DRUG AND CHEMICAL TOXICOLOGY, 14(1&2),
45-66 (1991)
TOXICITY AND CARCINOGENICITY STUDIES OF NALIDIXIC ACID IN RODENTS Richard E. Morrissey, Scot Eustis, Joseph K. Haseman, James Huff, John R. Bucher
Drug and Chemical Toxicology Downloaded from informahealthcare.com by Nyu Medical Center on 02/19/15 For personal use only.
National Toxicology Program National Institute of Environmental Health Sciences Research Triangle Park, NC 27709
ABSTRACT Toxicity and carcinogenicity studies of nalidixic acid, an antimicrobial agent used to treat bacterial infections of the urinary tract, were conducted in F344/N rats and B6C3F mice of each sex for 13 weeks or 2 years. In the 13-week studies, nalidixlc acid was administered at dietary concentrations ranging from 1,000 to 16,000 p p . Body weights of both rats and mice were reduced in the groups receiving diet containing 8,000 and 16,000 ppm, and feed consumption of rats in the highest treatment groups was approximately tw-thirds that of controls. Degeneration of the germinal epithelium in the seminiferous tubules of the testis was observed in male rats that received 16,000 ppm; no other compound-related histopathologic effects were observed in either species. !Wo-year studies were conducted by feeding diets containing 0, 2,000, or 4,000 ppm nalidixic acid to groups of 50 rats and mice/sex/group. The average daily feed consumption was slightly reduced compared to control groups and resulted in approximate daily doses of 82 or 175 mg nalidixic acidfig for low dose and high dose rats, and 220 or 475 mq/kg for l o w dose and high dose mice. Mean body weights of dosed rats and mice were lower than those of controls, except for groups of low dose female rats and male mice. The incidences of preputial gland neoplasms in dosed male rats and of clitoral gland neoplasms in dosed female rats were significantly increased compared to those in controls; responses in low dose groups were similar to those in high dose groups. There were decreased incidences of leukemia and mammary gland neoplasms in dosed female rats and of pituitary gland neoplasms in dosed male rats. Subcutaneous tissue fibrosarcomas were marginally increased in dosed male mice. There were no increased incidences of neoplasms in dosed female mice. under the conditions of these studies, the dietary administration of nalidixic acid was carcinogenic for rats, causing preputial gland or clitoral gland neoplasms in males and females, respectively. The association of subcutaneous neoplasms with administration of nalidixic acid to male mice was equivocal.
45 Copyright 0 1991 b y Marcel Dekker. I n c
MORRISSEY ET AL.
46
INTRODUCTION Nalidixic acid (l-ethyl-l,4-dihydro-7-methyl-4-oxo-l,8-naphthyridine-3carboxylic acid), an oral antimicrobial agent effective against gram-negative bacteria, is used to treat infections of the urinary tract in humans. It is effective against most Proteus strains, Kebsiella, Enterobacter, some Salmonella and Shigella strains, and Eschericia & (1); about 200,000 prescriptions were written in 1987 (2). Nalidixic acid selectively and
Drug and Chemical Toxicology Downloaded from informahealthcare.com by Nyu Medical Center on 02/19/15 For personal use only.
reversibly inhibits DNA synthesis in bacteria. It is an inhibitor of bacterial gyrase, an enzyme similar to topoisomerase I1 in eukaryotes ( 3 ) . The enzyme requires ATP to catalyze supercoiling of duplex DNA. Nalidixic acid is usually well tolerated in humans, but coxanon adverse reactions include gastrointestinal disturbances (41, skin lesions (5, 6), and neurologic reactions (1 for review).
