A. Toxicity Studies
The 36th Meeting of the Joint FAO/WHO Expert Committee on Food
Additives (JECFA) has evaluated the toxicology data for carbadox
and its major metabolites. These data clearly show that the endpoint
of toxicological concern for carbadox is carcinogenicity. Studies
relating to the carcinogenic potential of carbadox and its metabolites
are summarized.
Carbadox
Genotoxicity Studies
Carbadox was evaluated by the sponsor and others in standard genotoxicity
batteries. Positive results were seen in the Ames test with Salmonella
typhimurium TA1535, TA100, TA98 (+/- S9) and equivocal results
were seen in the Ames test in Salmonella typhimurium TA1536,
TA1537, TA1538 and C340. Mutagenicity Ames test was positive for
E. coli WP2hcr, TA100 (+/- S9), and TA 98 and negative for
TA1535, TA1537, and TA1538. The host-mediated assay was positive
for S. typhimurium and negative for TA1534 and TA1952. The
repair test (Bacillus subtillis [rec] and S. typhimurium
[urv]) and the fluctuation test (Klebsiella pneumoniae and
E. coli) were positive. The mutagenicity test (Saccharomyces
cerevisiae D4), micronucleus test (rat bone marrow), repair
test (Bacillus subtillis M45) and chromosomal damage tests
(mouse bone marrow and in vitro human lymphocytes) were positive.
The dominant lethal test in CD-1 mice was negative (Pfizer Central
Research, undated; Negishi et al., 1980; Ohta et al.,
1980; Voogd et al., 1980; Beutin et al., 1981; Yoshimura
et al., 1981; and Cihak and Srb, 1983).
Long-term/Carcinogenicity Studies
Rats
One hundred twenty Charles River C-D rats were divided into 6 groups
(10/sex/dose) and received carbadox in the diet at rates providing
100, 50, 25, 10, 5 and 0 mg carbadox/kg b.w./day for 26 months.
Hematology and urinalysis were evaluated in 5 rats/sex/dose at 3,
6, 12, 18 and 25 months. Rats were sacrificed at 14 and 112 weeks
and received gross necropsies. Tissues were examined histopathologically
(Stebbins, 1964).
At the interim sacrifice (14 weeks), rats in the 100 mg /kg b.w./day
group showed decreased weight gain and food consumption, reduced
hemoglobin, RBCs and neutropenia. Microscopic changes included pulmonary
hemorrhage and edema, adrenalcortical hemorrhage and degeneration,
splenic hemosiderin and thymic atrophy. Rats in the 50 mg /kg b.w./day
group showed decreased weight gain and food consumption. Microscopic
changes included pulmonary hemorrhage and edema, adrenal cortical
hemorrhage, necrosis and degeneration, splenic hemosiderin and renocorticomedullary
fatty metamorphosis. All of the rats in the 100 and 50 mg /kg b.w./day
groups were sacrificed at 14 weeks. In the remaining treatment groups,
3 rats/sex/dose were sacrificed at 14 weeks. In the 25 mg /kg b.w./day
group, rats had reduced weight gain, slight adrenal cortical atrophy,
degeneration/necrosis and renal tubular fatty change. Rats in the
5 and 10 mg /kg b.w./day groups had no clinical, gross or microscopic
changes reported at 3 months (Stebbins, 1964).
The remaining rats were evaluated from 3 months up to and including
the 26-month sacrifice. In the 25 mg /kg b.w./day group, one female
rat died at 51 weeks with no drug-related changes noted. All 13
remaining rats were sacrificed at 73 weeks due to palpable abdominal
masses. At necropsy, all rats had multiple hepatic nodules. Ten
of 13 rats were diagnosed microscopically as having benign nodular
hyperplasia while the remaining 3 rats were determined to have malignant
transformation bases on metastatic foci in other organs. In the
10 mg /kg b.w./day group, one rat died after 67 weeks with reticuloendothelial
neoplasia, a common tumor of aged rats. The remaining rats in the
group were sacrificed between the 64th and 112th
week. Eleven of 13 rats were found to have hepatic benign nodular
hyperplasia. In the 5 mg /kg b.w./day group, one male died at 20
weeks due to pulmonary abscesses. The remaining 13 rats died or
were sacrificed between the 61st and 112th
week. Five of the rats were found to have hepatic benign nodular
hyperplasia. One control rat was sacrificed at 33 weeks with a forestomach
papilloma and pulmonary atelectasis. A second male died at 93 weeks
with myocarditis, nephrosclerosis and peribronchitis. The remaining
rats were sacrificed between the 80th and 112th
week. None of the control animals were observed to have hepatic
benign nodular hyperplasia (Stebbins, 1964).
