residue and Metabolism
Studies
Tissue residue depletion and metabolism
studies in cattle were presented in the original NADA 128-620. Based on data
from those studies, an eight (8) day withdrawal time in edible tissues (muscle,
liver, fat, kidney) was established (48 FR 42809, September 20, 1983). The total
residue studies summarized below were submitted with this supplement to describe
fenbendazole residues in milk.
Milk Total Residue Study.
A study designed to measure residues
in milk from one untreated and five 14C fenbendazole-treated lactating
dairy cattle was conducted to determine the total residue profile as a function
of time, to identify metabolites of fenbendazole in milk, and to select a marker
substance to monitor residues in milk of lactating dairy cattle.
Study No. U.S. Dairy Cow Milk Residue Study LAV #1506
SVM (LSU Account # 166-60-6166)
Starting Date January 31, 1992
End Date August 5, 1993
Study Director Dr. Steven A. Barker
School of Veterinary Medicine
Louisiana State University
Baton Rouge, LA 70803
Identification of Substance and 14C-fenbendazole 1.89 mCi/g in aqueous
Dosage Form suspension
Species and Age Holstein, 33 months to 7 years
Number of Animals/weight Six lactating dairy cows; average 603 kg
Drug Level Tested 5.0 mg/kg body weight
Route of Administration Oral, administered once
After an acclimatization period, a morning milk sample was taken from each cow
prior to treatment. This sample served as a blank control for the study. Following
this milking, five cows received fenbendazole suspension by stomach tube. This
aqueous suspension of labeled (14C) fenbendazole and unlabeled fenbendazole
contained approximately 2 mCi activity/g. The amount of fenbendazole administered
to each cow by stomach tube was calculated to equal 5.0 mg/kg body weight. The
stomach tube was flushed with suspension solution to assure complete delivery.
A control cow received suspension solution which did not contain fenbendazole.
Morning and afternoon milk samples were collected for six days following drug
administration. After six days the residues were below the level of detection.
Total residues for each whole milk
sample from each cow were determined by scintillation counting. Each sample
was assayed in triplicate by dissolving 0.5 mL aliquots of blended sample in
12 mL of scintillation cocktail. Before counting, each sample was placed in
the dark for one hour to reduce contributions from chemiluminescence. Selected
whole milk samples were also centrifuged, and 0.5 mL aliquots of fat and water
portions were counted to determine label distribution.
For metabolic profiling, milk samples
were extracted by matrix solid phase dispersion (MSPD) techniques, and the absolute
recovery of total label was determined by scintillation counting of the extracts.
The distribution of the extracted label between remaining parent drug and metabolites
was determined by HPLC analyses using UV diode array and in-line radiolabel
detection. The identity of radiolabeled peaks was matched with known standards
for the metabolites of fenbendazole based on retention time and UV-diode array
spectra. Samples were also assayed quantitatively by HPLC using an internal
(mebendazole) standard and correcting for recovery.
The results from the radiolabel
assay for total residues in whole milk and the HPLC analyses of metabolites
in whole milk averaged for the five cows as a function of time are presented
in Table 1.
Table 1. Average Concentrations of Total Residues and Metabolites of
Fenbendazole in Whole Milk as a Function of Time Following Oral Administration
of 5.0 mg Fenbendazole/kg Body Weight to Five Lactating Dairy
Cows*.
