0 

FOREWORD 

The National Toxicology Program (NTP) is made upof four charter agencies of the U.S. Department of 

Health and Human Services (DHHS): the National Cancer Institute (NCI), National Institutes of Health; the 

National Institute of Environmental Health Sciences (NIEHS), National Institutesof Health; the National 

Center for Toxicological Research (NCTR), Food and Drug Administration; and the National Institute for 

Occupational Safety and Health (NIOSH), Centers for Disease Control. In July 1981, the Carcinogenesis 

Bioassay Testing Program, NCI, was transferred to the NIEHS. The NTP coordinates the relevant 

programs, staff, and resources from these Public Health Service agencies relating to basic and applied 

research and to biological assay development and validation. 

The NTP develops, evaluates, and disseminates scientific information about potentially toxic and hazardous 

chemicals. This knowledge is used for protecting the health of the American people and for the primary 

prevention of disease. 

The studies’described in this Technical Report were performed under the direction of the NIEHS and were 

conducted in compliance with NTP laboratory health and safety requirements and must meet or exceed all 

applicable federal, state, and local health and safety regulations. Animal care and use were in accordance 

with the Public Health Service Policy on Humane Care and Useof Animals. The prechronic and chronic 

studies were conducted in compliance with Food and Drug Administration (FDA) Good Laboratory Practice 

Regulations, and all aspects of the chronic studies were subjectedto retrospective quality assurance audits 

before being presented for public review. 

These studies are designed and conducted to characterize and evaluate the toxicologic potential, including 

carcinogenic-ictivity, of selected chemicals in laboratory animals (usuallytwo species, rats and mice). 

Chemicals selected for NTP toxicology and carcinogenesis studiesare chosen primarily on the bases of 

human exposure, level of production, and chemical structure. The interpretive conclusions presented in this 

Technical Report are based only on the results of these NTP studies. Extrapolation of these results to other 

species and quantitative risk analysesfor humans require wider analyses beyond the purview of these 

studies. Selection per se is not an indicator of a chemical’s carcinogenic potential. 

These NTP Technical Reports are availablefor sale from the National Technical Information Service, 

U.S. Department of Commerce, 5285 Port Royal Road, Springfield, VA 22161 (703-487-4650). Single 

copies of this Technical Report are available without charge while supplies last from NTP Central Data 

Management, NIEHS, P.O. Box 12233, MD El-02, Research Triangle Park, NC 27709 (919-541-3419). 

Listings of all published NTP reports.and ongoing studiesare also available from NTP Central Data 

Management. The Abstracts and other study information for 2-year studies are also available on the NTP’s 

World Wide Web site: http://ntp-server.niehs.nih.gov. 

4 Salicylazosulfapyridine, NTP TR 457 
CONTENTS 

ABSTRACT ............................................................. 5 

APPENDIX A Summary of Lesions in Male Rats in the 2-Year Gavage Study 

APPENDIX B Summary of Lesions in Female Rats in the 2-Year Gavage Study 

APPENDIX C Summary of Lesions in Male Mice in the 2-Year Gavage Study 

APPENDIX D Summary of Lesions in Female Mice in the 2-Year Gavage Study 

APPENDIX J Ingredients, Nutrient Composition, and Contaminant Levels 

APPENDIX L Chemical Disposition of Salicylazosulfapyridine 

EXPLANATION OF LEVELS OF EVIDENCE OF CARCINOGENIC ACTIVITY ............. 11 

TECHNICAL REPORTS REVIEW SUBCOMMITI'EE ............................... 12 

SUMMARY OF TECHNICAL REPORTS REVIEW SUBCOMMITFEE COMMENTS .......... 13 

INTRODUCTION ......................................................... 15 

MATERIALS AND METHODS ............................................... 23 

RESULTS .............................................................. 37 

DISCUSSION AND CONCLUSIONS ............................................ 69 

REFERENCES ........................................................... 75 

of Salicylazosulfapyridine ........................................ 85 

of Salicylazosulfapyridine ........................................ 145 

of Salicylazosulfapyridine ........................................ 183 

of Salicylazosulfapyridine ........................................ 217 

APPENDIX E Genetic Toxicology ............................................. 257 

APPENDIX F Organ Weights and Organ-Weight-to-Body-Weight Ratios .................. 275 

APPENDIX G Hematology, Clinical Chemistry, and Urinalysis Results .................... 283 

APPENDIX H Reproductive Tissue Evaluations and Estrous Cycle Characterization ........... 293 

APPENDIX I Chemical Characterization and Dose Formulation Studies ................... 297 

in NIH-07 Rat and Mouse Ration ................................... 313 

APPENDIX K Sentinel Animal Program ........................................ 317 

in F344/N Rats and B6C3Fi Mice ................................... 321 
5 

COOH

I 

CAS NO. 599-79-1 

Chemical Formula: C18H,,N405S Molecular Weight: 398.39 

Synonyms: 

Tradenames: 

2-Hydroxy-5-[[4-[2-(pyridinylamino)sulfonyl]phenyl]azo]benzoic acid; 4-(pyridyl-2-amidosuIfonyl)-3’-carboxy5-[p-(2-pyridylsulfamoyl)phenylazo]salicylic acid; salazosulfapyridine; sulfasalazine; sulphasalazine 

4’-hydroxyazobenzene;5-[4-(2-pyridylsulfamoyl)phenylazo]-2-hydroxybenzoic acid; 

Azopyrin, Azulfidine, Benzosulfa,Colo-Pleon, Reupirin, Salazopyrin 

Salicylazosulfapyridine is widely used for the treatment of ulcerative colitis and Crohn’s disease. It has 

been beneficial inthetreatment of psoriasis and 

rheumatoid arthritis, and it has been used in veterinary medicine for the treatmentofgranulomatous 

colitis. Salicylazosulfapyridine was nominatedfor 

toxicity and carcinogenicity testing bytheNational 

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 neoplasmsin rats. Toxicology and 

carcinogenicity studies were conductedin F344/N rats 

and B6C3F, mice. Rats and mice were administered 

salicylazosulfapyridine (96 % to 98 % pure) incorn 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 

and in vivo in rat and mouse bone marrow and mouse 

peripheral blood cells. 

n6-mY STUDY IN RATS 

Groups of five male and five female rats were administered 0, 675, 1,350, or 2,700 mg salicylazosulfapyridinekg body weightin corn oil by gavage for 

16 days excluding weekends. All rats survived to the 

end ofthe study. Withthe exception of the 

675 mgkg male group, the final mean body weights 

of all dosed groups of males and females were significantly lower than thoseof 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 triiodothyronineand thyroxine concentrations and 

increased thyroid-stimulating hormone concentrations, 

occurred in 2,700 mgkg male and female rats. The 

absolute and relativethymus weights of male rats 

typhimurium and cultured Chinese hamster ovary cells receiving 1,350 or 2,700 mgkg and female rats 
6 

receiving 2,700 mg/kg were siRnificantly 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/kghad red, 

enlarged thyroid glands. Chemical-relatedmicroscopic 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 maleand 

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 thhroid gland follicular 

cell hyperplasia and an increase in thyroid-stimulating 

hormone producing cells in the pars distalis of the 

pituitary gland. 

16-DAY STUDYINMICE 

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 

for16, 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 notedfor 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 mgkg had enlarged cecae/large 

intestines. There were .no biologically significant 

histopathologic lesions attributedto salicylazosulfapyridine administration. 

.':ISWEEK 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 malerats were 

similar to those of controls; the finalmeanbody 

weights and body weight gains of dosed females were 

significantly lower than those of controls. No 

chemical-related clinical findingswere noted in dosed 

Salicylazosulfapyridine,NTP TR 457 

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 

glandofmalesandfemalesinthe 337.5 mg/kg 

groups. The thyroid gland lesions observed were 

similar to those present in the16-day study. Nine 

male rats receiving 168.8 mg/kg and tenmaleand 

seven female rats receiving 337.5 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 thyroxineconcentrations 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 conce,ntrations were not affected in 

female rats. 

Sperm motility of all dosed groups of males was 

significantly lower than that of controls. Vaginal 

cytology parametersof 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 weightin corn oil by gavage for 

13 weeks. All mice survived to the end of the study. 

The final meanbody 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 

malemice 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 13-week 
Salicylazosulfapyridine, NTP TR 497 7 

study. There were, however, no differences in tri- reverse triiodothyronine concentrationsof dosed males 

iodothyronine and thyroid-stimulating hormone con- and females were similar to those of controls. 

centrations between dosed and control animals. 

&&hQ&QQFhdh%@ 

Absolute and relative liver weights of all groups of Administration of salicylazosulfapyridine for 2 years 

dosed male and female mice were significantly greaterwas associated with transitional epithelial papilloma in 

than those of controls. There were no chemicalrelated 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 alterationinthe 

vaginal cytology parameters of female mice. 

%YEAR STUDY IIN RATS 

Groups of 60 male and 60 female rats were administered 84, 168, or 337.5 mg salicylazosulfapyridine/kg 

bodyweightin corn oilbygavage 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 mgkg salicylazosulfapyridine in corn oil bygavagefor 6 months, after 

which animals received the corn oil vehicleby 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; 10 animals from each core-study 

group were evaluated at 15 months. 

%WViVlJk, Body Weights, 

and Clinical Chemistpat 

Survival of 337.5 mg/kg malecore-study rats was 

significantly lower than thatof 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 

meanbodyweightsthatwerelowerthanthoseof 

controls. The serum thyroxineconcentrationin 

337.5 mg/kg core-study males atstudytermination 

was minimally lower than that of controls; the serum 

thyroid-stimulating hormone, triiodothyronine, and 

the urinary bladder of male rats and may have been 

associated with transitional epithelial -papilloma of the 

kidneyand of the urinary bladder of female rats. 

Nonneoplasticeffects 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 thatdeveloped transitional epithelial 

papillomasofthe 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 

didnot occur in dosed rats during the 2-year stopexposure 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. 

%YEAR STmY IIN MIICE 

Groups of 60 male and 60 female mice were administered 0, 675, 1,350, or2,700 mg .salicylazosulfapyridinekg body weight in corn oil by gavage for up 

to104weeks. Ten animals from each group were 

evaluated at 15 months. 

Supvival, Body Weights9 

and Clinical Chemistry 

Survival of all the dosed groups of male and female 

micewassimilar to that,. of controls. Meanbody 

weights of 675 and 1,350 mgkg male and female 

mice were similar to controls throughout the study. 

From week 12 to the end of the study, 2,700 mgkg 

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 mgkg female micehadmean 

body weights that were lower than those of controls. 
8 

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 incorn 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 mde and female mice. The 

incidences of forestomach squamous cell papilloma in 

females and forestomach hyperplasia in malesand 

females were decreased. 

GENETICTOXICOLOGY 

Salicylazosulfapyridine wasmutagenic not in 

Salmonella typhimuriumstrains TA97, TA98, TA100, 

or TA1535, and it didnotinduce sister chromatid 

exchanges or chromosomal aberrations in cultured 

Chinese hamster ovary cells. These in vitro assays 

were performed withandwithout S9 metabolic 

activation enzymes. 

Results from in vivo mousebone marrow chromosomal 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 13 weeks were positive. Micronucleus tests inmalemice 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 

Salicylazosulfapyridine, NTP TR 457 

rats yielded equivocal results. Overall, results of 

these in vivo assays indicate that, salicylazosulfapyridine is capable of inducing chromosomal damage, 

possiblyinthe form of aneuploidy, in mousebone 

marrow cells after multiple administrations. 

CONCLUSIONS 

Under the conditions of these 2-year gavage studies, 

there was some evidence of carcinogenic activity* 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 

inmalesand a low incidence of rare transitional 

epithelial papillomas of the kidney and of the urinary 

bladder in females. There ' was clear evidence of

carcinogenic activity of salicylazosulfapyridine in 

male and female 'B6C3F, 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 maleand 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. 

* Explanation of Levels of Evidence of Carcinogenic Activity is on page 11. A summary of the Technical Reports Review Subcommittee 

comments and the public discussion onthis Technical Report appears on page 13. 
Salicylazosulfaapyridine, NTP TR 457 9 

Summary d the %Year Carcinogenesis and Genetic ToxicologyStudies of Salicyliazosulffaayr~d~~e 

Male Female Male Female 

F3441N Rats F344/NRats B6C3F, Mice B6C3F, Mice 

i

!

I Doses 

337.5 mg/kg, or 

0,84,168, or 

337.5 mg/kg 

0, 84,168, or 2,700 mglkg 

0,675,1,350, or 

2,700 mg/kg 

0,675,1.350, or 

337.5 mglkg 

stop-exposure 

Body weights 

controls

Dosed groups similar to 

lower than controls 

337.5 mg/kg group 

lower than controls 

2,700 mglkg group 

lower than controls 

2,700 mg/kg group 

i 

i 

i

I 

1

I 

i 

I 

Survivalrates 

Nonneoplastic effects 

i 

23/50.30150 

35/50.33/50.31/50. 

Urinarv b~der: 

hydronephrosis (0/50, 

43/50. 4/50); 

(10/50,10/50,20/50, 

epithelial hyperplasia 

33/50, 0/50); transitional 

(0150, 1/50.13/50, 

Kidnev: concretion 

0/47) 

(0/50, 1/49, 2/50, 7/50. 

10/50, 0147); dilatation 

(0/50, 0/49,1/50. 

41/50.1/47); concretion 

(0/50,0149,14/50, 

mucosal hyperplasia 

,35/50, 34/50. 31/50, 

26/50 

44/50) 

(34/50, 36/50, 41/50, 

10150); nephropathy 

(0/50. 0/50,3/50, 

43/50); hydronephrosis 

(3/50, 7/50. 23/50. 

epithelial hyperplasia 

37/50); transitional 

(0/50,9/50. 34/50, 

Kidnev: concretion 

12/50) 

(2/49, 0150, 4/50, 

mucosal hyperplasia 

Urinarv bladder: 

46/50 

40/50, 41/50. 41/50, 

47/50) ' 

(2/50. 25/50. 32/49. 

hemosiderin pigment 

16/50.20/49,13/50); 

cell proliferation(1 1/50. 

Soleen:hematopoietic 

22/50) 

(6/50,19/50. 20/50, 

Liver: eosinophilicfoci 

38/50 

41/50. 41/50, 37/50, 

46/49) 

(14/50, 37/50. 39/50, 

hemosiderin pigment 

21/50.19/50.23/49); 

cell proliferation (7150, 

SDleen: hematopoietic 

19/49) 

(5/50, 17/50.15/50. 

Liver: eosinophilicfoci 

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) 

Ween: 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) 

Neoplastic effects 

transitional epithelial 

Urinarvbladder: 

epithelial papilloma 

Kidnev:transitional 

adenoma (13/50, 32/50. -

Liver: hepatocellular 

adenoma (12/50, 28/50. 

Liver: hepatocellular 

I

I 

I

I 2/50, 6/50. 0/47) 

papilloma (0/50, 0/49, 

2/50, 0150) 

papilloma (0149, 0150, 

transitional epithelial 

Urinarv bladder: 

(0150, 0150, 0/50, 2/50) 

38/50, 38/50. 4/50) 

or carcinoma (24/50, 

hepatocellular adenoma 

28/50, 42/50); 

adenoma or carcinoma 

9/49); hepatocellular 

(2/50,10150,10150, 

hepatocellular carcinoma 

25/50. 28/49); 

(14/50, 32/50. 28/50. 

29/49) 

L 
10 Salicylazosulfapyridine, NTP TR 457 

Summary of the 2-Year Carcinogenesis and Genetic Toxicology Studies of Salicylazosulfapyridine (continued) 

12/50. 3/50, 10150) 

Male Female Male Female 

F344lN Rats F344/NRats B6C3FIMice B6C3F, Mice 

' 

Decreased incidences Multiole Jvlultiole oreans; oreans: Forestomach; Forestoma& squamous

mononuclear mononuclear cellcell hyperplasia (18/50, cell papilloma (5150,

leukemia (13150. 18/50, leukemia (14/50. 9/50. , 11/50,10150) 11/49, 1/50, 1/50. 0150); . 

8/50. 3/50) hyperplasia (12/50, 

6/49, 4/50, 4/49) . , 

Level of evidence of Some evidence Some evidence Clear evidence Clear evidence I 

carcinogenic activity .. 