No epidemiologic studies or case reports
examining the relationship between exposure to nalidixic acid and human cancer incidences have been located. After oral administration to humans, nalidixic acid is rapidly absorbed from the gastrointestinal tract, partially metabolized in the liver, and rapidly excreted through the kidneys (1). Absorption from the gastrointestinal tract is estimated to exceed 95%, with minimal fecal excretion. More than 90% of the nalidixic acid and 60% of its hydroxylated metabolite are protein bound in blood. both
A
steady-state serum concentration of
parent compound and metabolite is reached after 3 days of dosing
(half-life of each is about 2-3 hours). The recommended daily adult dose of 4 g (administered as 1 g four times per day for 1 or 2 weeks, average daily
dose of about 65 mg/kg) produces peak serum levels of active drug that average approximately 20-40 pg/ml (7, 8 ) .
Unchanged nalidixic acid (2%-3% of a dose)
appears in the urine along with an active metabolite, hydroxynalidixic acid (13%), which also has some antibacterial activity (9) reviewed in 1. Peak
urine levels of the active drug are approximately 150-300 pg/ml, 3-4 hours after administration ( 8 , 10). More than 80% of a dose is excreted as biologically inactive glucuronide and dicarboxylic derivatives of the parent
47
TOXICITY AND CARCINOGENICITY OF NALIDIXIC ACID
compound and the metabolite. In rats and mice, oral doses are rapidly absorbed; peak plasma concentrations are detected about 1 hour postdosing; elimination is via the kidneys and peaks at about 6 hours after dosing (11). A
series of oral studies (12-15) evaluated the potential toxicity of
nalidixic acid in rats, dogs, and monkeys. In rats, doses of 40 mg/kg/day for 1 year were reported not to cause hematologic, gross or pathologic changes. Similar findings were reported from a 131month study of dogs administered up
Drug and Chemical Toxicology Downloaded from informahealthcare.com by Nyu Medical Center on 02/19/15 For personal use only.
to 50 mg/kg, six times per week.
In adult rhesus monkeys receiving doses of
25, 75, or 225 mg/kg once per day for 1 year, a slightly lower growth rate, compared with controls, was the only observed effect in dosed monkeys.
In the
1-year monkey study, normal values were obtained in routine hematologic and serum biochemical analyses and urinalyses, and no pathologic tissue changes were observed.
A
1-year study conducted with juvenile monkeys administered
100 mg/kg had similar findings (15). There have been two reports addressing the potential carcinogenicity of nalidixic acid in mice. Kanisawa et al. (16) administered dietary concentration of 500 ppm to 20 ICR mice for 36 weeks. One hepatic adenoma was reported. Kurokawa et al. (17) exposed groups of 51 CFD, mice of each sex to nalidixic acid at dietary concentrations of 800 and 1,600 ppm for 7 6 weeks. All surviving animals were killed at week 85, after 9 weeks on a basal diet. Body weights were slightly laver in the high dose groups and in the low dose males compared to controls. No significant differences in the incidences of neoplasms in any organ were reported between dosed and control mice of either sex, Our studies were performed at the request of the Food and Drug Administration to investigate potential long-term health effects. Nalidixic acid exerts its antibacterial effect by inhibiting DNA synthesis and, at the time our studies began, no animal carcinogenicity data were available on this compound. The estimated human exposure to this drug is moderate-to-high and use is long-term in some persons. This paper reports the findings from our short-term toxicity and long-term carcinogenicity studies in rats and mice.
48
MORRISSEY ET AL.
mTERIALS AM) NETHODS
Chemical. Nalidixic acid (CAS No. 389-08-2; lot no. Hl20779) was obtained from Hilton-Davis Chemical Company (Cincinnati,OH) and its identity confinned
by infrared, ultravioletfiisible, and nuclear magnetic resonance spectroscopy. This lot was found to be approximately 99% pure, as determined by elemental analysis, Karl Fischer water analysis, ptentiometric titration in pyridine solution of the carboxylic acid group with 0.1 N tetrabutylanunonium hydroxide (in rnethanol:2-propanol, 1:9), thin-layer chromatography, and high-performance Drug and Chemical Toxicology Downloaded from informahealthcare.com by Nyu Medical Center on 02/19/15 For personal use only.