In a study to determine the level of carbadox tolerated by rats
chronically, 120 Charles River rats received 2.5, 1.0 or 0 mg/kg
b.w./day of carbadox in the diet. Hematology, urinalysis and ophthalmoscopic
examinations were done at 3, 6, 12, 18 and 24 months on 5 rats/sex/dose.
Interim sacrifices of 5 rats/sex/dose were conducted at 54 weeks
with the remainder of the rats being sacrificed at 106 weeks. All
evaluated gross necropsies and microscopic parameters were within
normal limits at 54 weeks. At two years in the 2.5 mg /kg b.w./day
group, 7/27 rats displayed hepatic benign nodular hyperplasia and
peliosis hepatis. Additionally, the 2.5 mg /kg b.w./day group showed
an increase in total mammary tumors. In the 1.0 mg /kg b.w./day
group, 1/29 rats had hepatic benign nodular hyperplasia and 3/29
displayed peliosis hepatis. In the control group, 3/29 rats had
hepatic benign nodular hyperplasia and 2/29 displayed peliosis hepatis.
The 1.0 mg /kg b.w./day dose was tolerated by rats for 2 years with
no adverse effects (Sigler, 1969).
In a third study, rats (14/sex/group) were treated with 25 mg/kg
b.w./day carbadox or one of two lots of desoxycarbadox in the feed
for 10 months to compare the oncogenic activity of carbadox and
desoxycarbadox. An equal sized group served as untreated controls.
Two to four rats were sacrificed at 30, 60, 90, 191 and 309 days.
Animals received a gross necropsy at sacrifice and liver and adrenals
were evaluated histopathologically. A moderate decrease in body
weight occurred in both sexes receiving carbadox. At necropsy, all
groups treated for 10 months showed evidence of hepatic changes
including necrosis and nodule formation. Changes in the carbadox-treated
group were less severe than the changes in the desoxycarbadox groups.
All desoxycarbadox-treated rats and 2/18 of the carbadox-treated
rats showed evidence of hepatocellular carcinoma. There was a significant
increase in adrenal cortical hemorrhage in all treatment groups.
In this study, carbadox induced tumors in rats (King, 1976).
Monkeys
The long-term toxicity of carbadox in primates was assessed. Twenty-eight
monkeys were divided into 4 groups of 7 animals (3 or 4 per sex)
and were dosed with carbadox orally in gelatin capsules. Doses were
20 mg/kg b.w./day (as 5 mg/kg b.w./day QID), 10 mg/kg b.w./day (as
5 mg/kg b.w./day BID) or 5 mg/kg b.w./day and controls. Animals
were evaluated at 1, 3, 6, 12 and 24 months. Animals were sacrificed
at 3 months and 2 years. Elevated transaminase levels were detected
at the 3- and 6-month evaluations. The monkeys tolerated 20 mg/kg
b.w./day for 2 years with no adverse effects (Coleman, 1967).
Summary
Using data from these studies, a low-dose linear statistical model
was used to determine an S0 of 106 ppt for carbadox.
Desoxycarbadox
Genotoxicity Studies Desoxycarbadox was evaluated by the sponsor
in standard genotoxicity test batteries. Negative results were seen
in the Ames tests with TA1535, TA1537, TA1538, TA100, TA98, TA1537+S9
and TA1535 +S9. The host-mediated assay was negative for TA1950,
for TA1950 in mice and rats, and TA1535 in rats. The Ames test was
positive for TA1535+/- TA100S9(rat) and for TA1535+S from rat and
mouse. The Ames test was negative TA1535+S9 from hamster, dog and
monkey. The chromosomal damage test was negative for human lymphocytes
and rat bone marrow in a 5-day test. The chromosomal damage test
was positive for rat bone marrow in a 9 month feeding test. The
cell transformation test in BALB/C Swiss 3T3 was positive (Pfizer
Central Research, 1975; Holmes, 1976).