Genetic toxicology 

Salmonella iyphimurium gene mutations: Negative with and without S9 in strains TA97, TA98, TA100, and TA1535 . , Sister chromatid exchanges 

Cultured Chinese hamster ovary cells in vim: Negative with and without S9 

Chromosomal aberrations , 

Cultured Chinese hamster ovary cells in virro: Negative with and without S9 

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 

Rat bone marrow in vivo: Equivocal 

Mouse peripheral blood in vivo: Positive 
Sal,icylazosulhpyridine, NTP TR 457 

The National Toxicology Program describes the results of individual experiments on a chemical agent and notes the strength of the evidence 

for conclusions regarding each study. Negative results, in which the study animals do not have a greater incidence of neoplyia than control 

animals, do not necessarily mean that a chemical is not a carcinogen, inasmuch as the experiments are conducted under a limited set of 

conditions. Positive results demonstrate that a chemical is carcinogenic for laboratory animals under the conditions of the study and indicate 

that exposure to the chemical has the potential for hazard to humans. Other organizations, such as the International Agency for Research on 

Cancer, assign a strength of evidence for conclusions based on an examination of all available evidence, including animal studies such as those 

conducted by the NTP, epidemiologic studies, and estimates of exposure. Thus, the actual determination of risk to humans from chemicals 

found to be carcinogenic in laboratory animals requires a wider analysis that extends beyond the purview of these studies. 

Five categories of evidence of carcinogenic activity are used in the Technical Report series to summarize the strength of the evidence 

observed in each experiment: two categories for positive results (clear evidence and some evidence); one category for uncertain findings 

(equivocal evidence); one category for no observable effects (no evidence); and one category for experiments that cannot be evaluated 

because of major flaws (inadequate study). These categories of interpretative conclusions were first adopted in June 1983 and then revised in 

March 1986 for use in the Technical Report series to incorporate more specifically the concept of actual weight of evidence of carcinogenic 

,activity. For each separate experiment (male rats, female tits, male mice, female mice), one of the following five categories is selected to 

describe the findings. These categories refer to the strength of the experimental evidence and not to potency or mechanism. 

0 Clear evidence of carcinogenic activity is demonstrated by studies that are interpreted as showing a dose-related 

(i) increase of malignant neoplasms, (ii) increase of a combination of malignant and benign neoplasms, or (iii) marked increase of 

benign neoplasms if there is an indication from this or other studies of the ability of such tumors to progress to malignancy. 

0 Some evidence of carcinogenic activity is demonstrated by studies that are interpreted as showing a chemical-related increased 

incidence of neoplasms (malignant, benign, or combined) in which the strength of the response is less than that required for clear 

evidence. 

0 Equivocal evidence of carcinogenic activity is demonstrated by studies that are interpreted as showing a marginal increase of 

neoplasms that may be chemical related. 

0 No evidence of carcinogenic activity is demonstrated by studies that are interpreted as showing no chemical-related increases in 

malignant or benign neoplasms. 

0 Inadequate study of carcinogenic activity is demonstrated by studies that, because of major qualitative or quantitative limitations, 

cannot be interpreted as valid for showing either the presence or absence of carcinogenic activity. 

When a conclusion statement for a particular experiment is selected, consideration must be given to key factors that would extend the actual 

boundary of an individual category of evidence. Such consideration should allow for incorporation of scientific experience and current 

understanding of long-term carcinogenesis s&dies in laboratory animals, especially for those evaluations that may be on the borderline 

between two adjacent levels. These considerations should include: 

0 adequacy of the experimental design and conduct; 0 occurrence of common versus uncommon neoplasia; 0 progression (or lack thereof) from benign to malignant neoplasia as well as from preneoplastic to neoplastic lesions; 0 some benign neoplasms have the capacity to regress but others (of the same morphologic type) progress. At present, it is 

impossible to identify the difference. Therefore, where progression is known to be a possibility, the most prudent course is to 

assume that benign neoplasms of those types have the potential to become malignant; 

0 combining benign and malignant tumor incidence known or thought to represent stages of progression in the same organ or tissue; 

0 latencyin tumor induction; 

0 multiplicityin site-specific neoplasia; 

0 metastases; 

0 supporting information from proliferative lesions (hyperplasia) in the same site of neoplasia or in other experiments (same lesion 

in another sex or species): 0 presence or absence of dose relationships; 0 statistical significance of the observed tumor increase; 0 concurrent control tumor incidence as well as the historical control rate and variability for a specific neoplasm: 0 survival-adjusted analyses and false positive or false negative concerns: 0 structure-activity correlations; and 0 in some cases, genetic toxicology. 
12 Salicylazosulfapyridine, NTP TR.457 

NATIONAL TOXICOLOGY PROGRAMBOARD OF SCIENTIFIC COUNSELORS 

TECHNICAL REPORTS REVIEW SUBCOMMITTEE 

The members of the Technical Reports Review Subcommittee who evaluated the draft NTP Technical Report on salicylazosulfapyridine on 

20 June 1995 are listed below. Subcommittee members serve as independent scientists, not as representatives of any institution, company, or 

governmental agency. In this capacity, subcommittee members have five major responsibilities in reviewing NTP studies: 

l to ascertain that all relevant literature data have been adequately cited and interpreted, l to determine if the design and conditions of the NTP studies were appropriate, .. 

* to ensure that the Technical Report presents the experimental results and conclusions fully and clearly, * to judge the significance of the experimental results by scientific criteria, and l to assess the evaluation of the evidence of carcinogenic activity and other observed toxic responses. 

Arnold L. Brown, M.D., Chairperson 

University of Wisconsin Medical School 

Madison, WI 

Thomas L. Goldsworthy, Ph.D., PrincipalReviewer 

Department of Environmental Pathology and Toxicology 

Chemical Industry Institute of Toxicology 

Research Triangle Park, NC 

Meryl-H. Karol, Ph.D. 

Department of Environmental Occupational Health 

University of Pittsburgh 

' Pittsburgh, PA 

Curtis D. Klaassen, Ph.D. 

Department of Pharmacology and Toxicology 

University of Kansas Medical Center 

Kansas City, KS 

Claudia S. Miller, M.D. 

University of Texas Health Sciences Center 

San Antonio, TX 

Janardan K. Reddy, M.D. 

Department of Pathology 

Northwestern University Medical School 

Chicago, IL 

Irma RUSSO, M.D., Principal Reviewer 

Fox Chase Cancer Center 

Philadelphia, PA 

Louise Ryan, Ph.D. 

Division of Biostatistics 

Dana-Farber Cancer Institute 

Boston, MA 

Robert E. Taylor, M.D., Ph.D. 

Department of Pharmacology 

Howard University College of Medicine 

Washington, DC 

Mary Jo Vodicnik, Ph.D. 

Lilly MSG Development Center . , .

Belgium 

Jerrold M. Ward, D.V.M., Ph.D., Principal Reviewer' 

National Cancer Institute 

Frederick, MD 
On 20 June 1995, the Technical Report on the toxicology and carcinogenesis studies of salicylazosulfapyridine received publicreview by the National 

Toxicology Program’s Board of Scientific Counselors’ 

Technical Reports Review Subcommittee. The review 

meeting was held at the National Institute ofEnvironmental Health Sciences, Research Triangle Park, NC. 

Dr. F.W. Kari, NIEHS, introduced the toxicologyand 

carcinogenesis, studies of salicylazosulfapyridine by 

discussing the uses of the chemical and the rationale 

for study, describing the experimental design, reporting on survival and bodyweight effects, and commenting on compound-related neoplastic and nonneoplastic lesions in rats and mice. Dr. Kari reported 

that the gross and morphological evidence of thyroid 

gland hyperplasia in the 16-day and 13-week studies 

in rats was confirmed by clinicalchemistryand 

indicated a derangement of the pituitary-thyroid axis. 

As a result, a stop-exposure study wasdesignedin 

male rats, which were exposed to the highest dose of 

salicylazosulfapyridine for six months and given the 

corn oil vehicle for the remainder of the 2-year term. 

The expected thyroid gland lesions were not seen in 

any of the animals carried to 2 years. The proposed 

conclusions for the 2-year studies weresome evidence 

of carcinogenic activity in male and female F344/N 

rats and clear evidence of carcinogenic activity in 

male and female B6C3F, mice. 

Dr. Ward, a principal reviewer, agreed withthe 

proposed conclusions. Hethoughthatmalerats 

might have tolerated a higher dose. Dr. Kari agreed. 

Dr. Ward said the stop-exposure studies had limited 

significance in that neither preneoplastic nor neoplastic lesions were shown to have occurred at 26 weeks. 

Dr. M.R. Elwell, NIEHS, commented that based on 

the hyperplasia and thyroid gland effectsat 13 weeks, 

it was reasonable to expect that they would be present 

at 26 weeks. 

Dr. Goldsworthy, the secondprincipal reviewer, 

agreed with theproposedconclusionsbuthad concerns with the dose selection for mice. Giventhat 

liver weight changes and centrilobular hypertrophy 

were observed in all doses in the 13-week studies, he 

questioned why a dose at which changes had not been 

seen was not chosen for the 2-year studies.

Dr. Elwell saidthat at the low dose in the 13-week 

studies, hypertrophy could not be detected morphologically in female mice, and there was only a 10% to 

15% increase in liver weight in males. 

Dr. Goldsworthy commented that the same rationale 

givenforstop-exposurestudiesin rats could have 

been applied to the mouse and mouse liver neoplasms. 

Dr. Kari reportedthat at the timethe studies were 

designed, the thyroid gland hyperplasia appeared to be 

the predominant effect from salicylazosulfapyridine, 

and further, there was great interest in theroleof 

goitrogenic compounds such as the aryl sulfonamides 

in endocrine disruption and thyroid gland neoplasia. 

Dr. Russo, the third principal reviewer, agreed with 

the proposed conclusions. She noted that review of 

the literature indicated the probability of fetaldamage 

in humansfrom salicylazosulfapyridine, as well as 

transplacental transport and secretion in milk. Lack 

of data on absorption and disposition of salicylazosulfapyridine in pregnant experimental animals suggests that future studies of transplacental effects would 

be desirable. 

A discussion ensued about the renal neoplasms and 

their association with bladder calculi (concretions) in 

rats. Dr. J.R. Bucher, NIEHS, noted that this association was strongly supported by the fact that every 

animal with a bladder or kidney papilloma also had 

grossly observable calculi. Dr. Reddy inquired as to 

the nature of the concretions. Dr. Kari responded that 

they were spiculated in nature, but were not chemically analyzed. The calculi were presumed to be 

precipitated drug and/or metabolites. Dr. Russo 

asked how thesefindingscouldbe extrapolated to 

humanswhenthedosesinanimals were so much 

greater. Dr. Kari saidthe experiments in rodents 

were designed to maximize the probability for identifying toxicityand carcinogenicity. He pointed out 

that blood levels in the rodents used in these and other 

studies were discussed in this Technical Report and 

allow comparison with blood levels reported after 

maintenance doses in humans. At least for some of 

the doses, comparable blood levels were observed 

between the species. 

Dr. A. Imondi, Director of Toxicology and Safety 

Assessment. Pharmacia, Inc., stated that after many 
14 Salicylazosulfapyridine,NTP TR 457 

years of extensive clinical use as a therapeutic agent pyridine did not produce increases in DNA adduct 

in inflammatory bowel disease and rheumatoidarthri- formation in target tissues. He concluded that clinical 

tis, there is no evidence that salicylazosulfapyridine experience indicated no relevance of the NTP neocauses neoplasia in humans. With regard to positive plasm findings to humans. 

micronucleus tests observed in mice by the NTP, he 

cited unpublished studies that indicatefolicacid Dr. Ward moved that the Technical Report on salicyldeficiency is involved and increased incidencesof azosulfapyridine be accepted with the revisions submicronuclei could be reducedor reversed by folic acid mittedbythe panelists and with the conclusions as 

supplements to drug treatment. Dr. Imondi described written. Dr. Russo seconded the motion, which was 

other unpublished studies showing that salicylazosulfa-accepted unanimously with ten votes. 
CAS No. 599-79-1 

Chemical Formula: C,,H,,N,OSS Molecular Weight: 398.39 

Synonyms: 

Trade names: 

2-Hydroxy-5-[[4-[2-(pyridinylamino)sulfonyl]phenylJazo]be~oic acid; 4-(pyridyl-2-amidosulfonyl)-3'-carboxy5-[p-(2-pyridylsulfamoyl)phenylazo]salicylic acid; salazosulfapyridinr;sulfasalazine; sulphasalazine 

4'-hydroxyazobenzene;5-[4-(2-pyridylsulfamoyl)phenylazo]-2-l~ydroxybenzoicacid; 

Azopyrin, Azulfidine, Benzosulfa,Colo-Pleon, Reupirin, Salazopyrin 

@lHUEX"@AILAND mUSI[@AIL~ROPERTI[ES 

Salicylazosulfapyridine is manufactured as minute, 

brownish-yellow, odorless crystals. Itisslightly 

soluble in alcohol and practically insoluble in water, 

benzene, chloroform, and ether. Salicylazosulfapyridine decomposes at 240" to 245" C and has 

absorption maxima at 237 and 359 nm (Merck Index, 

1976). 

rnODUCTIION, USE, 

AND lHh.J"N ~XPOSURE 

Salicylazosulfapyridine is synthesized by coupling 

diazotized 2-sulfanilamidopyridine with salicylic acid 

(U.S. PatentNo. 2,396,145). Two American firms 

(American Cyanamid and Salisbury Labs, Inc.) and 

one Swedish firm (Pharmacia, Inc.) account forall 

production of this drug. In 1977, United States 

production was estimated to be greater than 420 kg, 

and imports in 1978 were74,000 kg (Pharmacia, Inc., 

unpublished data). Itis estimatedthat Pharmacia, 

Inc., produces 80% of all salicylazosulfapyridine used 

in the United States. Salicylazosulfapyridine has not 

been foundinUnited States industrial effluents 

(USEPA. 1979), in drinking water (NAS, 1977), or in 

European water supplies (Commission of the European Communities, 1976). 

NationalFormulary grade salicylazosulfapyridine 

contains 93% to 103% active ingredient calculated on 

a dry weight basis, with a maximum of 0.5% ignitable 

residue, 0.4% sulfate, 0.014% chloride, 0.002% 

heavymetals, and 10% maximumweight loss on 

drying. Salicylazosulfapyridinewas approved as a 

drug (sulfasalazine) by the FDA in June 1950 for the 

treatment of ulcerative colitis. It has been marketed 

since then as National Formulary grade sulfasalazine 

tablets (500 mg) or as an oral suspension (50 mg/mL) 

containing 95% to 105 % of the labeled amountof the 

activeingredient (PDR, 1992). Enteric-coated tablets 

are also available. 

Salicylazosulfapyridine is widely used for the treatment of ulcerative colitis and Crohn's disease (Badley, 

1975; Caprilli et al., 1975; Goodman and Gilman 's, 

1975; Van Hees et al., 1981). In the treatment of 

mild to moderate ulcerative colitis it is used by itself, 
16 Salicylazosulfapyridine, NTP TR 457 

whereas in severe ulcerative colitis and Crohn's 

disease it is used as .an adjunct therapy (Gardner, 

1976; Goldstein et al., 1976).Recently, a new 

topical (enema) preparation ,of 5-aminosalicylic acid 

(mesalazine) has been introduced for the treatment of 

ulcerative colitis (Bruckstein, 1990). The effective 

therapeutic dose in the active/acute phase ofthese 

ailments is 3 to 6 grams per day, while theusual 

maintenance dose is 2 grams per day (Davies and 

Rhodes, 1978; Dronfield and Langman, 1978; Riis 

et al., 1979). Salicylazosulfapyridine has also been 

beneficial in the treatment of psoriasis (Farr et al., 

1990; Gupta et al., 1990), rheumatoid arthritis 

(McConkey et al., 1980; Bird et al., 1982), and in 

veterinary medicine for the treatment of granulomatous colitis (Merck Index, 1989). Neither age nor 

gender influences the efficacyofsalicylazosulfapyridine for treatment of rheumatoid arthritis (Capell 

et al., 1993; Wilkieson et al., 1993). In 1982, 

1.3 million prescriptions were written for salicylazosulfapyridine; 40 % of these were for ulcerative colitis, 

21 % were for regional enteritis, 18% were for 

gastroenteritis in humans at least 2 years old, and 9% 

were for diverticulitis. The cumulative oral exposure 

in the United States population is estimated to be 

33,997 kg/year (NCI/SRI Database, 1983). 