liquid chromatography (18). Stability of the bulk chemical during the course of the studies was monitored by nonaqueous titration of the carboxylic acid group and highperformance liquid chromatography. No notable degradation was observed during the studies. The homogeneity of formulated diets containing 8,000 ppm nalidixic acid was demonstrated to be within 1-3% of the target concentration in samples taken from three locations in the blender. Homogenous mixtures of nalidixic acid in NIH 07 rodent feed at a concentration of 8,000 ppm were shown to be stable for at least 2 weeks when held in the dark at temperatures up to 25OC. In the toxicity studies, the formulated diets were stored protected from light at roam temperature and used within 2 weeks of formulation. Animals. Four- to five-week-old male and female F344/N rats and 5- to 6-week-old male and female 86c3F, mice were obtained from Charles River Breeding Laboratories (Portage, MI) and were placed on study at 7-9 weeks of age. Animals were distributed to weight classes and assigned to cages and groups according to a table of random numbers. They were housed by sex, five per cage, in polycarbonate cages with filters and were provided with heat-treated hardwood chips as bedding. "he animals were maintained in a room that was kept at 20.0-26.7OC, relative hrnnidity of 30-74%, and a 12 hr fluorescent light cycle. Animals were observed twice per day; moribund animals were killed with carbon dioxide and necropsied. Feed consumption was measured one time per month by cage. Individual animal weights were recorded
TOXICITY AND CARCINOGENICITY OF NALIDIXIC ACID
09
one time per week up to 13 weeks, and thereafter in the 2-year study, every 4 weeks. Study Design. Dietary cpncentrations for the 13-week study were based on prior studies by the manufacturer (14). For the 13-week studies, groups of 10 rats and 10 mice of each sex were given diets containing 0, 1,000, 2,000, 4,000, 8,000, or 16,000 ppm nalidixic acid. Diets containing 0, 2,000, or 4,000 ppm nalidixic acid were fed to groups of 50 male and 50 female rats and
50 male and 50 female mice for 103 weeks. Control diets consisted of NIH 07 Drug and Chemical Toxicology Downloaded from informahealthcare.com by Nyu Medical Center on 02/19/15 For personal use only.
Rat or MOUSe Ration. Formulated or control diets and water were available ad libitum. Pathology.
A
necropsy was performed on all animals including those found
dead. During necropsy, all organs and tissues were examined for grossly visible lesions. Tissues were preserved in 10% neutral buffered formalin. embedded in paraffin, sectioned, and stained with hematoxylin and eosin. The following tissues were examined microscopically: adrenal glands, brain, cecum, colon, rib costochondral junction, duodenum, esophagus, eyes, gallbladder (mice),gross lesions, heart a d aorta, ileum, jejunum, kidneys, liver, lungs and bronchi, mammary gland, mandibular and mesenteric lymph nodes, nasal cavity and turbinates, pancreas, parathyroids, pituitary gland, preputial or clitoral gland, rectum, salivary gland, seminal vesicles/prostate/testes/or
ovaries/uterus, skin, spleen, femur including
marrow, stomach, thymus, thyroid gland, tissue masses and suspect tumors with regional lymph nodes, trachea, and urinary bladder. The following tissues were evaluated mic,coscopicallyin low dose animals: (male rats) adrenal glands, gross lesions, heart, nasal passage, pancreas, pituitary gland, preputial gland, and skin; (female rats) adrenal glands, clitoral gland, eyes, gross lesions, liver, nasal cavity, spleen, thymus, and uterus; (male mice) adrenal glands, gross lesions, kidneys, liver, and lungs. Statistical lethods: Organ and body weights were evaluated by Dunnett's test (19). Differences in survival were analyzed by life table methods (20). Tumor incidence data were analyzed by survival-adjusted procedures (21) and by
50
MORRISSEY ET AL.
the Fisher exact test and Cochran-Armitage trend test based on the overall proportion of tumor-bearing animals (22).
