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Abstract for TR-457

Toxicology and Carcinogenesis Studies of Salicylazosulfapyridine in F344/N Rats and B6C3F1 Mice (Gavage Studies)

CASRN: 599-79-1
Chemical Formula: C18H14N4 O5S
Molecular Weight: 398.39
Synonyms/Common Names: 2-Hydroxy-5-[[4-[2-(pyridinylamino)sulfonyl]phenyl]azo]benzoic acid; 5-[p- (2-pyridylsulfamoyl)phenylazo]salicylic acid; sulfasalazine; salazosulfapyridine; 5-[4-(2-pyridylsulfamoyl)phenylazo]-2-hydroxybenzoic acid; 4-(pyridyl-2-amidosulfonyl)-3'-carboxy-4'-hydroxyazobenzene; sulphasalazine
Report Date: May 1997

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Abstract

Salicylazosulfapyridine is widely used for the treatment of ulcerative colitis and Crohn's disease. It has been beneficial in the treatment of psoriasis and rheumatoid arthritis, and it has been used in veterinary medicine for the treatment of granulomatous colitis. Salicylazosulfapyridine was nominated for toxicity and carcinogenicity testing by the National Cancer Institute on the basis of its widespread use in humans and because it is a representative chemical from a class of aryl sulfonamides. Salicylazosulfapyridine is a suspect carcinogen because reductive cleavage of the azo linkage yields a p-amino aryl sulfonamide (sulfapyridine), and a related p-amino aryl sulfonamide (sulfamethoxazole) has been shown to produce thyroid neoplasms in rats. Toxicology and carcinogenicity studies were conducted in F344/N rats and B6C3F1 mice. Rats and mice were administered salicylazosulfapyridine (96% to 98% pure) in corn oil by gavage for 16 days, 13 weeks, or 2 years. The gavage route of administration was selected for these studies because it approximates the typical route of human exposure to the chemical. Genetic toxicology studies were conducted in vitro in Salmonella typhimurium and cultured Chinese hamster ovary cells and in vivo in rat and mouse bone marrow and mouse peripheral blood cells.

16-Day Study in Rats

Groups of five male and five female rats were administered 0, 675, 1,350, or 2,700 mg salicylazosulfapyridine/kg body weight in corn oil by gavage for 16 days excluding weekends. All rats survived to the end of the study. With the exception of the 675 mg/kg male group, the final mean body weights of all dosed groups of males and females were significantly lower than those of controls. Mean body weight gains of all dosed groups were less than those of controls. Clinical findings included ruffled fur and distended abdomens in male and female rats receiving 2,700 mg/kg.

Hypothyroidism, evidenced by decreased serum triiodothyronine and thyroxine concentrations and increased thyroid-stimulating hormone concentrations, occurred in 2,700 mg/kg male and female rats. The absolute and relative thymus weights of male rats receiving ,350 or 2,700 mg/kg and female rats receiving 2,700 mg/kg were significantly lower than those of controls.

At necropsy, all dosed rats had enlarged cecae/large intestines. Male rats receiving 1,350 mg/kg and male and female rats receiving 2,700 mg/kg had red, enlarged thyroid glands. Chemical-related microscopic lesions were present in the forestomach, thymus, thyroid gland, and pituitary gland. Minimal to mild hyperplasia of the forestomach mucosa was present in the 1,350 and 2,700 mg/kg male and female groups. Lymphoid depletion was observed in the thymus of three male and three female rats in the 2,700 mg/kg groups. Male and female rats receiving 1,350 and 2,700 mg/kg had thyroid gland follicular cell hyperplasia and an increase in thyroid-stimulating hormone producing cells in the pars distalis of the pituitary gland.

16-Day Study in Mice

Groups of five male and five female mice were administered 0, 675, 1,350, or 2,700 mg salicylazosulfapyridine/kg body weight in corn oil by gavage for 16 days excluding weekends. There were no chemical-related deaths, and final mean body weights of dosed mice were similar to those of controls. No chemical-related clinical findings were noted for male or female mice. There were no differences in triiodothyronine, thyroxine, or thyroid-stimulating hormone concentrations between dosed and control mice. There were no biologically significant differences in absolute or relative organ weights between dosed and control male and female mice. At necropsy, male mice receiving 2,700 mg/kg had enlarged cecae/large intestines. There were no biologically significant histopathologic lesions attributed to salicylazosulfapyridine administration.