Day, Milking Total Residue FBZ-SO (± SD) FBZ-SO2 (± FBZ/Total
(± SD) µg/mL µg/mL n = 5 SD) µg/mL n = Residue x 100
n = 5 (Sulfoxide) 5 (Sulfone) Ratio %
1, am (Time 0) 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000
1, pm 0.060 ± 0.043 0.026 ± 0.025 0.000 ± 0.000 43.333
2, am 0.482 ± 0.076 0.232 ± 0.045 0.018 ± 0.011 48.133
2, pm 0.526 ± 0.111 0.186 ± 0.005 0.024 ± 0.013 35.361**
3, am 0.408 ± 0.102 0.158 ± 0.026 0.062 ± 0.016 38.725
3, pm 0.298 ± 0.086 0.088 ± 0.034 0.046 ± 0.033 29.530
4, am 0.186 ± 0.080 0.030 ± 0.030 0.046 ± 0.024 16.129
4, pm 0.108 ± 0.044 0.006 ± 0.013 0.014 ± 0.017 5.555
5, am 0.054 ± 0.030 0.000 ± 0.000 0.010 ± 0.017 0.000
5, pm 0.024 ± 0.015 0.000 ± 0.000 0.000 ± 0.000 0.000
6, am 0.012 ± 0.008 0.000 ± 0.000 0.000 ± 0.000 0.000
6, pm 0.000 ± 0.000 0.000 ± 0.000 0.000 ± 0.000 0.000
*All residue levels were below the target of 0.83 ppm for the 1X tracer study
(one-half the 1.67 ppm established safe concentration). No residues were
detected in milk from the placebo (control) cow.
**Ratio percent used to calculate tolerance level.
At all times following administration
of fenbendazole to lactating dairy cattle, residues in milk of fenbendazole
and its metabolites were below the established safe concentration, and the total
residue was evenly distributed between the fat and aqueous fractions of the
whole milk.
Metabolic profiling of the total
residues indicated that the concentration of parent drug in milk was negligible.
The sulfoxide and sulfone metabolites of fenbendazole were the compounds that
contributed to milk residues. The sulfoxide metabolite of fenbendazole was established
to be the marker residue as it was present at levels significantly higher than
parent fenbendazole or its sulfone metabolite. No other metabolites of fenbendazole
were found in milk.
Milk Tolerance Calculation.
In Table 1 above, the ratio percent
value of fenbendazole sulfoxide, the marker residue, to total residues was 35.4%
at 36 hours following fenbendazole administration. At this time total residues
in milk were greatest. The tolerance was calculated by multiplying the ratio
percent of fenbendazole sulfoxide to total residues by the safe concentration
(1.67 ppm). The tolerance was established to be 0.6 ppm (600 ppb).
D. Calf Tissue Total Residue Study.
A study was conducted to measure
residues in calves born to 14C fenbendazole-treated dairy cattle.
Total residue profiles in calf liver, kidney, fat and muscle were measured to
provide data demonstrating the extent to which fenbendazole and its metabolites
are transferred to and retained by the tissues of calves born to fenbendazole-treated
cows.
Study No. U.S. Dairy Calf Tissue Residue Study LAV
#1507 SVM (LSU Account # 166-60-6167)
Starting Date April 21, 1992
End Date August 5, 1993
Study Director Dr. Steven A. Barker
School of Veterinary Medicine
Louisiana State University
Baton Rouge, LA 70803
Identification of Substance and 14C-fenbendazole, 1.89, 1.95 and 2.09 mCi/g
Dosage Form in aqueous suspension
Species and Age Holstein, 3 years old or older
Number of Animals/weight Six pregnant dairy cows; average 616 kg
Drug Level Tested 5.0 mg/kg body weight
Route of Administration Oral, administered once to the cow
Eight days prior to anticipated
calving, six pregnant dairy cows were moved to an approved facility for acclimation
and for study conduct. Three days after the start acclimation, each cow was
administered fenbendazole by stomach tube at a dose calculated to equal 5.0
mg/kg body weight. The drug was administered as an aqueous suspension of labeled
(14C) and unlabeled fenbendazole and contained approximately 2 mCi
activity/g. One of the six cows was administered carrier only and was the control
for the study. The calf from the control cow and calves from three cows receiving
fenbendazole were delivered by cesarean surgery approximately 70 hours after
dosing; the other two calves from treated cows were delivered by natural birth
at 4 and 25 hours post-dosing. One calf died 5 hours after delivery, three treated
calves and the control calf were sacrificed 24 hours after delivery, and one
treated calf was sacrificed 48 hours after delivery. Surviving calves received
colostrum from treated dams and milk replacer as needed for 24 to 48 hours after
birth and prior to sacrifice.
Total residues for the described
tissues were determined by oxidation of 0.5 g tissue samples in triplicate and
scintillation counting (Table 1). Each sample was placed in the dark for one
hour to reduce contributions from chemiluminescence.