RESULTS
Thirteen-Week Study: Rats One female rat that received 16,000 p p nalidixic acid died before the end
Drug and Chemical Toxicology Downloaded from informahealthcare.com by Nyu Medical Center on 02/19/15 For personal use only.
of the study (Table 1); no other deaths occurred. Final mean body weights of rats that received 8,000 or 16,000 ppm were 23% or 49% lower than that of the controls for males and 11% or 31% lower for females. Feed consumption at 16,000 ppm was approximately two-thirds that by controls. Liver weight to
body weight ratios for males that received 2,000 or more and females that received 8,000 ppm or more were significantly greater than those for controls. Degeneration of the germinal epithelium in the seminiferous tubules of the testis was severe in all male rats that received 16,000
but was not
observed in male rats that received 8,000 ppm. 'Ityo-YearStudy: Rats Body Weights, Feed Consumption, and Clinical Signs: Mean body weights of high dose male rats were 7%-ll% lower than those of controls from week 0 to week 10 and 11%-23% lower thereafter (Table 2). Mean boay weights of low dose male rats were 6%-11% lower than those of controls from week 24 to the end of the study. Mean body weights of high dose female rats were 7%-12% lower than those of controls from week 0 to week 20 and 12%-19% lower thereafter. Mean body weignts of low dose and control female rats were comparable. The average daily feed consumption per rat by low dose or high dose rats was 95% or 90% that by controls. The average amount of nalidixic acid consumed per day was approximately 78 or 164 n@g for low dose or high dose male rats and 86 or 185 mg/kg for low dose or high dose female rats. No significant differences
in survival and no compound-related clinical signs were observed between any groups of either sex (Table 2).
0
+53 2 3 +51 ?: 4 +58 f 3 +52 3 +42 2 3' +3 f 2"
:;
+138 f 3 +121 f 4 +145 f 5 +119 f +74 f 4.. -14 2 4
Mean Body Weight Gain (grams)"
101 100 95 89 69
96 98 93 77 51
Final Weight Relative to Controls ( % )
t *
P
45-66 (1991)
TOXICITY AND CARCINOGENICITY STUDIES OF NALIDIXIC ACID IN RODENTS Richard E. Morrissey, Scot Eustis, Joseph K. Haseman, James Huff, John R. Bucher
Drug and Chemical Toxicology Downloaded from informahealthcare.com by Nyu Medical Center on 02/19/15 For personal use only.
National Toxicology Program National Institute of Environmental Health Sciences Research Triangle Park, NC 27709
ABSTRACT Toxicity and carcinogenicity studies of nalidixic acid, an antimicrobial agent used to treat bacterial infections of the urinary tract, were conducted in F344/N rats and B6C3F mice of each sex for 13 weeks or 2 years. In the 13-week studies, nalidixlc acid was administered at dietary concentrations ranging from 1,000 to 16,000 p p . Body weights of both rats and mice were reduced in the groups receiving diet containing 8,000 and 16,000 ppm, and feed consumption of rats in the highest treatment groups was approximately tw-thirds that of controls. Degeneration of the germinal epithelium in the seminiferous tubules of the testis was observed in male rats that received 16,000 ppm; no other compound-related histopathologic effects were observed in either species. !Wo-year studies were conducted by feeding diets containing 0, 2,000, or 4,000 ppm nalidixic acid to groups of 50 rats and mice/sex/group. The average daily feed consumption was slightly reduced compared to control groups and resulted in approximate daily doses of 82 or 175 mg nalidixic acidfig for low dose and high dose rats, and 220 or 475 mq/kg for l o w dose and high dose mice. Mean body weights of dosed rats and mice were lower than those of controls, except for groups of low dose female rats and male mice. The incidences of preputial gland neoplasms in dosed male rats and of clitoral gland neoplasms in dosed female rats were significantly increased compared to those in controls; responses in low dose groups were similar to those in high dose groups. There were decreased incidences of leukemia and mammary gland neoplasms in dosed female rats and of pituitary gland neoplasms in dosed male rats. Subcutaneous tissue fibrosarcomas were marginally increased in dosed male mice. There were no increased incidences of neoplasms in dosed female mice. under the conditions of these studies, the dietary administration of nalidixic acid was carcinogenic for rats, causing preputial gland or clitoral gland neoplasms in males and females, respectively. The association of subcutaneous neoplasms with administration of nalidixic acid to male mice was equivocal.