ABSORPTION,DISTRIBUTION, 

METABOLISM, AND EXCRETION 

Experimental Animals plasma the concentration-time curve of salicylazoInSPrague-DawleY rats given SalicYlazosulfaPYridine sulfapyridine was 2-fold greater in. male rats than in 

in feed, the majority of theCompoundisreductivelYmalemice.whilethe svstemic bodv clearance and 

cleavedby gut bacteria to two biologically active 

moieties: 5-aminosalicylic acid and sulfapyridine 

(Peppercorn and Goldman, 1972). Riboflavinhas 

been shown to stimulate the azoreduction of orally 

administered salicylazosulfapyridine andthus increase 

the bioavailability of these metabolites inmale CD 

rats (Chungi et al., 1989). Sulfapyridine is well 

absorbed and eventually excreted via urine, but 

5-aminosalicylic acid is poorly absorbed and excreted 

mainly in feces (Bachrach, 1988). 

Inmaleand female B6C3Fi mice, salicylazosulfapyridine rapidly disappeared from blood following 

intravenous administration of a 5 mg/kg dose with a 

mean residence time of 0.45 to 0.78 hours (Zheng 

et al., 1993). The toxicokinetics of salicylazosulfapyridine were also determined following gavage 

administration of 67.5, 675, 1,350, or 2,700 mg/kg to 

.male and female mice.Bioavailabilitywas 16% to 

18% at a dose of 67.5 mg/kg and approximately 3 % 

to 9% for doses of 675 to 2,700 mg/kg. Following 

oral administration of salicylazosulfapyridine, both 

sulfapyridine and N-acetylsulfapyridine were identifiedin plasma. The areas under the plasma 

concentration-time curves of sulfapyridine at all four 

oral doses were 21- to 32-fold higher than those of 

salicylazosulfapyridine in male mice and5- to 25-fold 

higher in femalemice. The maximum plasma concentration (CmJ of sulfapyridine was 6 to 8 times 

higher than C, of salicylazosulfapyridine in male 

mice and up to 4 times higher in female mice. Elimination rate constants were greater for salicylazosulfapyridine; thus, sulfapyridine was eliminated more 

slowly than the parent compound, and sulfapyridine 

accumulated in maleandfemalemice following 

multiple doses of salicylazosulfapyridine (675, 1,350, 

and 2,700 mg/kg). 

In a companion study with male F344/N rats (NTP, 

1994a; Appendix L), plasma elimination rate constants and elimination half lives following a 5 mg/kg 

intravenous dose were similar to those reported for 

male B6C3Fi mice. The elimination half-lives (t,,J 

were 0.53 and 0.54 hours for rats and mice respec- tively, and the plasma elimination rate constants were 

1.47 hr-l and 1.28 hr-' respectively. The area under 

apparent volume of distribution were 2-fold higher in 

male mice compared to male rats. These data indicate 

that male F344/N rats clear salicylazosulfapyridine 

more slowly andthus retain salicylazosulfapyridine 

longer than male B6C3F, mice. Salicylazosulfapyridine and itsmetabolites were below detectable 

limits following low dose (67.5 mg/kg) oral administration. Following oral administration of 675 mg/kg 

salicylizosulfapyridine,the parent compound was detected in plasma at early time points but was undetectable at 12 hours. 

Nodata on salicylazosulfapyridine absorption and 

disposition in pregnant experimental animals were 

found in the peer-reviewed literature. 
I

t Salicylazosulfapyridine,NTP TW 457 

I 

WsdrntB-ZS 

The clinical pharmacokinetics of salicylazosulfa- glucuronidatedmetabolites 1 to 2.5 months after 

pyridine and its primary metabolites havebeen re- maternal dosing. 

viewed by Tett (1993), and they are summarized here. 

i The metabolism of salicylazosulfapyridine in humans 

i is similar to that previously described in rodents. The 

i metabolicpathway for salicylazosulfapyridineis I presented in Figure 1. i

I Studies in healthy volunteers as well as in patients 

with ulcerative colitis given single 2 to 4 g oral doses 

of salicylazosulfapyridine showed detectableblood i

! concentrations of salicylazosulfapyridine within 

1.5 hours of dosing and peak concentrations at 3 to 

5 hours post-dose (Schroder and Campbell, 1972; Das 

and Dubin, 1976). Both5-aminosalicylicacidand

I sulfapyridine (as well as their acetylated derivatives) 

appear in serum within 3 to 24hoursfollowing a 

single oral dose. Themajority of sulfapyridine is 

i 

I absorbed from the large intestine and is metabolized I extensively prior to urinary excretion; in contrast, I only a small percentage of 5-aminosalicylic acidis 

absorbed from the colon, and theremainderis excreted in the feces (Das and Dubin, 1976). 5-Aminoi salicylic acid is metabolized to acetylaminosalicylate 

byN-acetylation prior toitsurinary excretion 

I 

I (Schroder and Campbell, 1972). The rate at which i sulfapyridine is acetylated and hydroxylated is under I genetic control (Das and Eastwood, 1975). Patients 

i with a “slow” acetylator phenotype tendtohave I significantly higher serum concentrations of sulfa- I

I pyridine and itsnonacetylatedmetabolites. Serum i 

1 concentratiocs of sulfapyridine greater than50 pg/mL

appear to be associated with adverse reactions (Azad I Khan and Truelove, 1980). In patients with ulcerative I

I colitis, absorption and urinary excretionof salicylazosulfapyridine are not significantly changed compared 

to healthy controls, while these are reduced for i sulfapyridine (Azad Khan et al., 1982).

i

I Salicylazosulfapyridine and its two primary metabo- 1 lites can cross the placenta as evidenced by detection 

inumbilical cord blood andamnioticfluids(Azad 

Kahn and Truelove, 1979; Jarnerot et al., 1981). A 

small amount of salicylazosulfapyridine andsulfaMECrnNISM OF ACTION 

The mechanism of action of salicylazosulfapyridine is 

stillunderinvestigation. Originally, it was thought 

that salicylazosulfapyridine had a particular affinity 

for colonic connective tissue and serosal membrane 

(Svartz, 1942). Later, it was shown that both salicylazosulfapyridine and 5-aminosalicylic acid have such 

an affinity, but sulfapyridine does not (Hanngren 

et al., 1963). Following identification of the prodrug 

nature of salicylazosulfapyridine due to its cleavage 

into active products (Peppercorn and Goldman, 1972), 

it was proposed that the therapeutic effect was derived 

from the antibacterial action of sulfapyridine and the 

anti-inflammatoryaction of 5-aminosalicylic acid. 

Even though measurementof sulfapyridine serum concentrations has proven useful in dosage adjustment for 

salicylazosulfapyridine in patients and the serum concentrations correlate well with beneficial clinical 

response, the current evidence suggests that 5-aminosalicylicacid is the active therapeutic moiety of 

salicylazosulfapyridine (Azad Khanet al., 1977; Klotz 

et al., 1980; Van Hees et al., 1980). There is no evidence for antibacterial actionof sulfapyridine because 

the intestinal microflorae of patients taking salicylazosulfapyridine remain unchanged (Goodman and 

Gilmn’s, 1975).Ulcerativecolitis,theprimaryindication for salicylazosulfapyridine therapy, is characterized by an acute mucosal inflammation dominated 

bytherandommigrationand accumulation of 

polymorphonuclear leukocytes (Hermanowicz and 

Nawarska, 1981). Because salicylazosulfapyridine 

and 5-aminosalicylic acid inhibit such random migrations as well as phagocytosis and cellular oxidative 

metabolism (Molin and Stendahl, 1979), it has been 

hypothesized that the therapeutic action of 5-aminosalicylic acid could be due to its inhibition of prostaglandin biosynthesis and breakdown (Bakhle, 1980; 

Hillier et al., 1982) or to its immunosuppressant properties (Molin and Stendahl, 1979; Stenson and Lobos, 

pyridine was also detected in breast milk (Jarnerot and1982). Ultimately, salicylazosulfapyridine may be a 

Into-Malmberg, 1979; Berlin andYaffe,1980). No vehicle that only delivers high concentrations of both 

5-aminosalicylic acid has been detected in maternal 5-aminosalicylic acid and sulfapyridine to the colon 

plasma or milk or infant urine. Infantshavebeen (Das and Dubin, 1976; Goldman and Peppercorn, 

shown to excrete sulfapyridine and its acetylated and 1973). 

I! 
18 Salicylazosulfapyridine,NTP TR 457 

Salicylazosulfapyridine 

5-Aminosalicylic Acid Sulfapyridine 

5'-Hydroxysulfapyridine N4 -Acetylsulfapyridine 

. . 

0 

0 

N4-Acetyl-5'-hydroxysulfapyridine

OH HO 

5'-Hydroxysulfapyridine-O-glucuronide 

0 

OH HO 

FIGURE 1 

Pathways of Salicylazosulfapyridine Metabolism from Das .and Dubin(1976) I 
Salicylazosulhpyridine, NTP TW 457 

Toxrrcrrlru 

Exp@rim@ntal Animals 

In mice, the oral LD,, for 4-aminosalicylic acid is 

reported as 4 g/kg body weightand15 g/kg for 

sulfapyridine (Merck Index, 1976). 

Toxic effects of salicylazosulfapyridinetherapyin 

dogs have been reported. Keratoconjunctivitis sicca 

developed during a 4-year period in 13 dogs; discontinuance of the drug partially resolved the problem 

(Morgan and Bachrach, 1982). 

HUmanS 

It is estimated that more than 25% of patients given 

39 mgkg or more per day of salicylazosulfapyridine 

develop adverse/toxic effects during therapy. The 

n9 

Donovan et al., 1990).Becausemanyofthe side 

effects of salicylazosulfapyridine treatment of inflammatory bowel disease have been associated with the 

sulfapyridine moiety, 5-aminosalicylic acid was 

introduced as a new therapeutic agent (mesalazine) 

(Brimblecombe,1990). 

mPRoDuclrrrvIE 

rnDEvIELoPMENlrAL Toxrrcnlru 

Experimental Animals 

When male Sprague-Dawley rats were administered 

doses up to 617 mg salicylazosulfapyridine/kgbody 

weight per day for 8 weeks, dose-dependent, reversible infertility with a significant reduction in offspring 

litter size wasobserved (O’Morhin et al., 1984). 

side effects commonly associated with salicylazosulfa-Male rats exposed to sulfapyridine in feed sired smallpyridine therapy (both in adultsand children) have 

been anorexia, nausea, headache, fever, vomiting, 

gastric distress, general hypersensitivity reactions 

(rash, liver function abnormalities), and various 

hematological abnormalities (Svartz, 1942; Das and 

Dubin, 1976; Felson et al., 1990). In patients with 

ulcerative colitis, individuals with the “slow” acetylator phenotype are reported to have increased incidences of salicylazosulfapyridine-induced adverse 

reactions (Das et al., 1973), whereas inrheumatoid 

arthritis patients, acetylator phenotype is unrelated to 

adverse therapeutic outcomes (Chalmerset al., 1990). 

Several clinical (case) reports have reportedother 

toxidsevere adverse reactions tosalicylazosulfapyridine including pancreatitis (Block et al., 1970), 

renal and hepatic toxicity (Chesteret al., 1978), lupus 

syndrome (Carr-Locke. 1982), folate deficiency 

(Swinson et al., 1981), male infertility (Tooveyet al., 

1981), decreased pulmonary function (Eade etal., 

1980), neurotoxicity (Smithet al., 1982; Alloway and 

Mitchell, 1993), lymphadenopathy (Carr-Locke and 

Ali, 1982), and various hemolytic anemias (aplastic, 

Heinz body, etc.) (Davies and Palek, 1980; Vilaseca 

et al., 1980). Most of these adversereactionswere 

reported to be reversible within a few days to several 

weeks after the drug .was discontinued. Patients with 

ulcerative colitis or rheumatoid arthritis who benefit 

from salicylazosulfapyridine therapy butdevelop 

hypersensitivity responses are able to resume salicyler sized litters when mated to untreated virgins than 

did untreated males. There was no effect on litter size 

for males exposed to 5-aminosalicylic acid in feed. 

No adverse effects on seminal characteristicsor libido 

were observed in 4-year-old beagle dogs duringor for 

6 weeks after a 6-week oral treatment with 50 mg 

salicylazosulfapyridine/kg bodyweight per day 

(England and Allen, 1993). 

HUmanS 

Salicylazosulfapyridine-induced male infertility has 

been identified (Levi etal., 1979) and confirmed 

(Toth, 1979; Freeman et al., 1982; O’Morhin et al., 

1984) in a number of patients being treated for inflammatory bowel diseases. This adverse effect is reversible upon discontinuance of the drug. Although the 

mechanism of toxicity is not understood,an evaluation 

of human sperm quality (density, motility, and morphology) in time-course studies indicated that the 

metabolite sulfapyridine may be directly toxic to 

developingspermatozoa (Toovey et al., 1981). 

Embryotoxicityof salicylazosulfapyridine has been 

suggested in one case in which a young woman given 

salicylazosulfapyridine during pregnancy delivered 

(prematurely) an infant with bilateral cleft lip, cleft 

palate, and severe hydrocephalus (Craxi and 

Pagliarello, 1980). Other clinical studies, however, 

have reported no adverse effect of salicylazosulfapyriazosulfapyridine therapy once they have been desensi-dine on the human fetus following in utero exposure 

tizedvia a short withdrawal period (Holdsworth, (Levy et al., 198 1 ; Mogadam et al.. 1981; Vender 

1981; Farr et al., 1982; Taffet and Das.1982; and Sprio, 1982). 
20 

CARCINOGENICITY 

Experimental Animals . . 

. . 

There is no published literature regarding the evaluation of salicylazosulfapyridine or its metabolites for 

chronic toxicity or carcinogenicity in rodents, but 

there area few carcinogenicity studies of .related 

sulfonamide drugs. In rats, exposure to sulfamethoxazole in feed for 52 or 60 weeksinducedthyroid 

gland neoplasms with lung metastases (Swarm et al., 

1973). Withdrawal of sulfamethoxazole from the diet 

after 6 or 13 weeks of administration resulted in restoration of normal follicular structure in the gland. In 

contrast, sulfisoxazole was not carcinogenic to Fisher 

344 rats or B6C3F, mice (NCI, 1979). It is believed 

that various experimental animals (rat, mouse, and 

dog) are sensitive to a goitrogenic action of sulfonamides,, whereas the thyroid gland ofmonkeysand 

humans may not be sensitive to this effect (Swarm 

et al., 1973; Hill et al., 1989; McClain, 1992). 

Humans 

No epidemiology studies or case reports relating 

~ salicylazosulfapyridine to .human cancer have been 

reported in the literature. However, one woman 

treated with salicylazosulfapyridine and prednisone for 

' ulcerative colitis developed adenocarcinoma of the 

small intestine 18 years later (Schuringa, 1973). It is 

known that patients with chronic ulcerative colitis are 

at increased risk for the development of colon cancer 

(Reddy et al., 1977). 

GENETICTOXICITY 

There have been a limited number of in vitro studies 

conducted with salicylazosulfapyridine and/or its 

metabolites, and in general, the results indicate that 

noneofthese compounds are mutagenic in vitro. 

Neither salicylazosulfapyridine nor its two main 

metabolites, sulfapyridine and 5-aminosalicylic acid, 

were active inSalmonella typhimurium gene mutation 

assays, either with or without induced mouse or rat 

liver S9 enzymes (Voogd et al., 1980; Zeiger et al., 

1988). .Salicylazosulfapyridine, sulfapyridine, and 

5-aminosalicylic acid have also been tested for induction of chromosomal damage in culturedChinese 

hamster ovary cells in vitro. No increases in sister 

chromatid exchanges or chromosomal aberrations 

Salicylazosulfapyridine, NTP TR 457 

1990) or 5-aminosalicylic acid. (Witt et al., 1992a), 

'either with or without rat liver S9. Sulfapyridine, on 

the other hand, has been reported to induce small but 

statistically significant increases in sister chromatid 

exchanges in the absence of S9; with S9, no increase 

in sister chromatid exchanges was noted (Witt et al., 

1992a). Sulfapyridine did not induce chromosomal 

aberrations in cultured Chinese hamster ovary cells, 

either with or without rat liver S9. I . 