Long-term/Carcinogenicity Studies
Rats
A long-term study was conducted by the sponsor to determine the
tumorigenic potential of desoxycarbadox, a carbadox metabolite.
Four hundred Charles River C-D rats were divided into groups of
50/sex/dose. Desoxycarbadox was administered continuously in the
diet at doses of 0, 5, 10 and 25 mg/kg b.w./day. Although treatment
originally was scheduled for 2 years, test material was withdrawn
from all rats in the 25 mg/kg b.w./day and 50% of the rats of each
sex in the other two treatment groups on day 350 due to high morbidity
and mortality. Administration of desoxycarbadox was stopped completely
on day 416. The study was terminated on day 447 (Reinert, 1976).
Clinical examinations revealed a number of treatment-related signs
including tumors in the mammary region in both sexes, small cutaneous
nodules, and enlargement of the liver with nodules preceded by weight
loss and polyphagia. There was a dose-related decrease in weight
gain and a dose-related decrease in survival. Clinical chemistry
parameters were evaluated. Desoxycarbadox resulted in increases
in plasma enzyme activity, urea and bilirubin, abnormalities consistent
with hepatic disorders. The results were highly variable and nonreversible
following drug withdrawal. A dose related hypoglycemia was noted
in both sexes. Hematological parameters were evaluated in 10 rats/sex/group
at day 413 and for the remainder on day 447. A moderate hypochromic
microcytic anemia was seen in the 25 mg/kg b.w./day groups and a
slight anemia was seen in the 10 mg/kg b.w./day. A neutrophilia
also was observed in the 25 mg/kg b.w./day (Reinert, 1976).
All rats received a gross necropsy. Histopathological examinations
were performed on all grossly abnormal tissue and routinely on a
standard array of tissues. There was a dose-related increase in
pigmentation of the renal tubules and nephrosis. An increased tumor
incidence was seen in all treatment groups. Desoxycarbadox is a
potent hepatocarcinogen and there were dose-related increases in
other tumors (Reinert, 1976).
Summary
Using data from these studies, a low-dose linear statistical model
was used to determine an S0 of 61 ppt for desoxycarbadox.
Hydrazine
Genotoxicity Studies
Hydrazine was evaluated by several researchers in standard genotoxicity
batteries. Positive results were seen in the Salmonella typhimurium
Ames test, the mouse lymphoma cell test and the bacterial test using
E. coli WP2 uvr A trp (Ames, 1971; von Wright and Tikkanen,
1980; and Rogers and Back, 1981).
Long-term/Carcinogenicity Studies
Mice
A number of long-term/carcinogenicity studies have been conducted
in mice to evaluate the toxicity of hydrazine. These published studies
are briefly summarized for completeness.
Oral administration of hydrazine to BALB/C female mice at 1.13
mg/day for 46 weeks produced a 100% incidence of lung tumors (Biancifiori
and Ribacchi, 1962).
A 46% incidence of lung tumors was seen in female Swiss mice treated
with hydrazine at a dose level of 0.25 mg/day for 5 days/week for
46 weeks. Control mice had a 10% incidence (Roe et al., 1967).
In a study with CBA/Cb/Se mice, hydrazine was administered by gavage
at a dose of 1.13 mg/day for 36 weeks. Treated mice had an incidence
of 76% and 90% lung tumors for males and females, respectively.
Control mice had an incidence of 3%. Hepatomas were found in 62%
of males and 71% of the females treated with hydrazine. Control
mice had an incidence of 11% and 4% for males and females, respectively
(Severi and Biancifiori, 1968).
CBA mice were divided into groups of 40-59 mice /sex. Hydrazine
was administered by gavage daily for 150 days at rates of 45, 22,
11, 5.6 and 0 mg/kg b.w./day. Mice were examined at natural death
or following sacrifice when moribund. Control males had a hepatoma
incidence of 10% while females had a 3.4% rate. Treated males had
hepatoma rates of 60, 48, 28 and 3.8%, at 45, 22, 11, 5.6 mg/kg
b.w./day, respectively. Treated females had hepatoma rates of 62.5,
66.6, 8 and 0%, respectively (Biancifiori, 1970).