45 Copyright 0 1991 b y Marcel Dekker. I n c
MORRISSEY ET AL.
46
INTRODUCTION Nalidixic acid (l-ethyl-l,4-dihydro-7-methyl-4-oxo-l,8-naphthyridine-3carboxylic acid), an oral antimicrobial agent effective against gram-negative bacteria, is used to treat infections of the urinary tract in humans. It is effective against most Proteus strains, Kebsiella, Enterobacter, some Salmonella and Shigella strains, and Eschericia & (1); about 200,000 prescriptions were written in 1987 (2). Nalidixic acid selectively and
Drug and Chemical Toxicology Downloaded from informahealthcare.com by Nyu Medical Center on 02/19/15 For personal use only.
reversibly inhibits DNA synthesis in bacteria. It is an inhibitor of bacterial gyrase, an enzyme similar to topoisomerase I1 in eukaryotes ( 3 ) . The enzyme requires ATP to catalyze supercoiling of duplex DNA. Nalidixic acid is usually well tolerated in humans, but coxanon adverse reactions include gastrointestinal disturbances (41, skin lesions (5, 6), and neurologic reactions (1 for review).
No epidemiologic studies or case reports
examining the relationship between exposure to nalidixic acid and human cancer incidences have been located. After oral administration to humans, nalidixic acid is rapidly absorbed from the gastrointestinal tract, partially metabolized in the liver, and rapidly excreted through the kidneys (1). Absorption from the gastrointestinal tract is estimated to exceed 95%, with minimal fecal excretion. More than 90% of the nalidixic acid and 60% of its hydroxylated metabolite are protein bound in blood. both
A
steady-state serum concentration of
parent compound and metabolite is reached after 3 days of dosing
(half-life of each is about 2-3 hours). The recommended daily adult dose of 4 g (administered as 1 g four times per day for 1 or 2 weeks, average daily
dose of about 65 mg/kg) produces peak serum levels of active drug that average approximately 20-40 pg/ml (7, 8 ) .
Unchanged nalidixic acid (2%-3% of a dose)
appears in the urine along with an active metabolite, hydroxynalidixic acid (13%), which also has some antibacterial activity (9) reviewed in 1. Peak
urine levels of the active drug are approximately 150-300 pg/ml, 3-4 hours after administration ( 8 , 10). More than 80% of a dose is excreted as biologically inactive glucuronide and dicarboxylic derivatives of the parent
47
TOXICITY AND CARCINOGENICITY OF NALIDIXIC ACID
compound and the metabolite. In rats and mice, oral doses are rapidly absorbed; peak plasma concentrations are detected about 1 hour postdosing; elimination is via the kidneys and peaks at about 6 hours after dosing (11). A
series of oral studies (12-15) evaluated the potential toxicity of
nalidixic acid in rats, dogs, and monkeys. In rats, doses of 40 mg/kg/day for 1 year were reported not to cause hematologic, gross or pathologic changes. Similar findings were reported from a 131month study of dogs administered up
Drug and Chemical Toxicology Downloaded from informahealthcare.com by Nyu Medical Center on 02/19/15 For personal use only.
to 50 mg/kg, six times per week.
In adult rhesus monkeys receiving doses of
25, 75, or 225 mg/kg once per day for 1 year, a slightly lower growth rate, compared with controls, was the only observed effect in dosed monkeys.
In the
1-year monkey study, normal values were obtained in routine hematologic and serum biochemical analyses and urinalyses, and no pathologic tissue changes were observed.