Mackay et al. (1989) tested salicylazosulfapyridine, 

sulfapyridine, 5-aminosalicylic acid, and four acetylated and/or hydroxylated metabolites of sulfapyridine 

and .5-aminosalicylic acid for their ability to induce 

sister chromatid exchanges and micronuclei in cultured human lymphocytes. In contrast to the results 

obtained in cultured Chinese hamster ovary cells, 

salicylazosulfapyridine induced both sister chromatid 

exchanges and micronuclei in human lymphocytes at 

the same dose concentrations which yielded negative 

results; in cultured Chinese hamster ovary cells in the 

absenceof S9. Furthermore, sulfapyridine and two 

acetylated sulfapyridine metabolites induced only 

sister chromatid exchanges, and 5-aminosalicylic acid 

(along with its acetylated metabolite) induced neither 

sister chromatid exchanges nor micronuclei. Thus, 

salicylazosulfapyridine appears to be clastogenic in 

cultured human cells but notin cultured Chinese 

hamster ovary cells. 

Salicylazosulfapyridine also appears to have clastogenic activity in human lymphocytesin vivo, although 

studies in humans have suffered from various design 

flaws and the results cannot be considered unequivocal. Mitelman et al. (1980, 1982) concluded that 

patients undergoing salicylazosulfapyridine therapy 

exhibited evidence of chromosomal damage after one 

or more months of treatment. However, interpretation of the results from these studiesis complicated by 

concomitant exposures to other drugs such as metronidazole, lack of pretreatment baseline data, and other 

confounding factors. More recently, Fox et al. 

(1 987) reported that salicylazosulfapyridine induced 

significant increases in the frequency of sister chromatid exchanges and micronuclei in lymphocytes of 

patientsreceiving chronic salicylazosulfapyridine 

therapy compared to healthy controls. Complicating 

factors were present in this study as well, including 

were observed in cultured Chinese hamster ovary cellsconsiderable interindividual variability in the fretreated with salicylazosulfapyridine (Bishop et al., quency of micronuclei. 

P 
Salicylazosulhpyridine, FdTP TR 457 21 

Several studies in mice haveprovided further evi- In summary, salicylazosulfapyridine has not been 

dence of salicylazosulfapyridine-inducedchromosomal shown to be mutagenic in vitro, but it does appear to 

damage in vivo. Specifically, salicylazosulfapyridine induce chromosomal damage, perhaps in the form of 

has been reported to induce increased frequencies of aneuploidy, in mammalian cells in vivo. The genetimicronucleated erythrocytes in peripheral blood cells cally active subunitofthe salicylazosulfapyridine 

ofmaleandfemalemicedosed for 13 weeks by molecule is sulfapyridine; 5-aminosalicylic acid, the 

gavage (Bishop et al., 1990). Furthermore, it induced therapeutically active subunit, does not induce genetic 

dose-related increases in micronuclei in bone marrow alterations in vitro or in vivo. 

cells of male mice dosed for 2 or 3 days, by gavage 

(Bishop et al., 1990; Witt et al., 1992b). The two 

primary metabolites of salicylazosulfapyridine were 

also tested for induction of micronuclei in mouse bone 

marrow erythrocytes; sulfapyridine, but not 5-aminosalicylic acid, induced a strong, dose-related increase %INJlIDY kiTlIONAlLE 

in micronucleated polychromatic erythrocytes when Salicylazosulfapyridine was nominated for study by 

administered either by intraperitoneal injection (Witt theNational Cancer Institute on the basis of its 

et al., 1992a) or by gavage mitt et al., 1992b). The extended use by a sizable human population and as a 

majority of micronucleated erythrocytes induced by representative chemical from a class of aryl sulfonasalicylazosulfapyridine and sulfapyridine were shown mides. Salicylazosulfapyridine was a suspected

to contain kinetochores (Witt et al., 1992b). The carcinogen because reductive cleavage of the azo 

presence of a kinetochore within a micronucleus is linkage yields sulfapyridine, a p-amino aryl sulfonpresumed to indicate that the micronucleus contains an amidepossessing a heterocylic aromatic nitrogen 

entire chromosome, resulting froma failure of mitotic substituent on the sulfonamide nitrogen. Sulfamethchromosomal segregation, rather than a chromosomal oxazole (a related p-amino aryl sulfonamide) has been 

fragment resulting from chromosome breakage. shown to produce thyroid gland neoplasms in rats 

These results imply that salicylazosulfapyridine and (Swarm et al., 1973). Accordingly, salicylazosulfasulfapyridine induce aneuploidyinmousebone pyridine was recommended to the NTP for carcinomarrow erythrocytes. genicity testing. 
Salicylazosulfapyridine,NTP TR 457 
rnO@rnMlENT 

ANID @l€lIAM@TEIWI[ZATlION 

OF ~A~~I@PI~AZOS~LFAPPIWI[DINE 

Salicylazosulfapyridine was obtained from Salisbury 

Labs, Inc., (Charles City, IA), in one lot (1089), 

whichwasused during the 16-dayand13-week 

studies. For the 2-year studies, one lot (61838) was 

obtained from Pharmacia, Inc. (Piscataway, NJ). 

Identity, purity, and stability analyses were conducted 

by the analytical chemistry laboratory and confirmed 

by the study laboratories. Reports on analyses performedin support of thesalicylazosulfapyridine 

studies are on file at the National Institute of Environmental Health Sciences. The methods and results of 

these studies are detailed in Appendix I. 

Both lots of the chemical, an orange powder, were 

identified as salicylazosulfapyridine by infrared, ultraviolet/visible, and nuclear magnetic resonance spectroscopy. The purity of lots 1089 and 61838 was 

determined by elemental analyses, Karl Fischer water 

analysis, functional group titration, thin-layer chromatography, and high-performance liquid chromatography. For both lots, elemental analyses for carbon, 

hydrogen, nitrogen, and sulfur agreed with theoretical 

values. Karl Fischer water indicated analysis 

0.50% k 0.04% water in lot 1089 and 0.5% k 0.1% 

water in lot 61838. Functional group titration for 

lot 1089 by two methodsindicatedpurities of 

99.3% f 0.6%and 100.6%f 0.8%. Functional 

group titration for lot61838 by two methods indicated 

purities of 100.6%k 0.3%and 101.0%k 0.4%. 

For lot 1089, thin-layer chromatography by two 

systems indicated a major spot and a trace impurity. 

For lot 61838, thin-layer chromatography indicated a 

major spot with a trace and two slight trace impurities 

by one system. A second systemindicated a major 

spot with two trace impurities and a slighttrace 

impurity. Using a detection wavelength of 254 nm, 

high-performance liquid chromatography revealed a 

major peak and three impurities with a combined area 

of 2.3% relative to the major peak area for lot 1089. 

When using a detection wavelength of 365 nm, two 

impurities with a combined area of 1.9%relative to 

the major peakwere observed for lot 1089. Highperformance liquid chromatography using a detection 

wavelength of 254 nm resolved a major peak and two 

impurity peaks with a combined area of 1.7% relative 

to the major peak area for lot 61838. When 365 nm 

was used as the detection wavelength, three impurity 

peaks were observed with a combined area of 1.2% 

relative to the major peak for lot 61838. The overall 

purity was determined to be 96%to 97% for lot 1089 

and approximately98% for lot 61 838. 

The analytical chemistry laboratory analyzed lot 1089 

to determine if it met United States PharmacopeiaXX 

(USP) purity requirements. The complete battery of 

USP analyses was performed as a supplement to the 

chemical characterization of salicylazosulfapyridine. 

The chemical metheUSP requirements for the 

identification tests, whichincluded infrared and 

visible absorption spectroscopy. The weight loss on 

drying was 0.30% f 0.01%,andthe residue on 

ignition was0.18% f 0.04%,both of which met the 

USP requirements. The chemical metthe USP requirements of the chloride, sulfate, and heavy metals 

tests. The USP purityassayindicated that lot 1089 

had a purity of 96.5% k 1.7% , calculated on the 

anhydrous basis, relative to the USP salicylazosulfapyridine reference standard, which was consistent 

with USP requirements. 

Each lot was concomitantly analyzed with the USP 

standard for salicylazosulfapyridine content using a 

high-performanceliquid chromatography system. 

RelativetotheUSP standard, lot 1089 was determined to contain 96.3% k 0.7% salicylazosulfapyridine, andlot 61838 was found to contain 

97.0%k 0.9%salicylazosulfapyridine. 

Stabilitystudies of the bulkchemical for lot 1089 

performedbytheanalytical chemistry laboratory 

indicated that salicylazosulfapyridine was stable as a 

bulk chemical for 2 weeks when stored protected from 

light at temperatures up to 60"C. To ensure stability, 

the bulk chemical was stored in sealed amber plastic 
24 Salicylazosulfapyridine, NTP TR 457 

bags at 5" C during the 16-day and 13-week studies of disease. Groups of five male and five female rats 

and in, metal cans protected. from light at room tem- and mice received salicylazosulfapyridine in corn oil 

perature during the 2-year studies. by gavage for 12 days at doses of 0, 675, 1,350, or 

2,700 mg/kg. Feed and water were available 

ad libitum. Rats were housed five per cage and mice 

were housed individually. Clinical findings were PREPARATION AND ANALYSIS 

recorded daily for rats and mice. The animals were OF DOSEFORMULATIONS weighed initially, at 1 week, and at necropsy. Details 

The dose formulations were Prepared by mixing of the study design and. animal maintenance are 

salicylazosulfapyridine With CornOil to give the summarizedin Table 1. . . required concentrations (Table 11). The dose formulations were prepared weekly during the 16-day At the'end of the 16-day studies, animals were anesstudies and every 2 weeks during the 13-weekand thetizedwith an ultra short-acting barbiturate 2-year studies and stored for up to 3 weeks at (Bio-Tal), and blood was collected from the external 

0" 4 5" C in clear (16-day studies) or amber-glass jugular vein of all animals for clinical chemistry (1 3-week and 2-year studies) bottles. analysis. Serum thyroid hormone levels were measured and quantitated using the NML Tri-Tab RIA Kit Periodic analyses of the dose formulations of salicyl- (Nuclear Medical Laboratories, Dallas, TX). Reazosulfapyridine were conducted at the study labora- agents were provided bythe National' Institute of 

tories and analytical chemistry laboratory using high- Arthritis, Metabolism and Digestive Diseases. The .performance liquid chromatography. The dose clinical pathology parameters measured are listed in 

formulations were analyzed at the beginning of the Table 1. The-brain, heart; right kidney, liver, lung, 16-day studies, and all formulations were within 10% right testis, and thymus were weighed. One-halfof 

ofthe target concentrations (Table 12). During the the pituitary gland and one-half of the thyroid gland 13-week studies, the dose formulations were analyzed were fixed in a cold fixative suitable for electron 

at the beginning, midpoint, and end of the studies, andmicroscopic evaluation. Tissues for microscopic all formulations were within 10% of the target con- examination were fixed and preserved in 10 % neutral 

centrations (Table 13). During the 2-year studies, the buffered formalin, processed and trimmed, embedded 

dose formulations were analyzed every 6 to 12 weeks. in paraffin, sectioned to a thickness of 4 to 6 pm, and 

Of the dose formulations analyzed for the 2-year stained with hematoxylin and eosin. A necropsy and 

studies, 98%(103/105) were within 10% of the target complete histopathologic examination was performed 

concentrations (Table 14): The dose formulations on all rats and mice. Table 1 lists the tissues and 

differing by more than 10% from the target concentra- organs routinely examined. 

tions were remixed, and thenew dose formulations 

were found to be within 10%of the target concentrations. Results of the periodic referee analyses per- 13-WEEK STUDIES 

formed by the analytical chemistry laboratory were in The 13-week studies were performed to evaluate the 

agreement with the results obtained bythestudy cumulative toxic effects of repeated exposure to sali- laboratory (Table 15). cylazosulfapyridine and to determine the appropriate 

doses to be used in the 2-year studies. . . 

DAY STUDIES Male and female F344/N rats and B6C3Fl mice were 

Male and female F344/N rats and B6C3Fl mice were obtained from Simonsen Laboratories, Inc. (Gilroy, 

obtained from Frederick Cancer Research Facility CA). On receipt, the rats and mice were 4 weeks old. 

(Frederick, MD). On receipt, the rats and mice were Rats were quarantined for 11 days (males) or 13 days 

4 weeks old. Animals were quarantined for 10 to (females); mice were quarantined for 18 days (males) 

14 days and were 6 weeks old on the first day of the or ' 20 days (females). Rats were approximately 

studies. Before initiation of the studies, b o male and 6 weeks old and mice were approximately7 weeks old 

' twofemale rats and mice were randomly selected for at the start of the studies. Before initiation of the 

parasite evaluation and gross observation for evidence studies, five male and five female rats and mice were 
S~nliCyB~QSnlh~y,yk~~~~@, NTP TR 457 25 

randomly selected for parasite evaluation and gross 

observation for evidence of disease. 

Groups of 10 male and 10 female rats received 

salicylazosulfapyridine in corn. oil bygavagefor 

13 weeks at doses of 0, 84,. 168.8, or 337.5 mg/kg. 

Groups of 10 male and 10 female micereceived 

salicylazosulfapyridine in corn oilbygavage for 

13 weeks at doses of 0, 675, 1,350, or 2,700 mg/kg. 

Additional groups of 10 male a.nd 10 femalemice 

received salicylazosulfapyridine incorn oil by gavage 

for 13 . weeks at doses of 0, 675, 1,350, or 

2,700 mg/kg and were designated for clinical pathology evaluations. Feedandwaterwereavailable 

ad libitum. Rats were housed five per cage and mice 

were housed individually. Clinical findings and body 

weights were recorded weekly for each animal. 

Details of the study design and animal maintenance 

are summarized in Table 1. 

After 12 weeks of treatment, animals were placed in 

individual metabolism cages for a 16-hour urine collection period. Hematology analyses were conducted 

during week 12 for rats and mice. At the end of the 

13-week studies, animals were anesthetized with an 

ultra short-acting barbiturate (Bio-Tal), and blood was 

collected from the external jugular vein of rats and 

core studymiceand from special studymice for 

thyroidhormone analyses. Serum thyroid hormone 

levels were measured and quantitated using the NML 

Tri-Tab RIA Kit.Reagentswereprovided by the 

National Institute of Arthritis, Metabolism and Digestive Diseases. Clinical pathology parameters which 

were evaluated are listed in Table 1. 

At the end of the13-week studies, samples were 

collected for sperm morphology and vaginal cytology 

evaluations from all rats and mice. The parameters 

evaluated are listed in Table 1. Methods used were 

those described in the NTP’s Sperm Morphology and 

Vaginal Cytology Evaluations Protocol (NTP, 1983a). 

For 7 consecutive days before the scheduled terminal 

sacrifice, the vaginalvaults of the femaleswere 

moistened with saline, if necessary, and samples of 

vaginal fluid and cells were stained. Relative numropsy. Male rats and mice were evaluated for sperm 

morphology, count, and motility. The right epididymis and right testis were isolated and weighed. The 

tail of the epididymis(cauda epididymis) was then 

removed from the epididymal body (corpus epididymis)andweighed. Test yolk (rats) or modified 

Tyrode’s buffer (mice)wasapplied to slides and a 

small incision was made at the distal border of the 

cauda epididymis. The sperm effluxing from the 

incision were dispersed in the bufferon the slides, and 

the numbers of motile andnonmotile spermatozoa 

were counted for five fields per slide by two observers. Following completion of sperm motility estimates, each right cauda epididymis wasplacedin 

buffered saline solution. Caudae were finely minced, 

and the tissue was incubated in the saline solution and 

then heat fixed at 65’ C. Sperm density was then 

determined microscopically with the aid of a hemacytometer. Four sperm morphology slides were prepared for each animal evaluated. An aliquot of killed 

sperm suspension was stained ina test tube, spread on 

a microscope slide under a coverslip, and examined. 

A necropsy was performed on all core study animals. 

The brain, heart, right kidney, liver, lung, right testis, 

and thymus were weighed. One-half of the pituitary 

gland and one-half of the thyroid gland were fixed in 

glutaraldehyde for evaluation by electron microscopy. 

Theremaininghalvesofthe pituitary and thyroid 

glands and all other tissues for microscopic examination were fned and preserved in 10% neutral buffered 

formalin, processed and trimmed, embeddedin 

paraffin, sectioned to a thickness of 4 to 6 pm, and 

stainedwithhematoxylinand eosin. A complete 

histopathologic examination was performed on all 

core study rats and mice. Table 1lists the tissues and 

organs routinely examined. 