13-Week Study in Rats

Groups of 10 male and 10 female rats were administered 0, 84, 168.8, or 337.5 mg salicylazosulfapyridine/kg body weight in corn oil by gavage for 13 weeks. All rats survived to the end of the study. The final mean body weights of dosed male rats were similar to those of controls; the final mean body weights and body weight gains of dosed females were significantly lower than those of controls. No chemical-related clinical findings were noted in dosed male or female rats during the 13-week study. No significant differences in hematology or urinalysis parameters between control and dosed rats were observed. The absolute and relative right kidney weights of 337.5 mg/kg females were significantly greater than those of controls.

At necropsy, some 337.5 mg/kg male rats had red, enlarged thyroid glands. Histopathologic changes were noted primarily in the thyroid gland and pituitary gland of males and females in the 337.5 mg/kg groups. The thyroid gland lesions observed were similar to those present in the 16-day study. Nine male rats receiving 168.8 mg/kg and ten male and seven female rats receiving .5 mg/kg had minimal but consistent changes in thyroid gland follicular cells. In the pituitary gland of 337.5 mg/kg males and females, the thyroid-stimulating hormone producing cells were enlarged and contained pale-staining cytoplasm and prominent Golgi complexes. Decreased serum triiodothyronine and thyroxine concentrations and increased thyroid-stimulating hormone concentration, similar to differences observed in the 16-day study, occurred in 337.5 mg/kg male rats; thyroid hormone concentrations were not affected in female rats.

Sperm motility of all dosed groups of males was significantly lower than that of controls. Vaginal cytology parameters of dosed groups of females were similar to those of controls.

13-Week Study in Mice

Groups of 10 male and 10 female mice were administered 0, 675, 1,350, or 2,700 mg salicylazosulfapyridine/kg body weight in corn oil by gavage for 13 weeks. All mice survived to the end of the study. The final mean body weights of dosed male and female mice were similar to those of controls. The mean body weight gains of 1,350 and 2,700 mg/kg male mice were less than that of controls. No chemical-related clinical findings were noted in dosed male or female mice during the 13-week study.

There was minimal evidence of a responsive anemia in mice in the 13-week study. The anemia was probably related to a methemoglobinemia. There were minimal decreases in thyroxine concentration in all dosed groups of male and female mice in the -week study. There were, however, no differences in triiodothyronine and thyroid-stimulating hormone concentrations between dosed and control animals.

Absolute and relative liver weights of all groups of dosed male and female mice were significantly greater than those of controls. There were no chemical-related gross lesions. Microscopic evaluation of the liver revealed centrilobular hypertrophy in five 1,350 mg/kg and all 2,700 mg/kg male mice.

The right cauda weight of the 1,350 mg/kg group and the right epididymis weights of all dose groups were significantly lower than those of controls. There was no evidence of chemical-related alteration in the vaginal cytology parameters of female mice.

2-Year Study in Rats

Groups of 60 male and 60 female rats were administered 84, 168, or 337.5 mg salicylazosulfapyridine/kg body weight in corn oil by gavage for up to 105 weeks. Groups of 70 male and 60 female rats were administered the corn oil vehicle by gavage for up to 105 weeks. A stop-exposure group of 70 male rats was administered 337.5 mg/kg salicylazosulfapyridine in corn oil by gavage for 6 months, after which animals received the corn oil vehicle by gavage for the remainder of the 2-year study. Ten animals from the vehicle control male group and 10 animals from the 337.5 mg/kg stop-exposure group were evaluated at 6 months; animals from each core-study group were evaluated at 15 months.

Survival, Body Weights, and Clinical Chemistry

Survival of 337.5 mg/kg male core-study rats was significantly lower than that of controls; survival of 84 and 168 mg/kg core-study males, all groups of dosed females, and the stop-exposure male group was similar to controls. Mean body weights of core-study males and stop-exposure males were similar to controls throughout the study. From week 45 to the end of the study, females in the 337.5 mg/kg group had mean body weights that were lower than those of controls. The serum thyroxine concentration in 337.5 mg/kg core-study males at study termination was minimally lower than that of controls; the serum thyroid-stimulating hormone, triiodothyronine, and reverse triiodothyronine concentrations of dosed males and females were similar to those of controls.