Hamsters
Golden hamsters were divided into three groups of 23, 35 and 56
animals. Animals received 60 doses of 3.0 mg hydrazine via intubation
over a 15-week period, 100 doses of 2.8 mg hydrazine via intubation
over a 20-week period, or no hydrazine, respectively. Hepatic lesions,
including fibrosis, reticuloendothelial cell proliferation and bile
duct proliferation, were found in 60-80% of the treated hamsters
but in none of the control animals (Biancifiori, 1970).
Negative tumorigenicity results were obtained in a chronic study
in golden hamsters. Fifty hamsters/sex received hydrazine in the
drinking water at 2.3 mg/day for a lifetime (Toth, 1972).
Rats
A chronic oral study was conducted in Cb/Se rats. Hydrazine was
administered daily by stomach tube at doses of 18 mg/rat to 14 males
and 12 mg/rat to 18 females. Dosing continued for 68 weeks. Lung
tumors were found in 21 and 27% of the dosed male and female rats,
respectively. Control groups (28M, 22F) had no lung tumors. Hepatic
tumors were found in 30% of the treated males while no hepatic tumors
were found in treated females or controls (Severi and Biancifiori,
1968).
Summary
Based on these published data, FDA has concluded that hydrazine
induces tumors in animals. Using a low-dose linear statistical model,
an S0 of 11 ppb is calculated.
Methyl Carbazate
Genotoxicity Studies
Methyl carbazate was evaluated in standard genotoxicity batteries.
Negative results were seen in the Ames tests and the chromosomal
damage test. Results in the host-mediated assay were equivocal (Pfizer
Central Research, 1975; Holmes, 1976).
Long-term/Carcinogenicity studies
Rats
The chronic oral toxicity of methyl carbazate, a metabolite of
carbadox, was studied by the sponsor in the rat. Wistar rats were
divided into groups of 12/sex/dose and received methyl carbazate
at target doses of 1 and 10 mg/kg b.w./day in feed for 10 months.
Necropsies were performed on all animals and histology was conducted
on a standard array of tissues and all tissues with grossly observed
tumors. Three males and three females in the high dose group died
before termination. No histopathological evidence of toxicity or
evidence of elevation in tumors was reported. The administration
of methyl carbazate to rats at dose levels of 1 and 10 mg/kg b.w./day
produced no evidence of carcinogenic potential (Rutty, 1972).
In a second study conducted by the sponsor, Wistar rats were divided
into groups of 24/sex and were treated with target doses of 0, 2.5,
5 and 10 mg/kg b.w./day in the feed for 710 days. Clinical examinations
were conducted weekly. Blood samples for clinical chemistry were
obtained at terminal sacrifice and from moribund animals. Hematology
was conducted on samples from moribund and dead rats as well as
from an interim sacrifice of 6 rats at 12 months. Rats were sacrificed
and necropsied at 710 days. Histopathology was performed on all
gross lesions and on a standard array of tissues. There were no
treatment-related effects noted in any parameters evaluated in the
study. There was histological evidence of widespread chronic respiratory
disease in all groups. Methyl carbazate had no adverse effect when
given to rats in the diet for 2 years (Ferrando, 1980).
Summary
On the basis of these studies, the agency has concluded that methyl
carbazate does not induce tumors in animals.
Quinoxaline-2-Carboxylic Acid (QCA)
Genotoxicity Studies
Quinoxaline-2-carboxylic acid was evaluated by the sponsor in a
standard genotoxicity battery. Negative results were seen in the
Ames tests with Salmonella typhimurium, TA1535, TA1537, TA1538
and TA1535+S9 tests. The chromosomal aberration test in in vitro
human lymphocytes also was negative (Pfizer Central Research, 1975).
Long-term/Carcinogenicity Studies
Mouse
Charles River CD mice (50/sex/dose) received QCA in feed for 19
months at levels to deliver 0, 25, 50 and 100 mg/kg b.w./day. Hematology
and clinical chemistry were evaluated once, prior to sacrifice.
Necropsies were performed on all animals and histopathological examinations
were conducted on a standard array of tissues. No treatment-related
effects were noted in any parameter during the study. Cumulative
mortalities and incidences of tumors were comparable for the control
and treatment groups. Oral administration of QCA to mice for 19
months produced no evidence of toxicity (Faccini et al.,
1979).