%YEAR STUJlDES 

Study Desigrm 

Groups of 60 male and 60 female rats were administered 84, 168, or 337.5 mg salicylazosulfapyridine/kg 

bodyweightin corn oil by gavage for 103 weeks 

(males) or 105 weeks (females). Groups of 70 male 

bers of leukocytes, nucleated epithelial cells, and largeand 60 female rats were administered the corn oil 

squamous epithelial cells were determined and used tovehicle by gavage for up to 105 weeks. Groups of 

ascertain estrous cycle stage (i.e., diestrus, proestrus, 60 male and 60 female micewere administered 0, 

estrus, and metestrus). All core study male rats and 675, 1,350, or 2,700 mg salicylazosulfapyridine/kg 

mice were processed for sperm morphology at nec- bodyweightin corn oil by gavage for 103 weeks 
26 Salicylazosulfapyridine,NTP TR 457 

(males) or 104 weeks(females).Tenmaleandten 

female rats and mice from each group were evaluated 

at . . 15 months. 

Stop-Exposure Evaluation 

A group of 70 male rats was administered 337.5 mg 

salicylazosulfapyridine/kg body weight in corn oil by 

gavage for 26 weeks; at 26 weeks,thesalicylazosulfapyridinewasreplacedwith corn oil for the 

remainder of the study. Ten vehicle control and ten 

rats in the 337.5 mg/kg group were evaluated at6 and 

15 months. The stop-exposure evaluation with recovery period was designed to allow for assessment of the 

potential for salicylazosulfapyridine-inducedlesions to 

progress or regress over the 2-year study. 

Source and Specification of Animals 

Male and female F344/N rats and B6C3F1 mice were 

obtained from Simonsen Laboratories, Inc. (Gilroy, 

CA), for use inthe2-yearstudies.Onreceipt,the 

animalswereapproximately 4 weeks old.Theanimals were quarantined for 1 1 or 13 days and were 

approximately 6 weeks oldonthefirstday ofthe 

studies. Before the initiation of the studies, five male 

A complete ' necropsy and microscopic examination

were peiformed on all rats Ad mice. At the 6-month 

stop-exposureinterimevaluation,theright'kidney, 

liver, spleen, righttestis,andthyroidglandwere 

weighed.Atthe15-monthinterimevaluation,the 

rightkidney, liver, spleen, left testis (rats); ' right 

testis, and thyroid gland were weighed. At necropsy, 

allorgansandtissueswereexamined for grossly 

visible lesions, and all major tissues were fixed and 

preservedin 10% neutral bufferedformalin,processed and trimmed, embedded in paraffin, sectioned 

to a thickness of 4 to 6 pm, and stained with hematoxylin and eosin for microscopic examination. For 

all paired organs (i.e., adrenal gland, kidney, ovary), 

samplesfromeachorganwereexamined.Tissues 

examined microscopically are listed in Table 1. 

At the 6-monthinterimevaluation,bloodwascollected for thyroid hormone assays from the retroorbital sinus of 10 male control rats and 10 male rats 

fromthe337.5mg/kgstop-exposure group. Upto 

10maleratsandmicefromthecontroland 

337.5 mg/kg (rats) and 2,700 mg/kg (mice) groups 

wereevaluated for thyroid hormonealterations at 

and five female rats and mice were randomly selected15 months. Upto10maleand.10femaleratsand 

for parasite evaluationandgrossobservation for 

evidence of disease. At18months,fivemaleand 

three female rats were selected for a similar health 

check. Up to five male and five female rats and mice 

wereused for serology screeningatapproximately 

6-monthintervals.Thehealth of the animalswas 

monitored during the studies according to the protocols of theSentinel Program NTP Animal

(Appendix K). 

Animal Maintenance 

Ratswerehousedfive per cage, and micewere 

housed individually. Feed and water were available 

ad libitum. Ratcageswerechangedtwiceweekly, 

and mouse cages were changed once weekly. Racks 

were rotated every 2 weeks. Further details of animal 

maintenance are given inTable, 1. Informationon 

feedcomposition and, contaminants isprovidedin 

Appendix J. 

Clinical Examinations and Pathology 

Animals were observed twice daily. Clinical findings 

and body weights were recorded at the beginning of 

mice from each group and 10 male rats from the stopexposure group were evaluated for thyroid hormone 

alterations at the end of the study. The groups evaluated are listed in Table 1. 

Microscopic evaluations were completed by the study 

laboratory pathologist, and the pathology data were 

entered into the Toxicology Data Management Systern.Themicroscopicslides,paraffinblocks,and 

residual wet tissues were sent to the NTP Archivesfor 

inventory,slide/blockmatch,andwettissueaudit. 

The slides, individual animal data records, and pathology tables were evaluated by an independent quality 

assessment laboratory. The individual animal records 

and tables were compared for accuracy, the slide and 

tissuecountswereverified,andthehistotechnique 

wasevaluated. For the 2-yearstudies, a quality 

assessment pathologist reviewed selected neoplasms 

and nonneoplastic lesions. 

The quality assessment report and the reviewed slides 

were submitted to the NTP Pathology Working Group 

(PWG) chairperson, who reviewed the selected tissues 

the 2-year studies, once weekly for the first 14 weeks, andaddressedanyinconsistenciesinthediagnoses 

monthly thereafter, and at terminal sacrifice. madebythelaboratoryandqualityassessment 
pathologists. Representative histopathology slides 

containing examples of lesions relatedto chemical administration, examples of disagreements in diagnoses 

between 'the laboratory and quality assessment pathologists, or lesions of general interest were presented by 

the chairperson to the PWG for review. The PWG 

consisted of the quality assessment pathologist and 

other pathologists experienced in rodent toxicologic 

pathoiogy. This group examined the tissues without 

any knowledge of dose groups or previously rendered 

diagnoses. When the PWG consensus differed from 

the opinion of the laboratory pathologist, the diagnosis 

was changed. Thus, the final diagnoses represent a 

consensus of qualityassessment pathologists, the 

PWG chairperson, and thePWG. Details of these 

review procedures have been described, in part, by 

Maronpot and Boorman (1982) and Boorman et al. 

(1985). For subsequent analyses of thepathology 

data, the diagnosed lesions for each tissue type were 

evaluated separately or combined according to the 

guidelines of McConnellet al. (1986). 
28 

i 

Salicylazosulfapyridine, NTP TR.457 

TABLE1 

ExperimentalDesignand'Materials and MethodsintheGavageStudiesofSalicylazosulfapyridine , . : 

16-Day Studies 13-Week Studies , I 2-Year Studies 

Study Laboratory 

EG&G Mason Research Institute EG&G Mason Research Institute Southern Research Institute 

(Worcester, MA) (Worcester,MA) (Birmingham, AL) 

Strain and Species . , .. 

Rats:F344lN Rats: F344lN Rats: F344lN 

Mice:B6C3F, . . Mice: B6C3FI Mice: B6C3Fl 

Animal Source 

Frederick Cancer Research Facility Simonsen Laboratories, Inc. Simonsen Laboratories, Inc. 

(Frederick, MD) (Gilroy, CA) (Gilroy, CA) 

Time Held Before Studies 

10-14 days Rats:11days(males) or 13days Stop-exposureevaluation:11days 

(females) Core study: 

Mice: 18 days (males) or 20days Rats:11days 

(females) Mice:11 or 13 days 

Average Age When Studies Began 

6 weeks Rats: 6 weeks Stop-exposure evaluation: 6 weeks 

Mice: 7 weeks Corestudy: 7 weeks 

Date of First Dose 

Rats:23September1985 Rats:31March(males) or 2 April Stop-exposure evaluation: 

Mice: 25September1985 (females)1986 12 December 1988 

Mice: 7 April (males) or 9 April Core study: 

(females)1986 Rats:12December1988 

Mice: 3 January 1989 

Duration of Dosing 

16 days, 5 dayslweek 13 weeks, 5 dayslweek Stop-exposureevaluation:26weeks, 

5 dayslweek; then corn oil gavage 

5 dayslweek for the remainderof the 

study 

Core study: 

Rats:103weeks (males),or 

105 weeks (females), 

5 dayslweek 

Mice: 103 weeks (males), or 

104 weeks (females), 

5 dayslweek 

Date of Last Dose 

Rats: 7 October or (males) 8 October Rats: 29-30(males)1-2Rats:November June orJuly30 (males) or

(females) 1985 (females)1986 12 December (females) 1990 

Mice: 9 October (males) or Mice: 7-8 (males)9-10DecemberMice:(males) JulyJuly 21or or 

10 October'(fema1e.s) 1985 (females)1986 28 December (females) 1990 
29 

TABLE n 

EwperhenntaU Design and MateriaUs rannd Methods in the Gavage Studies of SzaUicyUazosPnBffa~y~~dine (continued) 

1Q.Day Studies 1IWeek Studies 

Necropsy Dates 

Rats: 8 October (males) or 9 October Rats: 30 June-1 July (males) or

(females) 1985 July 2-3 (females) 1986 

Mice: 10 October (males) or Mice: 8-9 July (males) or 10-1 1 July 

11 October (females) 1985 (females) 1986 

Average Age at Necropsy 

9 weeks Rats: 19 weeks 

Mice: 20 weeks 

Size of Study Groups 

5 males and 5 females 10 males and 10 females 

2-Year Studies 

Stop-exposure evaluation: 

26-week interim evaluation: 

12 June 1989 

Core study: 

Rats: 

15-Month interim evaluation: 

12-13 March (males) or 

14-15 March (females) 1990 

Terminal sacrifice: 10-14 December 

(males) or 19-21 December 

(females) 1990 

Mice: 

15-Month interim evaluation: 3 April 

(males) or 4 April (females) 

1990 

Terminal sacrifice: 31 December 

1990-4 January 1991 (males) or 

7-10 January (females) 1991 

Stop-exposure evaluation: 

26-week interim evaluation: 

32 weeks . 

Core study: 

Rats: 

15-Month interim evaluation: 

72 weeks 

Terminal sacrifice: 110-1 11 weeks 

(males) or 11 1-112 weeks 

(females) 

Mice: 

15-Month interim evaluation: 

72 weeks 

Terminal sacrifice: 110-1 11 weeks 

(males) 11 1-112 weeks 

(females) 

Rats: 

Stop-exposure evaluation: 70 males 

(337.5 mglkg) 

Core study: 70 males and 60 females 

(0 mglkg); 60 males and 

60 females (84, 168, and 

337.5 mg/kg) 

Mice: 60 males and 60 females 
30 Salicylazosulfapyridine,NTP TR 457 

TAISLE 1 

Experimental Design and Materials and Methods'in the Gavage Studies of Salicylazosulfapyridine (continued) 

ldDay Studies 

Method of Distribution 

Animals were distributed randomly into 

groups of approximately equal initial 

mean body weights. 

Animals per Cage 

Rats: 5 

Mice.: 1 

Method of Animal Identification 

Toe clip 

Diet 

NIH-07 open formula meal diet (Zeigler 

Brothers Inc., Gardners, PA), available 

ad libitum, changed weekly 

Water Distribution 

Tap water (City of Worcester municipal 

supply) via automatic watering system 

(Edstrom Industries, Waterford, WI), 

available ad libitum 

Cages 

Polycarbonate (Lab Products Inc., 

Rochelle Park, NJ), changed twice 

weekly for rats and weekly for mice 

Bedding 

BetaChips (Northeastern Products 

Corp., Warrensburg, NY), changed 

weekly 

Cage/Rack Filters 

Nonwoven fiber cage filters (Snow 

Filtration, Cincinnati, OH), changed 

every 2 weeks . 

Rack 

Stainless steel (Lab Products Inc., 

Rochelle Park, NJ), rotated every 

2 weeks 

13-Week Studies 

Same as 16day studies 

Rats: 5 

Mice: 1 

Toe clip 

Same as 16day studies 

Same as 16day studies 

Polycarbonate (Lab Products Inc., 

Garfield, NJj, changed twice weekly 

for rats and weekly for mice 

BetaChips (Northeastern Products 

Corp., Warrensburg, NY),changed 

twice weekly for rats and weekly for 

mice 

Same as 16day studies 

Stainless steel (Lab Products Inc., 

Garfield, NJ), rotated every2 weeks 

%Year Studies 

'Same as 16day studies 

Rats: 5 

Mice: 1 

Rats: Tail tattoo 

Mice: Toe clip 

Same as 16day studies 

Tap water (City of Birmingham 

municipal supply) via automatic 

. watering system (Edstrom Industries, 

Waterford, WI), availablead libitum 

Polycarbonate (Lab Products Inc., 

Maywood, NJ), changed twice weekly 

for rats and weekly for mice 

Sani-Chips (P.J. Murphy Forest 

Products Corp., Montville, NJ), 

changed twice weekly for rats and 

weekly for mice 

Reemay" spun-bonded polyester rack 

filters (Andico, Birmingham, AL), 

changed every 2 weeks 

Stainless steel (Lab ProductsInc., 

Maywood, NJ), rotated every 2 weeks 

32 Salicylazosulfapyridine, NTP TR 457 

TAIBLE1 

Experimental Design and Materiaband Methods in the Gavage Studiesof Salicylazosulfapyridine(continued) 

16-Day Studies 13-Week Studies 2-year studies 

Clinical Pathology 

At necropsy, blood was collected from 

the jugular vein. 

Clinical Chemistry: Thyroidstimulating hormone, triiodothyronine, 

and thyroxine concentrations. 

Histopathology 

Complete histopathologic examinations 

were performed on all rats and mice. 

In addition to gross lesions, tissue 

masses, and associated lymph nodes, 

the tissues examined included: adrenal 

gland, brain, clitoral gland (rats), 

esophagus, femur with marrow, 

gallbladder (mice), heart, large intestine 

(cecum, colon, rectum), small intestine 

(duodenum, jejunum, ileum), kidney, 

liver, lungs and mainstem bronchi, 

lymph nodes, (mandibular and 

mesenteric), mammary gland, nose, 

ovary, pancreas, parathyroid gland, 

pituitary gland, preputial gland (rats), 

prostate gland, salivary gland, skin, 

spleen, stomach (forestomach and 

glandular stomach), testes (with 

epididymis and seminal vesicle), 

thymus, thyroid gland, trachea, urinary 

bladder, and uterus. 

After 12 weeksof study, 16-hour urine 

samples were collected and blood,was 

collected from tail the for analysis of 

hematology parameters. At necropsy, 

blood was collected from the jugular 

vein for clinical chemistry. 

Hemdology: Hematocrit, hemoglobin 

concentration, erythrocyte and 

reticulocyte counts, mean cell volume, 

mean cell hemoglobin, mean cell 

hemoglobin concentration, leukocyte 

count and differential, and 

methemoglobin concentration 

Clinical Chemistry: Urea nitrogen, 

creatinine, glucose, total protein and 

albumin concentrations, alanine 

aminotransferase, alkaline phosphatase, 

and aspartate aminotransferase 

concentrations, thyroid-stimulating 

hormone, triiodothyronine, and 

thyroxine concentrations. 

Urinalysis: Volume, specific gravity 

Complete histopathologic examinations 

were performed on 0 and 337.5 mglkg 

rats and 2,700 mg/kg mice. In addition 

to gross lesions, tissue masses, and 

associated lymph nodes, the tissues 

examinedincluded: adrenal gland, 

brain, clitoral gland (rats), esophagus, 

femur with marrow, gallbladder (mice), 

heart, large intestine (cecum, colon, 

rectum), small intestine (duodenum, 

jejunum, ileum), kidney, liver, lungs 

and mainstem bronchi, lymph nodes, 

(mandibular and mesenteric), mammary 

gland, nose, ovary, pancreas, 

parathyroid gland, pituitary gland, 

preputial gland (rats), prostate gland, 

salivary gland, skin, spleen, stomach 

(forestomach and glandular stomach), 

testes (with epididymis and seminal 

vesicle), thymus, thyroid gland, 

trachea, urinary bladder, and uterus. 

Additionally, the pituitary gland and 

thyroid gland from all rats at all dose 

levels were evaluated, and kidney tissue 

was evaluated for all rats administered 

168.8 mg/kg. Gross lesions in mice 

administered 675 or 1,350 mg/kg were 

evaluated microscopically. 