Pathology Findings

Administration of salicylazosulfapyridine for 2 years was associated with transitional epithelial papilloma in the urinary bladder of male rats and may have been associated with transitional epithelial papilloma of the kidney and of the urinary bladder of female rats. Nonneoplastic effects in the urinary bladder and kidney of male and female rats and in the spleen of male rats were also observed. Dosed male and female rats had increased incidences of grossly and microscopically observed urinary bladder concretions (diagnosed grossly as calculi at necropsy); male and female rats that developed transitional epithelial papillomas of the urinary bladder had grossly observed concretions (calculi) in the urinary bladder at necropsy. The microscopic neoplastic and nonneoplastic urinary bladder and kidney effects observed in dosed male rats during the 2-year continuous study did not occur in dosed rats during the 2-year stop-exposure study, nor were there gross observations of concretions (calculi) at necropsy. The incidences of mononuclear cell leukemia in male and female rats were decreased. The thyroid gland hyperplasia seen in the 13-week study was not observed in the 2-year study, and there was no evidence of chemical-related thyroid gland follicular cell adenomas or carcinomas.

2-Year Study in Mice

Groups of 60 male and 60 female mice were administered 0, 675, 1,350, or 2,700 mg salicylazosulfapyridine/kg body weight in corn oil by gavage for up to 104 weeks. Ten animals from each group were evaluated at 15 months.

Survival, Body Weights,and Clinical Chemistry

Survival of all the dosed groups of male and female mice was similar to that of controls. Mean body weights of 675 and 1,350 mg/kg male and female mice were similar to controls throughout the study. From week 12 to the end of the study, 2,700 mg/kg male mice had mean body weights that were lower than those of controls. From week 14 to the end of the study, the 2,700 mg/kg female mice had mean body weights that were lower than those of controls. There were no chemical-related differences in triiodothyronine, reverse triiodothyronine, thyroxine, or thyroid-stimulating hormone concentrations between dosed and control mice at the 15-month evaluation. 

Pathology Findings

Exposure of mice to salicylazosulfapyridine in corn oil by gavage for 2 years was associated with increased incidences of hepatocellular neoplasms in males and females. Nonneoplastic effects in the liver and spleen were also observed in male and female mice. The incidences of forestomach squamous cell papilloma in females and forestomach hyperplasia in males and females were decreased.

Genetic Toxicology

Salicylazosulfapyridine was not mutagenic in Salmonella typhimurium strains TA97, TA98, TA100, or TA1535, and it did not induce sister chromatid exchanges or chromosomal aberrations in cultured Chinese hamster ovary cells. These in vitro assays were performed with and without S9 metabolic activation enzymes.

Results from in vivo mouse bone marrow chromo somal aberration tests were uniformly negative, while results of micronucleus assays performed on male or female mice exposed to salicylazosulfapyridine for periods ranging from 3 days to weeks were positive. Micronucleus tests in male mice for shorter exposure times (1 to 2 days) yielded negative or very weakly positive results. A three-treatment (72-hour exposure time) micronucleus test performed in male rats yielded equivocal results. Overall, results of these in vivo assays indicate that salicylazosulfa pyridine is capable of inducing chromosomal damage, possibly in the form of aneuploidy, in mouse bone marrow cells after multiple administrations.

Conclusions

Under the conditions of these 2-year gavage studies, there was of carcinogenic activity some evidence of salicylazosulfapyridine in male and female F344/N rats based on increased incidences of neoplasms in the urinary tract. There was an increased incidence of transitional epithelial papilloma of the urinary bladder in males and a low incidence of rare transitional epithelial papillomas of the kidney and of the urinary bladder in females. There was of carcinogenic activity clear evidence of salicylazosulfapyridine in male and female B6C3F1 mice based on increased incidences of hepatocellular neoplasms.

Increased incidences of nonneoplastic lesions of the urinary bladder and kidney in male and female rats and of the spleen in male rats were observed. Increased incidences of nonneoplastic lesions of the liver and spleen in male and female mice were observed.

Decreased incidences of mononuclear cell leukemia in male and female rats were related to salicylazosulfapyridine administration. Decreased incidences of forestomach squamous cell papilloma in female mice and forestomach hyperplasia in male and female mice were related to salicylazosulfapyridine administration.