Rats
Nine male and 9 female Charles River C-D rats were divided into
groups of 3 rats/sex/dose. Rats received QCA in the feed for 2 years
at levels to provide 100, 50 or 0 mg/kg b.w./day. Rats received
clinical examinations weekly and routine ophthalmoscopic, hematology
and urinalysis examinations. Terminal sacrifice was performed on
day 735 of the study. All rats received gross necropsies and standard
tissues were evaluated microscopically. No treatment-related changes
were reported and QCA was tolerated at up to 100 mg/kg b.w./day
when given to rats via feed (Coleman, 1971).
In a study to determine whether QCA has tumorigenic potential,
Charles River Sprague-Dawley rats (20/sex/dose) were treated with
QCA in the diet for two years at doses to provide 0, 10, 25, and
50 mg/kg b.w./day. Rats received clinical examinations weekly and
routine ophthalmoscopic, hematology and urinalysis examinations.
At 12 months, 5 rats/sex/dose and at 24 months all remaining rats
were sacrificed, received a gross necropsy and standard tissues
were examined histopathologically. No treatment-related effects
were noted for any of the evaluated parameters. Cumulative tumor
rates were comparable for control and treated rats. QCA in doses
of 10, 25, and 50 mg/kg b.w./day over a two year period does not
produce any toxicity or elevated tumor incidence (Pfizer Central
Research, 1971).
Summary
Thus, quinoxaline-2-carboxylic acid is not a carcinogen in animals.
B. Definition of "No Residue"
Neither an ADI nor a safe concentration of total residues is calculated
for carbadox. Carbadox, and its metabolites desoxycarbadox and hydrazine,
were determined to be carcinogenic in animals. Methyl carbazate
and quinoxaline-2-carboxylic acid were also tested in short-term
genotoxicity assays and carcinogenicity studies and these compounds
are not carcinogens.
Pursuant to 21 CFR § 500.84(c)(2), FDA considers that "no
residue" of a compound remains in edible tissue when the residue
of carcinogenic concern in the total diet of people does not exceed
S0. The S0- is defined in 21 CFR §§
500.82(b) and 500.84 (c)(1) as the concentration of total residue
of carcinogenic concern of the test compound in the total diet of
test animals that corresponds to a maximum lifetime risk of cancer
in the test animals of 1 in 1 million. For carbadox and each of
the carcinogenic metabolites, an S0 was calculated using
a low-dose linear extrapolation statistical model. An S0
of 106 parts per trillion, 61 parts per trillion, and 11 parts per
billion was calculated for carbadox, desoxycarbadox, and hydrazine,
respectively. Based on these results, an S0 of 61 parts
per trillion (ppt) is determined for the total residues of carcinogenic
concern for carbadox in the total diet. As provided in 21 CFR §
500.82(b), this concentration, i.e., the S0, represents
no significant increase in risk of cancer to people.