Stop-exposure evaluation: At 26 weeks 

and the end of the study, blood was 

collected from the retroorbital sinus 

for. serum thyroid hormone assays. 

Clinical Chemistry: Thyroidstimulating hormone, 

triiodothyronine, reverse 

triiodothyronine, and thyroxine 

concentrations. 

Core study: At 15 months (male rats 

and mice) and the end of the study, 

blood was collected from the 

retroorbital sinus for serum thyroid 

hormone assays. 

Clinical Chemistry: Thyroidstimulating hormone, 

triiodothyronine, reverse 

triiodothyronine, and thyroxine 

concentrations. 

Complete histopathologic examinations 

were performed on all rats and mice. 

In addition to gross lesions, tissue 

masses and associated lymph nodes, the 

tissues examined included: adrenal 

gland, brain, clitoral gland, esophagus, 

femur with marrow, gallbladder (mice), 

heart, large intestine (cecum, colon, 

rectum), small intestine (duodenum, 

jejunum, ileum), kidney, liver, lungs 

and mainstem bronchi, lymph nodes, 

(mandibular and mesenteric), mammary 

gland, nose, ovary, pancreas, 

parathyroid gland, pituitaiy gland, 

preputial gland, prostate gland, salivary 

gland, skin, spleen, stomach 

(forestomach and glandular stomach), 

testes (with epididymis and seminal 

vesicle), thymus, thyroid gland, 

trachea, urinary bladder, and UtCNS. 
ec 
34 

STATISTICALMETHODS 

Survival Analyses 

The probability of survival was estimatedbythe 

product-limit procedure of Kaplan and Meier (1958) 

and is presented in the form of graphs. Animals 

found dead of other than natural causes were censored 

from the survival analyses; animals dying from 

nahlrd causes were not censored. Statistical analyses 

for possible dose-related effects on survival used 

Cox's (1972) method for 'testing two groups for 

equality and Tarone's (1975) life table test to identify 

dose-related trends. All reported P values for the 

survival analyses are two sided. 

Calculation of Incidence 

The incidences of neoplasms or nonneoplastic lesions 

as presented in Tables Al, A5, B1, B5, C1, C5, Dl, 

and D5 are given as the number of animals bearing 

such lesions at a specific anatomic site and the number 

of animals with that site 'examined microscopically. 

For calculation of statistical significance, the incidences of most neoplasms (Tables A3, B3, C3, and 

D3) and all nonneoplastic lesions are given as the 

numbers of animals affected at each site examined 

microscopically. However, when macroscopic 

examination was required to detect neoplasms in 

certain tissues (e.g., harderian gland, intestine, 

mammary gland, and skin) before microscopic 

evaluation, or when neoplasms had multiple potential 

sites of occurrence (e.g., leukemia or lymphoma)., the 

denominators consist of the number of animals on 

which a necropsy was performed. Tables A3, B3, 

C3, and D3 also give the survival-adjusted neoplasm 

rate for each group and each site-specific neoplasm, 

i.e. ~ the Kaplan-Meier estimate of theneoplasm

incidence that would have been observedat the end of 

the study in the absence of mortality from all other 

competing risks (Kaplan and Meier, 1958). 

Analysis of Neoplasm Incidences 

The majority of neoplasms in these studies were 

considered to be incidental to the causeof death or not 

rapidly lethal. Thus, the primary statistical method 

used was logistic regression analysis, which assumed 

that the diagnosed neoplasms were discovered as the 

result of death from an unrelated cause and thus did 

not affect the risk of death. In this approach, 

neoplasm prevalence wasmodeled as a logistic 

function of chemical exposure and time. Both linear 

Salicylazosulfapyridine, NTP TR 457 

and quadratic terms intime were incorporated 

initially, and the quadratic term was eliminated if the 

fit of the model was not significantly enhanced. The 

neoplasm incidences of exposed and control groups 

were 'compared .on .the basis of the likelihood score 

test for the regression coefficient of dose. This 

method of adjusting for intercurrent mortality is the 

prevalence analysis of Dime and Lagakos (1983), 

further described and illustrated by Dime and 

Haseman (1986). When neoplasms are incidental, this 

comparison of the time-speciiic neoplasmprevalences 

also provides. a comparison of the time-specific 

neoplasm incidences (McKnight and Crowley, 1984). 

In addition to logistic regression, other methods of 

statistical analysis were used, and the results of-these 

tests are summarized in the appendixes. These methods include the life table test (Cox, 1972; Tarone, 

1975), appropriate. for rapidly lethal neoplasms, and 

the Fisher exact test and the Cochran-Armitage trend 

test (Armitage, 1971; Gart et al., 1979), procedures 

based on the overall proportion of neoplasm-bearing 

animals. 

Tests of significance included pairwise comparisons of 

each exposed group with controls and a test for an 

overall dose-related trend. Continuity-corrected tests 

were used in the analysis of neoplasm incidence, and 

reported P values are one sided. The procedures 

described in the preceding paragraphs were also used 

to evaluate selected nonneoplastic lesions. For further 

discussion of these statistical methods, refer to 

Haseman (1984). 

Analysis of Nonneoplastic Lesion Incidences 

Because all nonneoplastic lesions in this study were 

considered to be incidental to the cause of death or not 

rapidly lethal, the primary statistical analysis used was 

a logistic regression analysis. in which nonneoplastic 

lesion prevalence was modeled as a logistic function 

of chemical exposure and time. For lesions detected 

at the interim evaluation, the Fisher exact test was 

used, a procedure based on the overall proportion of 

affected animals. 

Analysis of Continuous Variables 

Two approaches were employed to assess the significance of pairwise comparisons between exposed and 

control groups in the analysis of continuous variables. 

Organ and body weight data, which . have
S~~nPiCyl~QSmnff~]~~~~~~, Nm 11‘B457 35 

approximately normal distributions, were analyzed 

using the parametric multiple comparison procedures 

of Dunnett (1955) and Williams (1971, 1972). 

Hematology, clinical chemistry, andepididymal 

spermatozoa data, whichavetypicallyskewed 

distributions, were analyzed using the nonparametric 

multiple comparison methods of Shirley (1977) and 

D~nn(1964). Jonckheere’s test (Jonckheere, 1954) 

was used to assess the significance of the dose-related 

trends and to determine whether a trend-sensitive test 

(Williams’ or Shirley’s test) was more appropriate for 

pairwise comparisons than a test that does not assume 

a monotonic dose-related trend (Dunnett’s or D~nn’s 

test). Prior to analysis, extreme values identified by 

the outlier test of Dixon and Massey(1951)were 

examined by NTP personnel, and implausible values 

were eliminated from the analysis. Average severity 

values were analyzed for significance using the MannWhitney U test (Hollander and Wolfe, 1973). 

Because the vaginal cytology dataare proportions (the 

proportion of the observation period that an animal 

was in a given estrous stage), an arcsine 

transformation was used to bring the data into closer 

conformance with a normality assumption. Treatment 

effects were investigated by applying a multivariate 

analysis of variance (Morrison, 1976) to the 

transformed data to test for simultaneous equality of 

measurements across dose concentrations. 

msuorican caprmdrapn ID~U~ 

Although the concurrent control group is always the 

first and most appropriate control group used for 

evaluation, historical control datacan be helpful in the 

overall assessment of neoplasm incidence in certain 

instances. Consequently, neoplasm incidencesfrom 

the NTP historical control database, which is updated 

yearly, are included in the NTP reports for neoplasms 

appearing to show compound-related effects. 

QuJALnTY ASSWCE rnTWODS 

The 13-week and 2-year studies were conducted in 

compliance with Food and Drug AdministrationGood 

Laboratory Practice Regulations (21 CFR, Part 58). 

In addition, as records from the 2-year studies were 

submitted to the NTP Archives, these studieswere 

audited retrospectively by an independentquality 

assurance contractor. Separate audits covering completeness and accuracy ofthepathology data, 

pathology specimens, final pathology tables, and a 

draft of this NTP Technical Report were conducted. 

Audit procedures and findings are presented in the 

reports and are on file at NIEHS. The audit findings 

were reviewed andassessedby NTP staff, so all 

commentshad been resolved or were otherwise 

addressed during the preparation of this Technical 

Report. 

GErnTIIC ToxneoLoGY 

The genetic toxicityof salicylazosulfapyridine was 

assessedbytesting the ability of the chemical to 

inducemutationsin various strains of Salmonella 

typhimurium, sister chromatid exchanges and chromosomal aberrations in cultured Chinese hamster ovary 

cells, changes in the frequency of micronucleated 

erythrocytes in mouse peripheral blood and bone 

marrow (with or without kinetochores) and in rat bone 

marrow, and chromosomal aberrations in mouse bone 

marrow cells following in vivo treatment. The 

protocols for these studies and the results are given in 

Appendix E. 

The genetic toxicity studies of salicylazosulfapyridine 

are part of a larger effort by the NTP to develop a 

database that would permit the evaluation of carcinogenicity in experimental animals from the structure 

and responses of the chemical in short-term in vitro 

and in vivo genetictoxicity tests. These genetic 

toxicitytestswereoriginally developed to study 

mechanisms of chemically induced DNA damage and 

to predict carcinogenicity in animals, based on the 

electrophilic theory ofchemical carcinogenesis and 

the somatic mutation theory (Miller and Miller, 1977; 

Straus, 1981; Crawford, 1985). 

There is a strong correlation between a chemical’s 

potential electrophilicity (structural alert to DNA 

reactivity), mutagenicity in Salmonella, and carcinogenicity in rodents. The combination of electrophilicity and Salmonella mutagenicity is highly ‘correlated 

with the induction of carcinogenicity in rats and mice 

and/or at multiple tissue sites (Ashby and Tennant, 

1991). Other in vitro genetic toxicity tests do not 

correlate well with rodent carcinogenicity (Tennant 

et al., 1987; Zeigeret al., 1990), although these other 

tests can provide information on the types of DNA 

and chromosome effects that can be induced by the 

chemical being investigated. Data from NTP studies 

showthat a positive response in Salmonella is 
36 

currently the most predictive in vitro test for rodent 

carcinogenicity (89% ofthe Salmonella mutagens 

were rodent carcinogens), and that there is no complementarity among thein vitro genetic toxicity tests. 

That is, no battery oftests that includedthe r 

Salicylazosulfapyridine, NTP TR 457 

Salmonella test improved the predictivity of the 

Salmonella test alone. The predictivity for carcinogenicityof a positive response in bone marrow 

chromosome aberration or micronucleus tests is not 

yet defined. 
mTs 

n6-nDAY STUDY 

All rats survived to the end of the study (Table 2). 

With the exception of 675 mgkg male rats, 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. Clinicalfindings 

included ruffled fur and distended abdomens in male 

and female rats receiving2,700mgkg salicylazosulfapyridine. Female rats receiving 2,700 mg/kg salicylazosulfapyridine appeared to be thinner than controls. 

Hypothyroidism, evidencedby decreased serum 

triiodothyronine and thyroxine concentrations and 

increased thyroid-stimulating hormone concentrations, 

occurred in 2,700 mgkg maleandfemale rats 

(Table Gl). These thyroid hormone changes were 

consistent with theknown goitrogenic effects of 

sulfonamides. 

The absolute and relative thymus weightsof male rats 

receiving 1,350 or 2,700 mgkg salicylazosulfapyridine were significantly lower than those of controls (Table Fl). The absolute andrelativethymus 

weights of 2,700 mg/kg females werelowerthan 

those of controls. Absolute liver and heart weights of 

male rats and absolutelungweightsofmaleand 

female rats were significantly decreased, and the 

decreases were considered to be secondary to lower 

body weights in dosed rats. At necropsy, all dosed 

rats had enlarged cecaellarge intestines. Male rats 

receiving 1,350 mgkg and maleandfemale rats 

receiving 2,700 mg/kg had red and slightly 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 four 

males and one female in the 1,350 mgkg groups and 

in three males and two females in the 2,700 mgkg 

groups. Lymphoid depletion was observed in the 

thymus of three male and three female rats in the 

2,700mgkg groups. All male and female rats in the 

1,350 and 2,700 mgkg groups had minimal to mild 

thyroid gland follicular cell hyperplasia. Follicles 

varied in sizeand were lined by columnar cells. 

Colloid (PAS positive) was present in some but not in 

all follicles. Ultrastructural examination of the 

thyroidgland from male andfemale rats receiving 

2,700mgkg revealed distention of rough endoplasmic 

reticulum with colloid, decreased numbers of cytoplasmic dense bodies and cytoplasmic colloid droplets, fusion of apical microvilli, increased numbers of 

dense secretory granules, increased size and numbers 

of mitochondria, and increased numbers of free ribosomes when compared to controls. Morphometric 

analysis of the pituitary gland revealed a significant 

increase in the number (P <0.03) but not the size of 

thyroid-stimulatinghormone producing cells in the 

pars distalis of the pituitary gland of the 1,350 and 

2,700 mgkg groups. The gross, microscopic, and 

ultrastructural changes in thyroid glands of dosed rats 

are suggestive of increased functional activity and are 

most likely due to the reported goitrogenic effects of 

salicylazosulfapyridine. 

Dose Selection Rationale: Because of the lower mean 

body weights of male and female rats in each dose 

group, 337.5 mgkg was selected as the high dose for 

the 13-week study. 
38 Salicylazosulfapyridine, NTP TR 457 

TABLE2 

Survival and Body Weights of Rats in the 16-Day Gavage Studyof Salicylazosulfapyridine 

Mean Body Weightb (g) Final Weight 

Initial Survivala Dose Final Change Relativeto Controls 

, Male 1 

Ve:hicle Control 515 135 f 3 215 f 5 80 & 4 

675 515 138 f 3 204 f 4 662* 95 

1,350 

2,700 

515 

515 

133 f 4 

137 f 3 147 f 4** 

160 f 4** 26 f 2** 

9 f 4** 68 

74 

. Female 

Vehicle Control 

, 675 

1,350 

2,700 

515 

515 

515 

515 

,108 f 3 

111 f 3 

lo9 f 3 

110 f 4 111 f 3** 

129 f 4** 

133 f 2** 

147 i 3 

20 f 2** 

21 f 2** 

40*1 

1 f 2** 75 

87 

90 

a Number of animals surviving at 16 dayslnumber initially in group 

** PsO.01 

* Significantlydifferent(PsO.05)from thecontrolgroup by Williams’ or Dunnett’stest 

Weights and weight changes, are given as mean f standard error. 

. . 
SdlqP~QS,slalBsa~~~~~~~, 39 Nm 1 457 

nwvEEK STUDY 

All rats survived to the end of the study (Table 3). 

The final mean body weights of dosed male rats were 

similar to that of controls; the finalmeanbody 

weights and body weight gains of dosed females were 

significantly lower than those of controls. No 

chemical-related clinical observations were noted in 

dosed rats during the 13-week study. No significant 

differences in hematology or urinalysis parameters 

between control and dosed rats wereobserved 

(Table G2). The absolute and relativerightkidney 

weightsof 337.5 mg/kg females weresignificantly 

greater than those of controls (Table F2). 

At necropsy, some 337.5 mgkg male rats had red, 

enlarged thyroid glands. Histopathologicchanges were noted primarilyin the thyroid gland and pituitary 

gland of males and females receiving 337.5 mg/kg. 

The thyroid gland lesions observed were similar to 

those present in the 16-day study. Ninemale rats 

receiving 168.8 mgkg and ten male and seven female 

rats receiving337.5 mgkg had minimal but consistent 

changes in thyroid gland follicular cells. There was 

addition, the cytoplasmof follicular cells of the dosed 

rats appeared to be more vacuolated than that of controls. In the pituitary gland of337.5 mgkg males and 

females, the thyroid-stimulating hormone producing 

cells were enlarged and contained pale-staining cytoplasmand prominent Golgi complexes. Decreased 

serum triiodothyronine and thyroxine concentrations 

and increased thyroid-stimulating hormone concentration, similar to that which occurred in the 16-day 

study, occurred in 337.5 mgkg male rats; thyroid 

hormone concentrations were not affected in female 

rats (Table G2). 

Sperm motility of all dosed groups of maleswas 

significantly lower than that of controls (Table Hl). 

Vaginal cytology parameters of dosed groups of 

females were similar to those of controls. 