Studies

Summary of the 2-Year Carcinogenesis and Genetic Toxicology Studies of Salicylazosulfapyridine
  Male F344/N Rats Female F344/N Rats Male B6C3F1 Mice Female B6C3F1 Mice
Doses 0, 84, 168, or 337.5 mg/kg, or 337.5 mg/kg stop-exposure 0, 84, 168, or 337.5 mg/kg 0, 675, 1,350, or 2,700 mg/kg 0, 675, 1,350, or 2,700 mg/kg
Body weights Dosed groups similar to controls 337.5 mg/kg group lower than controls 2,700 mg/kg group lower than controls 2,700 mg/kg group lower than controls
Survival rates 35/50, 33/50, 31/50, 23/50, 30/50 35/50, 34/50, 31/50, 26/50 40/50, 41/50, 41/50, 46/50 41/50, 41/50, 37/50, 38/50
Nonneoplastic effects Urinary bladder: mucosal hyperplasia (0/50, 0/49, 14/50, 41/50, 1/47); concretion (0/50, 0/49, 1/50, 10/50, 0/47); dilatation (0/50, 1/49, 2/50, 7/50, 0/47)
Kidney: concretion (0/50, 1/50, 13/50, 33/50, 0/50); transitional epithelial hyperplasia (10/50, 10/50, 20/50, 43/50, 4/50); hydronephrosis (0/50, 1/50, 1/50, 28/50, 0/50); mineralization (3/50, 10/50, 11/50, 13/50, 3/50); renal tubule dilatation (0/50, 1/50, 1/50, 11/50, 3/50)
Spleen: hemosiderin pigment (14/50, 15/50, 15/50, 30/50, 13/50); hematopoietic cell proliferation (14/50, 15/50, 17/50, 23/50, 14/50
Urinary bladder: mucosal hyperplasia (2/49, 0/50, 4/50, 12/50)
Kidney: concretion (0/50, 9/50, 34/50, 37/50); transitional epithelial hyperplasia (3/50, 7/50, 23/50, 43/50); hydronephrosis (0/50, 0/50, 3/50, 10/50); nephropathy (34/50, 36/50, 41/50, 44/50)
Liver: eosinophilic foci (6/50, 19/50, 20/50, 22/50)
Spleen: hematopoietic cell proliferation (11/50, 16/50, 20/49, 13/50); hemosiderin pigment (2/50, 25/50, 32/49, 47/50)
Liver: eosinophilic foci (5/50, 17/50, 15/50, 19/49)
Spleen: hematopoietic cell proliferation (7/50, 21/50, 19/50, 23/49); hemosiderin pigment (14/50, 37/50, 39/50, 46/49)
Neoplastic effects Urinary bladder: transitional epithelial papilloma (0/50, 0/49, 2/50, 6/50, 0/47) Kidney: transitional epithelial papilloma (0/50, 0/50, 0/50, 2/50)
Urinary bladder: transitional epithelial papilloma (0/49, 0/50, 2/50, 0/50)
Liver: hepatocellular adenoma (13/50, 32/50, 28/50, 42/50); hepatocellular adenoma or carcinoma (24/50, 38/50, 38/50, 44/50) Liver: hepatocellular adenoma (12/50, 28/50, 25/50, 28/49); hepatocellular carcinoma (2/50, 10/50, 10/50, 9/49); hepatocellular adenoma or carcinoma (14/50, 32/50, 28/50, 29/49)
Decreased incidences Multiple organs: mononuclear cell leukemia (13/50, 18/50, 12/50, 3/50, 10/50) Multiple organs: mononuclear cell leukemia (14/50, 9/50, 8/50, 3/50) Forestomach: hyperplasia (18/50, 11/50, 11/49, 10/50) Forestomach: squamous cell papilloma (5/50, 1/50, 1/50, 0/50); hyperplasia (12/50, 6/49, 4/50, 4/49)
Level of evidence of carcinogenic activity Some evidence Some evidence Clear evidence Clear evidence
Genetic Toxicology of p,p'-dichlorodiphenylSulfones
Assay Test System Results
Bacterial mutagenicity Salmonella typhimurium gene mutations: Negative with and without S9 in strains TA97, TA98, TA100, and TA1535
Sister chromatid exchanges Cultured Chinese hamster ovary cells in vitro: Negative with and without S9
Chromosomal aberrations Cultured Chinese hamster ovary cells in vitro Negative with and without S9
Chromosomal aberrations Mouse bone marrow in vivo: Negative in standard and nonstandard protocols
Micronucleated erythrocytes Mouse bone marrow in vivo: Positive in two standard protocol assays and in kinetochore assay
Micronucleated erythrocytes Rat bone marrow in vivo: Equivocal
Micronucleated erythrocytes Mouse peripheral blood in vivo: Positive