Because the total human diet is not derived from food producing
animals, a correction for food intake is made in determining the
concentration of residues of carcinogenic concern that will be permitted
in edible animal tissue, 21 CFR § 500.84(c)(2). FDA designates
as Sm the permitted concentration of residues of carcinogenic
concern in a specific edible product, 21 CFR §§ 500.82(b)
and 500.84 (c)(2). Given a 1500 g total daily diet in humans, it
is assumed that up to 500 g is due to the consumption of meat. Of
this 500 g total daily meat consumption, 300 g is assumed to be
comprised of muscle, 100 g comprised of liver, 50 g comprised of
kidney, and 50 g comprised of fat. Thus, for any calculated S0,
one fifth of the total diet ( or 300g muscle/1500g total
diet) would be comprised of muscle. For an S0
of 61 parts per trillion (ppt), the concentration of total residues
of carcinogenic concern, Sm-muscle, would then be calculated
as 61 ppt ¸ 1/5 or 305 ppt. The Sm for each edible
tissue is calculated as follows:
Table 1: Consumption values and calculated Sm
for residues of carcinogenic concern in edible swine tissues
| Tissue |
Consumption Factor |
Fraction of Total Diet |
S0 |
Sm |
| muscle |
300 g |
1/5 |
61 ppt |
305 ppt* |
| liver |
100 g |
1/15 |
61 ppt |
915 ppt |
| kidney |
50 g |
1/30 |
61 ppt |
1830 ppt or 1.83 ppb** |
| fat |
50 g |
1/30 |
61 ppt |
1830 ppt or 1.83 ppb** |
* parts per trillion
** parts per billion
C. Residue and Metabolism Studies
Introduction
The sponsor and academic researchers have conducted numerous studies
evaluating the fate of carbadox in animals. These residue depletion
data are summarized in FAO Food and Nutrition Paper 41/3 (Food and
Agriculture Organization (FAO) of the United Nations, 1991) and
show that carbadox, desoxycarbadox and hydrazine do not persist
in edible tissue as detectable residues beyond 72 hours. The agency's
evaluation of these data, and the new information provided by the
sponsor, demonstrate that following administration, parent carbadox
is rapidly metabolized; that the metabolism of carbadox is similar
among species; that the in vivo metabolism of the compounds
of carcinogenic concern is also rapid and irreversible such that
the resulting metabolic products cannot regenerate compounds of
carcinogenic concern; that the unextractable residues are related
to noncarcinogenic compounds, quinoxaline-2-carboxylic acid and
quinoxaline-2-carboxaldehyde; and that quinoxaline-2-carboxylic
acid is the only residue detectable in the edible tissues beyond
72 hours postdosing. Thus, the agency concludes that the unextractable
bound residue is not of carcinogenic concern and that QCA is a reliable
marker residue for carbadox.
The sponsor has conducted two total residue and metabolism studies
in swine to establish the marker residue for carbadox in swine liver.
In the first radiotracer study, the swine developed an enteritis
on Day 2 of the treatment period that persisted for two days posttreatment.
Feed consumption was reduced in the sick animals. The second study
was conducted to address deficiencies in the first study resulting
from the presence of enteritis in the test animals.
Total Residues in Swine
1. A 14C-Carbadox Radiotracer Tissue Residue Study in
Growing Swine
The final report is No. 1525N-60-87-004, conducted by MJ Lynch,
Pfizer Central Research. The report is dated February 1988.
Ten preconditioned crossbred swine (5M and 5F) weighing 30 kg were
used in the study. The pigs were identified by ear tags and were
given ad libitum access to medicated feed containing 55 ppm
ring-labeled 14C-carbadox for five consecutive days. The radiolabeled
drug used to prepare the medicated feed had a specific activity
of 8.4 µCi/mg and a radiopurity (HPLC and TLC) of more than
99%. Following the treatment period, the swine were maintained on
a basal ration pending sacrifice at 30, 45 or 70 days withdrawal.
Two untreated swine served as controls. Excreta were collected during
the treatment period and for two days following return to the basal
ration. At necropsy, samples of the four edible tissues (500 g each)
were collected. Following homogenization, the tissue samples were
assayed for total and bound radioactivity and for major metabolites.
Total radioactivity was determined via combustion and liquid
scintillation counting. Bound residues were assessed following organic
extraction, with and without acid. Quinoxaline-2-carboxylic acid
(QCA), the only metabolite present at the sampling times, was determined
by thin-layer, gas chromatography reverse isotope dilution analysis,
following derivatization to methyl quinoxaline-2-carboxylate.
Table 1: Total radioactivity (ppm 14C-carbadox
equivalents) in tissues of swine following five days of feeding
14C-carbadox at 55 ppm
| Days Postdosing |
Liver |
Kidney |
Muscle |
Fat |
| 30 |
44.7±27.0 |
14.5±4.9 |
6.7±2.5 |
<2 |
| 45 |
12.3±3.8 |
4.0±2.0 |
1.7±0.6 |
<2 |
| 70 |
4.0±1.6 |
1.8±0.5 |
<1 |
<2 |
Table 2: Percentages of radioactivity remaining
in tissue following organic extraction without acid
| Days Postdosing |
Liver |
Kidney |
Muscle |
Fat |
| 30 |
93.8±1.1 |
93.0±1.3 |
95.4±3.2 |
N/A* |
| 45 |
94.2±1.8 |
94.6±4.7 |
92.1±3.2 |
N/A |
* N/A = Not assayed
Table 3: Percentages of radioactivity extracted
from tissue following digestion 1M HCl
| Days Postdosing |
Liver |
Kidney |
Muscle |
Fat |
| 30 |
2.3±0.9 |
1.8±0.3 |
2.3±0.7 |
N/A* |
| 45 |
3.0±0.2 |
7.2±5.5 |
3.5±3.5 |
N/A |
* N/A = Not assayed
2. A 14C-Carbadox Radiotracer Tissue Residue Study in
Growing Swine
The final report is No. 1525N-60-87-005, conducted by MJ Lynch,
Pfizer Central Research. The report is dated February 1988.