Table 1. Concentrations of Total Residues of Fenbendazole and Metabolites in
Calf Tissues Following Administration of 5.0 mg Fenbendazole/kg Body Weight to
Five Pregnant Dairy Cows.
Tissue Total Residue (µg/g)
liver 1.398 ± 0.998*
kidney 0.528 ± 0.383
fat 0.386 ± 0.400
muscle 0.306 ± 0.236
*mean ± SD; n = 5
For metabolic profiling, liver
tissue from one calf was extracted by matrix solid phase dispersion (MSPD) techniques,
and the absolute recovery of total label was determined by scintillation counting
of the extracts. The distribution of the extracted label between parent drug
and metabolites was determined by HPLC analyses using UV diode array and in-line
radiolabel detection. The identity of radiolabeled peaks was matched with known
standards for the metabolites of fenbendazole based on retention time and UV-diode
array spectra.
Results indicated that the label
was distributed between the sulfone (34%) and sulfoxide (58%) metabolites of
fenbendazole and parent fenbendazole (8%). No other radiolabeled metabolites
were observed in the liver. Profiles of kidney, fat and muscle tissue from all
calves using HPLC indicated the presence of the sulfoxide and sulfone metabolites.
The parent drug, fenbendazole, was present in trace quantities. No other metabolites
were indicated.
It was concluded from the residue
data above, that in calves born to and consuming colostrum from fenbendazole-treated
dams, residues of fenbendazole in liver, kidney, fat and muscle were below the
established safe concentrations. Residue levels in liver, kidney, fat and muscle
as a percent of the safe concentrations were 13.98%, 3.52%, 1.93% and 6.12%,
respectively. Therefore, meat from calves born to fenbendazole-treated dams
is safe even when fenbendazole is administered prior to parturition.
E. Milk Residue Tolerance Study.
A study with non-radiolabeled fenbendazole
was conducted to determine the total quantity of fenbendazole and its metabolites
in whole milk as a function of time and to expand the examination to include
use of the actual market formulation. A further objective was to determine whether
incurred fenbendazole residues or its metabolites demonstrate activity in three
commonly used milk antibiotic screening tests, Charm II assay, Delvotest P,
and Bacillus stearothermophilis disc assay. For this study fenbendazole
paste 10% at a rate of 5 mg/kg body weight was administered to ten lactating
dairy cows; an additional cow served as a control.
Study No. U.S. Dairy Cow Milk Residue Study LAV #1591
SVM (LSU Account # 166-60-6172)
Starting Date November 3, 1992
End Date July 16, 1993
Study Director Dr. Steven A. Barker
School of Veterinary Medicine
Louisiana State University
Baton Rouge, LA 70803
Identification of Substance and Fenbendazole, Safe-Guard® Paste 10% (100
Dosage Form mg/g)
Species and Age Holstein, > 20 kg milk per day
Number of Cows/weight Eleven lactating dairy cows; average 530 kgs
Drug Level Tested 5.0 mg/kg body weight
Route of Administration Oral, administered once
Animals used in this study were
selected from the Holstein herd maintained at the LSU Agricultural Experimental
Station. All lactating dairy cows were at least thirty days postpartum and were
producing a target minimum of 20 kg of milk per day. Animals were managed in
the same manner as the remaining cow herd. The ration consisted of concentrate
and corn silage, and the cows grazed Bermuda and rye grass. Cows were monitored
for reproductive function, were bred by artificial insemination, and were treated
for reproductive dysfunction according to standard herd practices.
Cows were weighed within twenty-four
(24) hours of drug administration. The ten treated cows received Safe-Guard®
Paste 10% in an amount to equal delivery of 5.0 mg fenbendazole/kg body weight.
The control cow was untreated.
Cows were machine milked in the
morning prior to treatment. Milk samples were collected at that milking and
were used as blank controls for the study. Milk samples (100 mL) were then collected
at the 4:00 AM and 4:00 PM milkings for seven days following fenbendazole treatment.
For metabolic profiling, milk samples
were extracted by matrix solid phase dispersion (MSPD) technique. The amount
of parent drug and metabolites was determined quantitatively by HPLC analyses
using UV diode array detection. The identity of peaks was matched with known
standards for the metabolites of fenbendazole based on retention time and UV-diode
array spectra.