Dose Selection Rationale: Based on the absence of 

biologically significant body weight differences andof 

potentiallylife-threatening thyroid gland lesions in 

dosed rats, the high dose selected for continuous 

slight follicular cell hypertrophy and hyperplasia withexposure in the 2-year rat study was 337.5 mgkg. 

apparent thickening of the follicular epithelium. A The 337.5 mgkg dose was selected for the 6-month 

few follicles were enlarged and had apparent ingrowthstop-exposure evaluation in male rats to examine the 

of the hyperplastic epithelium into the lumen.In progression or regression of thyroid gland lesions. 

Initial Survivala Dose 

(mg/kg) 

Male 

Vehicle Control 

84 

168.8 

337.5 

Female 

Vehicle Control 

84 

168.8 

337.5 

Mean Body Weightb(g) Final Weight 

Final ChangeRelative to Controls 

(%) 

~~ ~ ~ 

10110 

10/10 

123 f 4 

124 f 3 

353 f 4 

365 f 7 

231 f 2 

241 f 8 

97 

10110 123 f 3 354 f 5 231 f 3 97 

10110 123 f 4 352 f 6 229 f 4 97 

10110 103 f 1 202 f 3 99*3 

10110 

10/10 

105 f 1 

104 f 1 

191 f 2** 

193 f 2* 

86 f 3** 

89 f 2** 

95 

96 

10110 105 f 1 191 f 2** 85 f 2** 94 

~~~ 

* Significantly different (PsO.05)from the vehicle control group by Williams' or Dunnett's test 

** PiO.01 

a Number of animals surviving at 13 weekdnumber initially in group 

Weights and weight changes are given as mean f standard error. 
40 Salicylazosulfapyridine,NTP TR 457 

%'YEAR STUDY 

Su.wiva1 

Estimates of 2-year survival probabilitiesfor male and 

female rats &e shown in Table 4 and in the KaplanMeier survival curves in Figures 2 (core study) and 3 

(stop-exposure males). Survival of 337.5mg/kg corestudy male rats was significantly lower than that of 

controls, with most deaths occurring during the last 

8 weeks of the study. Survival of 84and 168 mg/kg 

core-study males, all groups of dosed females, and the 

stop-exposure male group was similar to controls. 

Body Weights, Clinical Findings, 

and Clinical Chemistry 

Mean body weights of core-study males and stopexposure males were similar to controls throughout 

the study (Figures 4 and 5 and Table 5). From week 

45 to week 89, females in the 337.5mg/kg group had 

mean body weights that were 9% to 15% lower than 

those of contiols; the final mean body weight of 

337.5 mg/kgfemaleswas 7% lower than that of 

controls (Table 6 and Figure 4). No chemical-related 

clinical observations were noted in male or female. 

rats. The serum thyroxine concentration in the 

337.5mg/kg core-study males at study termination 

was minimally lower than that of controls; +e serum 

thyroid-stimulating hormone, triiodothyronine, and 

reverse triiodothyronine concentrations of dosed male 

and female rats were similar to those. of controls 

(Table G3). . -
VehicleControl $4 mg/kg 168 mgkg 337.5 mglkg 337.5 mg/kga .. (Stop-Exposure) . . 

Male 

Animals initially in.study 70 60 60 60 70 

6-Month interim evaluationb 10 0 0 0 10 

15-Month interim evaluationb 10 10 10 10 10 

Accidental deathsb 1 4 1 2 3 

Moribund 13 11 16 15 13 

Natural deaths 1 2 2 10 4 

Animals surviving to study termination 35 33 31f 23 30 

Percent probability of survival 

' at end ofstudy' 71 72 64 48 65 

Mean survival (days)d 582 628 630 629 538 

Survival analysise P=O.O14 P= 1 .WN P=O.424 P=O.O36 3 

Survival analysis P10.416 

Female 

Animals initially in study 60 60 60 60 

15-Month interim evaluationb 10 10 10 10 

Accidental deathsb 1 1 0 9 

Moribund 12 13 14 11 

Natural deaths 2 2 5 4 

Animals surviving to study termination 35f 34f 31 26 

Percent probability of survival 

at end of study 72 69 62 65 

Mean survival (days) 696 674 715 625 

Survival analysis P=O.455 P=O.897 P=O.577 P=O.564 

~ ~~ ~~ ~~~~ ~~ 

a Ten male rats receiving 337.5 mg/kg salicylazosulfapyridine were evaluated at 6 months. The remaining 70 male rats received the corn oil 

vehicle until the end of the 2-year study, and 10 male rats were evaluated at 15 months. 

Censored from survival analyses 

Kaplan-Meier estimated neoplasm incidence at the end of the study after adjustment for intercurrent mortality 

Mean of all deaths (uncensored, censored, and terminal sacrifice) 

e The result of the life table trend test (Tarone, 1975) is in the vehicle control column, and the results of the life table pairwise comparisons 

(Cox, 1972) with controls are in the dosed columns. A lower mortality in a dose group is indicated by N. 

Includes one animal that died the last week of the study 

Stop-exposure group is not included in trend test analysis 
42 SaiicylaioSulfapyridine,'NTP TR 457 

1.o 

0.9 

-I 

E O=I 

2 

s 

i! 

a

5 0.6 

0 

L 

4 

m 

n 

a 

0 0.6 

0.5 

0.4 I 

I5 I so I I I 45 60 I5 

WEEKS ON STUDY 

1 

90 

I 

105 1 0 

FIGURE 2 

Kaplan-Meier Survival Curves for Male and Female Rats 

Administered Salicylazosulfapyridine by Gavage for 2 Years 
iII

I I

i 

.." .............. I 

i 

i 

....

IiI

I ..I. .............. I 

..;. .............. i 

I 

i IiI

I 

-

i 

1015 10 

WEEKS Old STUDY

I11III

i I

I 

i 

I I

1 

I 

! 
44 Salicylazosulfapyridine,NTP TR 457 

5a I 1 I I 1 I I 

0 IS so 4s a0 75’ 90 105 1 

WEEKS ON STUDY 

..... ...-......: ................. :............................... ..... :................. 2 ................. I.................................. .. 

.....^._.....:.... ............. :... ............................ “...;.......-........:.................I ............... -:................ 

................ 4 ................. 4 .................................... i ....... I......... .................. + ................. :................ 

................. j................. 6..... ........... i.......... ...... ...... 

: i

....... ................................... 

rn 0 YO/KO 

0 84 uc/Ko I 

1..._.........i.............. ...i................. i............. -...i .... ........-. i.......... A lMYO/KO ..... 0 sn3YC/KO 

- . I 1 I I I I I 

15 so Is EO 7s 90 1os 

WEEKS ON STUDY 

FIGURE 4 

Growth Curves for Male and Female Rats 

Administered Salicylazosulfapyridine by Gavagefor 2 Years 
Bralicy%~Qsnih~ygrridine, NTP TW457 

500 

450 

400 

590 

so0 

250 

200 

1so 

loo 

w 
46 Salicylazosulfapyridine, NTP TR 457 

TABLE 5 

Mean Body Weights and Survivalof Male Rats in the 2-Year Gavage Studyof Salicylazosulfapyridine 

Weeks Control Vehicle 84 &ke 168 mdke

on Av. Wt. No. of Av. Wt. wt. (% of No. of ' Av. Wt. wt. (% of No. of 

!Study (8) Survivors (8) controls) Survivors (8) controls) survivors 

1 102 70 102 100 60 102 100 60 

2 139 70 137 99 60 138 100 60 

3 171 70 176 103 60 170 99 60 

4 1 96 70 203 103 60 198 101 60 

5 216 70 217 100 60 216 100 60 

6 238 70 240 101 60 24 1 1W 60 

7 258 70 259 100 60 259 100 60 

8 273 70 273 100 60 274 100 60 

9 287 70 285 99 60 280 98 60 

10 299 70 300 100 60 296 99 60 

11' 307 70 304 99 60 301 98 60 

12 3 17 70 316 100 60 312 99 60 

13 324 70 324 100 59 324 100 60 

14 333 70 339 102 59 333 100 60 

18 360 70 366 102 59 363 101 60 

21 374 70 372 100 59 367 98 60 

25 393 69 3% 101 59 397 101 60 

29a 416 59 412 99 59 414 100 60 

33 424 59 423 100 59 423 100 60 

37 439 59 430 98 57 43 1 98 59 

41 447 59 437 98 57 439 98 59 

45 457 59 448 98 57 453 99 59 

49 46 1 59 460 100 57 458 99 59 

53 469 59 458 98 57 463 99 59 

57 471 59 465 99 57 464 99 59 

61 477 59 465 98 56 468 98 57 

65 477 59 469 98 55 469 98 55 

69a 474 48 471 99 44 470 99 45 

73 477 46 475 100 44 473 99 44 

77 472 45 468 99 43 468 99 43 

81 468 44 469 100 43 461 98 41 

85 467 44 467 100 43 465 100 40 

89 46 1 43 468 102 42 463 100 38 

93 453 42 464 102 41 462 102 37 

97 442 41 457 103 38 457 103 37 

101 422 39 424 101 35 430 102 34 

Mean for weeks 

1-13 24 1 24 1 100 239 99 

14-52 410 408 100 408 100 

53-101 464 463 100 463 100 

(continued) 
Weeks 337.5 mdkg 337.5 mg/kp (StOD-ExDosure) 

on Av. Wt. . Wt. (% of No. of Wt. Av. wt. (% of No. of

Study (8) Survivors controls) (8) 1 Survivors controls)

1 102 100 60 99 97 70 

2 135 97 60 133 96 70 

3 173 101 60 166 97 70 

4 195 100 60 193 98 70 

5 215 100 60 212 98 70 

6 240 101 60 237 100 70 

7 259 100 60 255 99 70 

8 275 101 60 270 99 70 

9 280 97 60 280 97 70 

10 296 99 60 292 97 70 

11 301 98 60 296 96 70 

12. 313 99 60 ,307 97 70 

13 316 97 60 3 10 96 69 

14 33 1 100 60 325 98 69 

18 358 99 60 355 99 68 

21 363 97 59 359 96 67 

25 386 98 59 387 99 67 

29a 400 96 59 41 1 99 57 

33 413 97 59 428 101 57 

37 423 96 58 432 98 56 

41 428 96 58 449 100 56 

45 439 96 58 459 100 56 

49 444 96 58 465 101 56 

53 451 96 58 468 100 55 

57 460 98 58 472 100 54 

61 453 95 58 475 100 54 

65 459 96 56 477 100 52 

69a 46 1 97 45 476 100 41 

73 463 97 45 474 99 41 

77 45 1 96 45 467 99 40 

81 449 96 45 470 101 38 

85 450 96 42 463 99 36 

89 444 96 41 462 100 36 

93 439 97 39 453 100 35 

97 437 99 34 442 100 33 

101 417 99 31 427 101 31 

Mean for weeks 

1-13 238 99 235 98 

14-52 399 97 407 99 

53-101 449 97 464 100 

a Interim evaluations occurred during week 27 (vehicle control and 337.5 stop-exposure groups only) and week 66 (vehicle control and all 

dosed groups). 
48 Salicylazosulfapyridine,NTP TR 457 

TABLE6 

Mean Body Weights and Survivalof Female Rats in the 2-Year Gavage Study of Salicylazosulfapyridine -

Vehicle ControlWeeks 84 mdke 168mdke 337.5 melke

01R Av. Wt. No. of Av. Wt. Wt. (% of No. of Av.Wt.Wt. (% of No. of Av. Wt. Wt. (7% of No. of 

Study (9) Survivors (g) controls) Survivors (g) controls) Survivors (g) controls) Survivors

1 98 60 99 101 60 98 100 60 98 100 60 

2 116 60 117 101 60 115 99 60 114 98 60 

3 132 60 132 100 60 131 99 60 129 98 60 

4 142 60 141 99 60 139 98 60 138 98 60 

5 151 60 148 98 60 147 97 60 144 95 60 

6 159 60 156 98 60 155 97 60 152 95 60 

7 167 60 165 98 60 161 96 60 158 94 60 

8 174 60 171 98 60 168 97 60 163 94 60 

9 175 60 171 98 60 171 97 60 163 93 60 

YO 179 60 178 99 60 175 98 60 171 96 60 

11 183 60 180 99 60 177 97 60 172 94 .60 

1% 187 60 183 98 60 179 96 60 171 91 60 

I3 190 60 187 99 60 184 97 60 178 94 60 

17 197 60 194 98 60 191 97 60 186 94 60 

21 20 1 60 197 98 60 196 98 60 189 94 , 59 

25 208 60 205 99 60 20 1 97 60 198 95 59 

29 215 60 21 1 98 60 206 96 60 20 1 94 59 

33 218 60 214 98 59 210 96 60 205 94 58 

37 221 60 213 97 59 212 96 60 207 93 58 

41 226 59 219 97 58 216 95 60 212 94 58 

45 233 59 224 96 58 220 95 60 209 90 58 

49 240 58 229 95 58 226 94 60 218 91 58 

53 248 58 236 95 58 233 94 60 219 88 56 

57 256 58 24 1 94 57 240 94 60 226 88 54 

61 262 58 248 95 56 245 93 60 226 86 51 

65 27 1 58 255 94 55 253 93 60 233 86 51 

figa 279 48 261 93 44 258 93 50 237 85 41 

73 284 47 27 1 95 43 270 95 50 242 85 37 

77 287 47 273 95 43 273 95 50 249 87 35 

El 287 47 28 1 98 42 273 95 49 252 88 35 

85 290 47 284 98 41 277 96 49 259 89 34 

89 293 45 283 97 41 279 95 49 264 90 32 

93 295 43 285 97 40 285 96 45 268 91 32 

97 290 40 283 97 39 282 97 44 270 93 30 

101 293 36 283 97 36 287 98 37 272 93 28 

Mean for weeks 

1-13 158 156 99 154 97 150 95 

14-52 218 212 97 209 96 203 93 

53-101 280 268 96 266 95 247 88 ~ 

~ ~~~ 

a Interim evaluation occurred during week 66. 
SsnPicyl~osaaPha~i~~~~, NTP TP 05'7 09 

Pathology and Statistical Analysis 

This section describes the statistically significant and 

biologically noteworthy changes in the incidences of 

neoplasms and/or nonneoplastic lesions of the urinary 

bladder, kidney, spleen, and other organs, and in the 

incidences of mononuclear cell leukemia. Summaries 

of the incidences of neoplasmsandnonneoplastic 

lesions, individual animal tumor diagnoses, statistical 

analyses of primary neoplasms that occurred with an 

incidence of at least 5% in at least one dose group, 

and historical incidences for the neoplasms mentioned 

in this section are presented in Appendix A for male 

rats and Appendix B for female rats. 

Urinury Bladder: In male rats, the incidence of transitional epithelial papilloma in the337.5 mgkg corestudy group was significantly greater than that in the 

control group; two of the 337.5 mgkg core-study 

males had multiple transitional epithelial papillomas. 

Two transitional epithelial papillomas occurred in 

each of the 168 mgkg male and female groupsas well 

(Tables 7, Al, and Bl). There were no transitional 

epithelial papillomas in the stop-exposure group at any 

of the evaluation timepoints. The incidences of transitional epithelial papilloma in 168 mgkg male and 

female rats and in 337.5 mgkg core-study male rats 

exceeded the ranges in historical controls from NTP 

2-year corn oil gavage studies (Tables 7, A4, and 

B4a). 

The incidences of mucosal(transitionalepithelial) 

hyperplasia in 168 and337.5 mgkg core-study males 

and in 337.5 mgkg females were significantly greater 

than those in controls (Tables 7, A5, and B5). 

Mucosal hyperplasia was present in one 337.5 mgkg 

stop-exposure male at the endofthe2-year study. 

Concretions were observed grossly at necropsy and 

diagnosed as calculi. The incidences of grossly observed concretions (calculi) were significantly greater 

inmale rats receiving 168 or 337.5 mgkg than in 

controls. However, fewer concretions were diagnosed 

were presumed to be lost during tissue processing. 

The incidence of dilatation in core-study males receiving 337.5 mgkg was significantly greater than that in 

controls. The neoplastic and nonneoplastic lesions 

observed during the 2-year continuous study did not 

occur during the 2-year stop-exposure study. Urinary 

bladder transitional epithelial papillomas were single 

or multiple, discrete masses that protruded from the 

mucosa into the lumen of the urinary bladder from 

narrow- or broad-based attachments (Plate 1). Papillomas consisted of a few to several layers of hyperplastic transitional epithelium which formed thick, 

highly convoluted folds or papillary frond-like structures supportedby a core of scant to moderate 

amounts of fibrovascular stroma (Plates 2 and 3). In 

general, the hyperplastic cells were cuboidal to polyhedral. However, focal areas of epithelial dysplasia 

composed of atypical spindle to pleomorphic cells 

were evident. Papillomas frequently were concurrent 

with, and appeared to be a neoplastic progression of, 

mucosal epithelial hyperplasia. 