Ten preconditioned crossbred swine (5M and 5F) weighing 30 kg were
used in the study. The pigs were identified by ear nOver The Counterh and were
given ad libitum access to medicated feed containing 55 ppm
ring-labeled 14C-carbadox for five consecutive days. The radiolabeled
drug used to prepare the medicated feed had a specific activity
of 8.4 µCi/mg and a radiopurity (HPLC and TLC) of more than
99%. Following the treatment period, the swine were maintained on
a basal ration pending sacrifice at 30, 45 or 70 days withdrawal.
One untreated female pig served as the control. Excreta were collected
during the treatment period and for two days following return to
the basal ration. At necropsy, samples of the four edible tissues
(500 g each) were collected. Following homogenization, the tissue
samples were assayed for total and bound radioactivity and for major
metabolites. Total radioactivity was determined via combustion
and liquid scintillation counting. Quinoxaline-2-carboxylic acid
(QCA), the only metabolite present at the sampling times, was determined
by thin-layer, gas chromatography reverse isotope dilution analysis,
following derivatization to methyl quinoxaline-2-carboxylate.
Table 4: Total radioactivity (ppm 14C-carbadox equivalents)
in tissues of swine following five days of feeding 14C-carbadox
at 55 ppm
| Days Postdosing |
Liver |
Kidney |
Muscle |
Fat |
| 30 |
74.5±30.5 |
15.3±5.1 |
5.0±1.4 |
2.3±1.0 |
| 45 |
20.0±2.8 |
5.0±1.0 |
3.0±1.0 |
<1 |
| 70 |
13.3±0.6 |
3.7±0.6 |
2.3±0.6 |
<1 |
Metabolic Profiling in Swine
The profiling of carbadox metabolites in swine was conducted with
tissues from a preliminary total residue study.
In swine treated once with 14C-carbadox following a
stress period with cold drug, peak radioactivity in plasma was observed
at 3 hours postdosing. In plasma collected five hours posttreatment,
carbadox (13% of total), desoxycarbadox (19% of total), aldehyde
(13% of total) and QCA (19% of total) were present. At eight hours
posttreatment, only desoxycarbadox (9% of total) was identified
in swine plasma. All four compounds had disappeared within 24 hours.
About two-thirds of the dose was eliminated in the urine, the remainder
in the feces. For the 0-24 hour postdosing interval, urine radioactivity
averaged 65.4% and fecal radioactivity averaged 7.6% for a total
average 0-24 hour excretion of 73.1%. For the 24-48 hour postdosing
interval, urine radioactivity averaged 2.4% and fecal radioactivity
averaged 10.2% for a total average 24-48 hour excretion of 12.6%
and a 0-48 hour excretion of 85.4%. A small percentage of radioactivity
was excreted over the 48-72 hour period (0.4%, urine, and 1.1% feces)
for a total 0-72 excretion of 88.2%. Urinary metabolites of carbadox
were assayed by TLC and radiography. Quinoxaline-2-carboxylic acid
was identified as the major metabolite in swine urine. It was present
in a free form and as its glycine conjugate. No N-oxides were found
in urine. In feces, 9% of the radioactivity was attributable to
QCA and no unchanged carbadox was detected.
When studies were conducted with carbonyl-labeled carbadox, methyl
carbazate is generated. Approximately 25% is excreted in the urine.
Most of the methyl carbazate is enzymatically cleaved to yield CO2
which is exhaled. Radioactivity in the liver decreased with a half-life
of two days. At five days postdosing, liver radioactivity corresponded
to 0.12 ppm methyl carbazate equivalents, consisting in part of
amino acids labeled by incorporation of 14CO2.