A
1-year study conducted with juvenile monkeys administered
100 mg/kg had similar findings (15). There have been two reports addressing the potential carcinogenicity of nalidixic acid in mice. Kanisawa et al. (16) administered dietary concentration of 500 ppm to 20 ICR mice for 36 weeks. One hepatic adenoma was reported. Kurokawa et al. (17) exposed groups of 51 CFD, mice of each sex to nalidixic acid at dietary concentrations of 800 and 1,600 ppm for 7 6 weeks. All surviving animals were killed at week 85, after 9 weeks on a basal diet. Body weights were slightly laver in the high dose groups and in the low dose males compared to controls. No significant differences in the incidences of neoplasms in any organ were reported between dosed and control mice of either sex, Our studies were performed at the request of the Food and Drug Administration to investigate potential long-term health effects. Nalidixic acid exerts its antibacterial effect by inhibiting DNA synthesis and, at the time our studies began, no animal carcinogenicity data were available on this compound. The estimated human exposure to this drug is moderate-to-high and use is long-term in some persons. This paper reports the findings from our short-term toxicity and long-term carcinogenicity studies in rats and mice.
48
MORRISSEY ET AL.
mTERIALS AM) NETHODS
Chemical. Nalidixic acid (CAS No. 389-08-2; lot no. Hl20779) was obtained from Hilton-Davis Chemical Company (Cincinnati,OH) and its identity confinned
by infrared, ultravioletfiisible, and nuclear magnetic resonance spectroscopy. This lot was found to be approximately 99% pure, as determined by elemental analysis, Karl Fischer water analysis, ptentiometric titration in pyridine solution of the carboxylic acid group with 0.1 N tetrabutylanunonium hydroxide (in rnethanol:2-propanol, 1:9), thin-layer chromatography, and high-performance Drug and Chemical Toxicology Downloaded from informahealthcare.com by Nyu Medical Center on 02/19/15 For personal use only.
liquid chromatography (18). Stability of the bulk chemical during the course of the studies was monitored by nonaqueous titration of the carboxylic acid group and highperformance liquid chromatography. No notable degradation was observed during the studies. The homogeneity of formulated diets containing 8,000 ppm nalidixic acid was demonstrated to be within 1-3% of the target concentration in samples taken from three locations in the blender. Homogenous mixtures of nalidixic acid in NIH 07 rodent feed at a concentration of 8,000 ppm were shown to be stable for at least 2 weeks when held in the dark at temperatures up to 25OC. In the toxicity studies, the formulated diets were stored protected from light at roam temperature and used within 2 weeks of formulation. Animals. Four- to five-week-old male and female F344/N rats and 5- to 6-week-old male and female 86c3F, mice were obtained from Charles River Breeding Laboratories (Portage, MI) and were placed on study at 7-9 weeks of age. Animals were distributed to weight classes and assigned to cages and groups according to a table of random numbers. They were housed by sex, five per cage, in polycarbonate cages with filters and were provided with heat-treated hardwood chips as bedding. "he animals were maintained in a room that was kept at 20.0-26.7OC, relative hrnnidity of 30-74%, and a 12 hr fluorescent light cycle. Animals were observed twice per day; moribund animals were killed with carbon dioxide and necropsied. Feed consumption was measured one time per month by cage. Individual animal weights were recorded
TOXICITY AND CARCINOGENICITY OF NALIDIXIC ACID
09
one time per week up to 13 weeks, and thereafter in the 2-year study, every 4 weeks. Study Design. Dietary cpncentrations for the 13-week study were based on prior studies by the manufacturer (14). For the 13-week studies, groups of 10 rats and 10 mice of each sex were given diets containing 0, 1,000, 2,000, 4,000, 8,000, or 16,000 ppm nalidixic acid. Diets containing 0, 2,000, or 4,000 ppm nalidixic acid were fed to groups of 50 male and 50 female rats and
50 male and 50 female mice for 103 weeks. Control diets consisted of NIH 07 Drug and Chemical Toxicology Downloaded from informahealthcare.com by Nyu Medical Center on 02/19/15 For personal use only.