The administration of fenbendazole
at a target dose of 5.0 mg/kg body weight as paste 10% to lactating dairy cows
produced residues in whole milk identifiable as fenbendazole sulfoxide, fenbendazole
sulfone and trace quantities of fenbendazole. Peak residue time in milk was
twenty-four (24) hours after administration, and the peak fenbendazole sulfoxide
marker level was 0.24 ± 0.03 µg/mL (Table 1). No residues of fenbendazole
were detected in the control cow.
Table 1. Concentrations of Fenbendazole and Marker Metabolites of Fenbendazole
in Whole Milk as a Function of Time Following Oral Administration of Paste 10%
(100 mg/gm) at a Rate of 5.0 mg Fenbendazole/kg Body Weight to Ten Lactating
Dairy Cows.
Time after FBZ Fenbendazole (± FBZ-SO (± SD), FBZ-SO2 (± SD),
Administration SD), µg/mL, n= 10 µg/mL, n= 10 µg/mL, n= 10
(Sulfoxide)** (Sulfone)
0 nd* nd nd
12 nd 0.15 ± 0.06 0.01 ± 0.00
24 nd 0.24 ± 0.03 0.08 ± 0.01
36 nd 0.19 ± 0.03 0.11 ± 0.01
48 nd 0.10 ± 0.02 0.11 ± 0.01
60 nd 0.03 ± 0.01 0.08 ± 0.01
72 nd 0.00 ± 0.00 0.03 ± 0.00
*No residues detected.
**Marker residue
Antibiotic residue test screening
was conducted on milk samples from three (3) treated cows chosen randomly. The
samples were collected at 12 hour intervals for 72 hours post-dose . Tests performed
included the Charm II assay, Delvotest P, and Bacillus stearothermophilis
disc assay. Zero time samples were included in all antibiotic screening
tests; Delvotest P and B. stearothermophilis disc assay also included
milk collected from the control animal at 12 hour intervals for 72 hours post-dose.
Examinations indicated that the incurred residues from cows receiving fenbendazole
suspension 10% at a rate of 5.0 mg/kg body weight had no discernible or consistent
effect on the assays in term of producing false positive or suspect sample results.
No sample from any cow examined gave a "positive" response to the
Delvotest P and Bacillus stearothermophilis disc assay. Assay results
of ten antibiotic classes indicated that fenbendazole and its metabolites do
not interfere or cross-react with any consistency in the Charm II assay.
It was concluded that the fenbendazole
sulfoxide marker residue level was below the tolerance level, and therefore,
total residues were below the established safe concentration for milk. A zero-day
withdrawal period was approved for use of fenbendazole paste 10% in dairy cattle
of breeding age. It was further concluded that use of fenbendazole does not
interfere with the practice of antibiotic drug screening.
F. Milk Discard and Slaughter Time
A zero (0) milk discard time is
established for fenbendazole in dairy cattle of breeding age. The milk residue
depletion studies described in Parts C and E above demonstrate that the maximum
levels of fenbendazole residues in milk are well below the 1.67 ppm safe concentration
and 0.6 ppm tolerance when lactating dairy cows are treated at the approved
dosing rate of 5 mg/kg body weight. Accordingly, no discard of milk is required
following treatment with fenbendazole.
An eight (8) day withdrawal time
in edible tissues (muscle, liver, fat and kidney) was established in the original
NADA 128-620 (48 FR 42809, September 20, 1983) and applies to dairy cows treated
with fenbendazole.
G. Regulatory Methods:
A regulatory milk assay method
is not required because of the establishment of a zero (O) milk withdrawal period
in lactating dairy cattle. However, an HPLC assay method is on file at FDA/CVM
in Rockville, MD.
A regulatory tissue method was
developed as part of the original fenbendazole approval. The method, entitled,
"Determination Procedure for the Measurement of Fenbendazole in Bovine
Liver Tissue", is on file at the FDA's Freedom of Information Office, 5600
Fishers Lane, Rockville, MD 20857.