Enzymatic hydrolysis of methyl carbazate implies but does not prove
the formation of hydrazine. No radiotracer method can demonstrate
the absence of hydrazine-related residues (i.e., hydrazine
= H2N-NH2). Chemical assays strongly suggest,
however, that hydrazine does not form a significant tissue residue.
In plasma, hydrazine is not detected by an assay sensitive to 0.1
ppm. This is not unexpected since several enzymatic processes are
known to destroy hydrazine.
Residues of QCA were determined in the tissues collected from studies
No. 1525N-60-87-004 and No. 1525N-60-87-005.
Table 5: Residues (ppb) of methyl quinoxaline-2-carboxylate
(expressed as ppb 14C-carbadox equivalents) in the tissues
of swine (No. 1525N-60-87-004)
| Days Postdosing |
Liver |
Kidney |
Muscle |
Fat |
| 30 |
9.3±6.7 |
<1 |
<1 |
N/A* |
| 45 |
2.7±0.7 |
<1 |
<1 |
|
| 70 |
0.3±0.1 |
<1 |
<1 |
N/A |
* N/A = Not assayed
Table 6: Residues (ppb) of methyl quinoxaline-2-carboxylate
(expressed as ppb 14C-carbadox equivalents) in the tissues
of swine (No. 1525N-60-87-005)
| Days Postdosing |
Liver |
Kidney |
Muscle |
Fat |
| 30 |
18.9±10.4 |
N/A |
N/A |
N/A* |
| 45 |
5.5±1.1 |
N/A |
N/A |
N/A |
| 70 |
1.3±0.5 |
N/A |
N/A |
N/A |
* N/A = Not assayed
D. Comparative Metabolism Studies
The metabolism of carbadox was studied in monkeys and rats. The
animals were treated orally with 14C-carbadox having
a specific activity of 1.97 µCi/mg. Rats were given 5 mg/kg
by stomach tube and one monkey was given 5 mg/kg in a capsule. Urine
and feces were collected and assayed for total radioactivity. Urinary
metabolites of carbadox were compared qualitatively by TLC and radiography.
The majority of the metabolites found in the pig also were found
in the rat or the monkey. One metabolite not found in the monkey
or the rat is the glycine conjugate of QCA; the other metabolite
represented only a few percent of the total urinary radioactivity.
Table 7: Excretion pattern (0-72 hrs) of radioactivity
(expressed as percent of dose) found in swine, monkey and rats,
after ingestion of 14C-carbadox.
| Species |
Dose |
Urine |
Feces |
| Swine |
3.5 mg/kg |
74.1 |
16.5 |
| Monkey |
5 mg/kg |
61.3 |
7.5 |
| Rats (6) |
5 mg/kg |
54.0 |
N/A* |
E. Designation of a Marker Residue and Tolerance
According to metabolic studies on carbadox, desoxycarbadox, and
hydrazine, which demonstrate that these compounds do not persist
in swine muscle, liver or kidney beyond 72 hours, the assignment
of quinoxaline-2-carboxylic acid (QCA) can serve as the marker residue
for the residues of carcinogenic concern. The assignment of a tolerance
of 30 ppb for QCA in swine liver assures that all residues of carcinogenic
concern are well below their respective S0 in all edible tissues.
Therefore, no residues of carcinogenic concern remain in the carcass
when using an assigned value of 30 ppb for the noncarcinogenic marker
residue (QCA).
The average percentage of total residues represented by quinoxaline-2-carboxylic
acid was determined from the two radiolabeled metabolism studies
in swine (see Section C above).
Table 8: The average percent of total residues represented
by QCA
| Days Postdosing |
Total Residue |
QCA |
%QCA |
| 30 |
75 ppb |
18.9ppb |
24.4 |
| 45 |
20 ppb |
5.5 ppb |
27.5 |
| 70 |
13 ppb |
1.3 ppb |
9.9 |
F. Regulatory Method
Residues of quinoxaline-2-carboxylic acid are determined using
an gas chromatographic assay with electron capture detection. The
method has a limit of quantification of 5 ppb. The method is on
file at the Center for Veterinary Medicine, Food and Drug Administration,
HFV-199, 5600 Fishers Lane, Rockville, Maryland 20855.