Rat or MOUSe Ration. Formulated or control diets and water were available ad libitum. Pathology.
A
necropsy was performed on all animals including those found
dead. During necropsy, all organs and tissues were examined for grossly visible lesions. Tissues were preserved in 10% neutral buffered formalin. embedded in paraffin, sectioned, and stained with hematoxylin and eosin. The following tissues were examined microscopically: adrenal glands, brain, cecum, colon, rib costochondral junction, duodenum, esophagus, eyes, gallbladder (mice),gross lesions, heart a d aorta, ileum, jejunum, kidneys, liver, lungs and bronchi, mammary gland, mandibular and mesenteric lymph nodes, nasal cavity and turbinates, pancreas, parathyroids, pituitary gland, preputial or clitoral gland, rectum, salivary gland, seminal vesicles/prostate/testes/or
ovaries/uterus, skin, spleen, femur including
marrow, stomach, thymus, thyroid gland, tissue masses and suspect tumors with regional lymph nodes, trachea, and urinary bladder. The following tissues were evaluated mic,coscopicallyin low dose animals: (male rats) adrenal glands, gross lesions, heart, nasal passage, pancreas, pituitary gland, preputial gland, and skin; (female rats) adrenal glands, clitoral gland, eyes, gross lesions, liver, nasal cavity, spleen, thymus, and uterus; (male mice) adrenal glands, gross lesions, kidneys, liver, and lungs. Statistical lethods: Organ and body weights were evaluated by Dunnett's test (19). Differences in survival were analyzed by life table methods (20). Tumor incidence data were analyzed by survival-adjusted procedures (21) and by
50
MORRISSEY ET AL.
the Fisher exact test and Cochran-Armitage trend test based on the overall proportion of tumor-bearing animals (22).
RESULTS
Thirteen-Week Study: Rats One female rat that received 16,000 p p nalidixic acid died before the end
Drug and Chemical Toxicology Downloaded from informahealthcare.com by Nyu Medical Center on 02/19/15 For personal use only.
of the study (Table 1); no other deaths occurred. Final mean body weights of rats that received 8,000 or 16,000 ppm were 23% or 49% lower than that of the controls for males and 11% or 31% lower for females. Feed consumption at 16,000 ppm was approximately two-thirds that by controls. Liver weight to
body weight ratios for males that received 2,000 or more and females that received 8,000 ppm or more were significantly greater than those for controls. Degeneration of the germinal epithelium in the seminiferous tubules of the testis was severe in all male rats that received 16,000
but was not
observed in male rats that received 8,000 ppm. 'Ityo-YearStudy: Rats Body Weights, Feed Consumption, and Clinical Signs: Mean body weights of high dose male rats were 7%-ll% lower than those of controls from week 0 to week 10 and 11%-23% lower thereafter (Table 2). Mean boay weights of low dose male rats were 6%-11% lower than those of controls from week 24 to the end of the study. Mean body weights of high dose female rats were 7%-12% lower than those of controls from week 0 to week 20 and 12%-19% lower thereafter. Mean body weignts of low dose and control female rats were comparable. The average daily feed consumption per rat by low dose or high dose rats was 95% or 90% that by controls. The average amount of nalidixic acid consumed per day was approximately 78 or 164 n@g for low dose or high dose male rats and 86 or 185 mg/kg for low dose or high dose female rats. No significant differences
in survival and no compound-related clinical signs were observed between any groups of either sex (Table 2).
0
+53 2 3 +51 ?: 4 +58 f 3 +52 3 +42 2 3' +3 f 2"
:;
+138 f 3 +121 f 4 +145 f 5 +119 f +74 f 4.. -14 2 4
Mean Body Weight Gain (grams)"
101 100 95 89 69
96 98 93 77 51
Final Weight Relative to Controls ( % )
t *
P