Mucosal (transitional epithelial) hyperplasia was generally a multifocal to diffuse change which varied 

from minimal to marked. The thickness of the epithelium was increased from the normal one or two cell 

layers to three to seven cell layers (Plate 4). Often 

there were focal areas of more prominent plaque-like 

thickening that exhibited both cellular dysplasia and 

atypia similar to that of papillomas. With increasing 

severity, focal areas of the hyperplastic epithelium 

formed thick complex folds that protruded into the lumen (Plate 5). Along the base of the mucosa in these 

areas, nodules of hyperplastic epithelium (some of 

which had central lumina) extended into the subjacent 

lamina propria. Microscopically, urinary bladder 

concretions were large, irregular masses composed of 

mostlyamphophilicspiculated material (Plate 6). 

Dilatation was primarily a gross change which presumablyresulted from obstruction of the lower 

microscopically than observed grossly at necropsy andurinary tract by concretions. 
50 Salicylazosulfapyridine, NTP TR 457 

TABLE7 

Incidences of Neoplasms and Nonneoplastic Lesionsof the Urinary Bladder in Rats 

in t.he 2-Year Gavage Studyof Salicylazosulfapyridine , , .. ,. 

-

Vehicle 84 mglkg 168 mglkg 337.5 mglkg 337.5 mglkg 

Control 

' 

(Stop-Exposure) 

Malle 

2-Year Study 

Number Examined Microscopically 50 49 50 50 47 

Calculus (gross diagnosis)c 

Mucosa Hyperplasia 

Dilatation 

Concretiona 

0 

0 

0 

0 

2 

0 

1 

0 

(3.0) 

1 (4.O)b 

12** 

14** (2.7) 

2 (3.5) 

28** 

41** (2.7) 

7* (3.6) 

lo** (3.1) 

0 

1 

0 

,o 

(2.0) 

Transitional Epithelial Papillomad 

Adjusted ratef 

Overall ratee 0150 (0%) 

0.0% ' 0.0% 

0149 (0%) 

6.5% 

2150 (4%) ; 

22.1% 

6/50 (12%) 

0.0% ' 

0147 (0%) 

Logistic regression test 

First incidence (days) 

Terminal rateg 

P<O.ool 

0135 (0%) -I 

0/33 (0%) 

P=O.212 

729 (T) 

2/31 (6%) 

P=O.O11 

653 

3/23 (13%) 0130 (0%) 

Female 

15-Month Interim Evaluation. 

Number Microscopically Examined 10 10 10 10

Mucosa Hyperplasia '. . 0 0 0 2 (1.5) 

2-Year Study 

50Microscopically Examined Number49 50 50

Concretion 0 0 1 (4.0) 0 

Hyperplasia Mucosa 2 (2.5) 0 4 (3.3)

diagnosis)(grossCalculus 1 0 4 1 

Transitional Epithelial Papillom$ 

Overall rate 0149 (0%) 0150 (0%) 2150 (4%) 0/50 (0%) 

* Significantly different (Pr0.05) from the vehicle control group by the logistic regression test 

** Fk0.01 

(T)?'erminal sacrifice 

a Numberofanimalswithlesion 

Average severity grade of lesions in affected animals: l=minimal, 2=mild, 3=moderate, 4=marked 

Number of animals with grossly observed urinary bladder concretions (diagnosed as calculi at necropsy) 

Historical incidencefor 2-year NTP corn oil gavage studies with vehicle control groups (meanf standard deviation): 11904 

(0.1% f 0.5%). range0%-2% 

e Number of animals with neoplasm per number of animals with urinary bladder examined microscopically 

Kaplan-Meier estimated neoplasm incidence at the endof the study after adjustment for intercurrent mortality 

Observed incidence at terminal kill 

Beneath the vehicle control incidenceare the P values associated with the trend test (does not include the stop-exposure group). Beneath 

the dosed group incidence are the P values corresponding to painvise comparisons between the vehicle controls and that dosed group. The 

logistic regression test regards neoplasms in animals dying prior t terminal kill as nonfatal. 

i Not applicable; no neoplasms in animal group 

J Historicalincidence:3/903 (0.3% f 0.8%). range 0%-2% 

12*(1.7) 
Kidney: The absolute kidney weight of 337.5 mgkg 

females was significantly greater than that of controls 

at the 15-month interim evaluation (Table F3). Papilloma of the renal pelvis transitional epithelium was 

observed in two female rats receiving 337.5 mgkg 

(Tables 8 and Bl). The incidence of transitional epithelial papilloma in 337.5 mgkg female rats exceeded 

the incidence in historical controls from NTP 2-year 

corn oil gavage studies (Tables 8 and B4b). 

There were increased incidences of nonneoplastic 

kidney lesions in dosedmaleandfemale rats at 

2 years and in 337.5 mgkg females at 15 months. 

The incidences of concretion and transitional epithelial 

hyperplasia in 168 and 337.5 mg/kg core-study male 

and female rats were significantly greater than those 

in controls at 2 years as were the incidencesin 

337.5 mgkg females at 15 months (Tables 8, A5, and 

TABLEI 

Male 

&Month Interim Evaluation 

Number Examined Microscopically 

Mineralizationa 

Nephropathy 

Renal Tubule Dilatation 

15-Month Interim Evaluation 

Number Examined Microscopically 

Mineralization 

Nephropathy 

Renal Tubule Dilatation 

Transitional Epithelial 

Hyperplasia 

%Year Study 

Number Examined Microscopically 

Concretion 

Hydronephrosis 

Mineralization 

Nephropathy 

Papillary Necrosis 

Renal Tubule Dilatation 

Transitional Epithelial 

Hyperplasia 

Atypical Transitional 

Epithelial Hyperplasia 

Calculus (gross diagnosis)c 

(continued) 

Vehicle M mg/kg 

Control 

10 

1 (l.O)b 

8 (1.0) 

0 

10 10 

0 0 

10(1.5) 10(1.0) 

1 (2.0) 0 

0 0 

50 50 

sn 

B5). In addition, focal atypical epithelial hyperplasia 

occurred in three core-study male rats receiving 

337.5 mgkg for 2 years. At 2 years, the incidences 

of hydronephrosis in 337.5 mgkg core-study males 

and females were significantly greater than those in 

controls. The incidence of renal tubule dilatation in 

337.5 mg&g core-study maleswassignificantly 

greater than thatin controls at 2 years. The incidences of mineralization in 84, 168, and 337.5 mg/kg 

males were significantly greater than that in controls 

at 2 years. 

Papillary necrosis was observed in four 337.5 mgkg 

core-study males andin one 337.5 mgkg female 

(Tables 8, A5, and B5). The average severity of nephropathy was greater in dosed males than in controls. 

The incidence of nephropathy in 337.5 mgkg females 

wassignificantly greater than that in controls. The 

141 mgkg 337.5 mgkg 337.5 mg/kg 

(Stop-Exposure) 

10 

J 9 

1 

(1.0) 

(1.0) 

1 (2.0) 

10 10 10 

10(1.1) 

0 

10(1.2) 

0 

10(1.1) 

2 (1.0) 

0 2 (2.0) 0 

0 1 (1.0) 0 

50 50 50 

0 1 (1.0) 13** (2.1) 33** (2.6) 0 

0 1 (2.0) 1 (2.0) 28** (2.2) 0 

3 (1.0) 10* (1.2) 11* (1.5) 13** (1.8) 3 (1.0) 

48(1.6) 47 (1.9) 50 (2.0) 48 (2.5) 46(1.5)

0 0 1 (3.0) 4 (2.5) 0 

0 1 (1.0) 1 (3.0) 11** (2.7) 3 (2.0) 

10(1.2) 10 (1.1) 20* (1.8) 43** (2.6) 4 (1.0) 

0 0 0 3 (3.0) 0 

0 0 3 18 0 
52 Salicylazosulfapyridine,NTP TR 457 

TAIBLE 8 

Incidences of Neoplasms and Nonneoplastic Lesionsof theKidneyinRatsinthe2-YearGavage,Study . 

- . . of Salicylazosulfapyridine(continued) -

Vehicle 84 mg/kg 168 mg/kg 337.5 mg/kg 337.5 mg/kg 

Control (Stop-Exposure) 

Female 

15-Month Interim Evaluation 

Number Examined Microscopically 10 10 10 . $

c 10 

Concretion 0 0 1 (1.0) 6** (2.7) Mineralization 9 (1.3) 10 (1.9) 8 (1.9) (2.1) 9 

Nephropathy (1.0)4 (1.0) 6 6 (1.0) lo** (1.0)

Renal Tubule Dilatation 0 0 0 0 

Transitional Epithelial 

Hyperplasia 

2-Year Study 

Number Examined Microscopically 

Concretion 

Hydronephrosis 

Mineralization 

Nephropathy 

Papillary Necrosis 

Renal Tubule Dilatation 

0 

50 

0
 

0 

44 

34 

0 

. 

(1.7) 

(1.1) 

0 

50 

9** (1.6) 

0 

47 (1.7) 

36 (1.2) 

0 

2 , (1.5) 

50 

34** (1.9) 

3 (3.0) ' ' 

43 (1.9) 

41. (1.2) 

0 

(2.0)2 (2.0)2 (3.0) 1

8** (2.4) 

50 

37** (2.9) 

lo** (2.0) 

41 (1.8) 

44** (1.4) 

1 (2.0)

6 (2.3)

Transitional Epithelial 

Hyperplasia (1.3)3 7 (1.3) 23** (1.5) , 43** (2.6) 

Calculus (gross diagnosis) 0 0 2 11 

Transitional Epithelial 

Papillomad 0 0 0 2 

.. 

* Significantly different (PsO.05) from the vehicle control group by the Fisher exact test (interim evaluations) or the logistic regression test 

(:Z-year study) 

** P50.01 

a Number of animals with lesion 

Average severity grade of lesions in affected animals: l=minimal, 2=mild, 3=moderate, 4=marked 

Plumber of animals with grossly observed kidney concretions (diagnosed as calculi at necropsy) 

Historical incidence for 2-year NTP corn oil gavage studies with vehicle control groups: 0/920 

incidence and severity of nonneoplastic kidney lesions fronds of thickened transitional epithelium supported 

in stop-exposure male rats were similar to those in 

controls. 

Microscopically, concretions consisted of one or more 

variably sized irregular aggregates of lightly eosinophilic to amphophilic spicules (often radially arranged) or granular materia$ within the lumen of the 

renal pelvis and/or incorporated within hyperplastic 

pelvic transitional epithelium (Plate 7). Focal mineralization was often a feature of concretions. 

Transitional epithelial papillomas were narrow- or 

broad-based exophytic masses that protruded into the 

lumen of the renal pelvis. They consisted of papillary 

by a core of scant to moderate amounts of fibrovascular tissue. Transitional epithelial hyperplasia was a 

multifocal to diffuse change. The hyperplastic epithelium frequently occurred as few to several nodular to 

papillary projections with scantcores of fibrovascular 

stroma (Plate 8), and/or as focal or focally extensive 

areas in which the epithelium was increased in thickness from two to five times the normal thickness due 

to an increase in the number of cell layers. Less frequently, thickening of the pelvic epithelium was diffuse and relatively uniform and there was concurrent 

hyperplasia of the epithelium of the proximal ureter. 

Atypical transitional epithelial hyperplasias were 

single, epithelial-lined, nodular lesions, 0.5 to 1 .Ocm 
53 

in diameter, that consisted of densely packed poly- Multiple Organs: The incidences of mononuclear cell 

gonal to spindle cells that projected aboveor occurred leukemia in malecore-study .(13/50, 18/50, 12/50, 

in the submucosa lamina propria immediately beneath3/50, 10150; Table A3) and female (14/50, 9/50, 

the mucosa (Plate 9). 

Papillary necrosis was consideredan ischemic change 

most likelythe result of pressure from the concretions 

lodged within the urinary pelvis rather than from 

precipitation of compound or a metabolite into the 

collecting tubules. The entire renal papillawas 

generally affected and appeared hypoeosinophilic with6/50; Table A3). The incidences of mammary gland decreased cellular detail and cells that lacked nuclei 

(Plate 10). 

Minimal to mild nephropathy consisted of scattered 

clusters of small diameter regenerative tubules lined 

by basophilic cuboidal epithelial cells and often surrounded by a slightly thickened basement membrane, 

and dilated tubules filled with eosinophilic proteinaceous fluid. With increasing severity, more tubules 

were involved, and interstitial fibrosis with variable 

mononuclear cell infiltrates and focal tubule suppuration and variable glomerular sclerosis were widespread. Mineralization consisted of round to irregular 

particulate and/or laminated bodies that occurred in 

the inner cortex or near the corticomedullary junction. 

Mineralization inthis location is a common, spontaneous finding in rats, particularly in female rats. The 

increased incidence of mineralization noted in dosed 

male rats may be associated with the increased severityof nephropathy noted intheseanimals.Renal 

tubule dilatation was characterized by widespread 

distension of renal tubules to approximately twice the 

normal diameter. Hydronephrosis was characterized 

by mild to moderate dilatation of the renal pelvis. 

Spleen: The incidences of splenic hematopoietic cell 

proliferation [vehicle control, 14/50; 84 mg/kg, 

15/50; 168 mg/kg, 17/50; 337.5 mgkg (continuous 

exposure), 23/50; 337.5 mgkg (stop-exposure), 

14/50] and hemosiderin pigmentation (14/50, 15/50, 

15/50, 30/50, 13/50) were significantly greater. in 

core-study male rats receiving 337.5 mgkg than in 

controls (Table A5). These increases may be suggestive of increased erythrocyte toxicity and destruction 

(hemolysis). 

8/50, 3/50; Table B3) rats receiving 337.5 mgkg 

were significantly less than in controls. 

Other Organs: The combined incidences of benign 

and malignant pheochromocytomaof the adrenal 

medulla were significantly less in dosed groups of 

males than that in controls (16/50, 7/49, 7/50, 8/49, 

fibroadenoma (2260, 9/50,20/50, 5/50) and pituitary 

gland (pars distalis) adenoma (26150, 20/49, 24/50, 

13/50) in dosed groups of females were less than in 

controls (Table B3). 

Incidences of pancreatic acinar cell cytoplasmic alteration (1150, 1/50, 3/50, 15/50,3/48), forestomach 

ulcer (1150, 1/50, 1/50, 7/50,0/50 ), and forestomach 

mucosalhyperplasia (0/50,0/50,0/50,9/50, 0/50) 

were significantly greater in core-study males administered 337.5 mgkg than in controls (Table A5). The 

incidence of mammary gland ectasia was significantly 

greater in 168 mgkg females than that in controls 

(lobo, 10/50, 20150, 4/50;Table B5). 

Incidences of accessory adrenal cortical nodules 

(19150, 19/50, 13/50, 8/50, 12/50), chronic inflammation of the preputial gland (24/50, 18/50, 16/50, 

9/50, 25/50), and corpora amylacea ofthe prostate 

gland (28/50, 28/50, 19/50, 11/50, 25/50) were significantly less in males administered337.5 mgkg than 

in controls (Table A5). Incidences of pituitary gland 

(pars distalis) cysts infemales administered 168 or 

337.5 mgkg were significantly less than in controls 

(29/50, 26/49, 18/50, 15/50; Table BS). 

The biological significance of these incidences is uncertain but may be related to treatment or changes in 

body weight. In general, 1) the lesions represent 

usual age-related changes encountered in chronic 

studies, 2) the increased or decreased incidences were 

marginal and did not have a clear dose-related trend, 

or 3) there was usually no similar effect in animals of 

the other sex. 
54 

MICE. 

16i-DAY STUDY 

There were no chemical-related deaths, and final 

mean body weights of dosedmice were similar to 

those of controls .(Table 9): No chemical-related 

clinical observations were noted for male or female 

mice. There were no.differences between dosed and 

control mice in triiodothyronine, thyroxine, or 

thyroid-stimulating hormone concentrations 

(Table G4). There were no biologically significant 

differences in absolute or relative organ weights 

TABLE9 

Salicylazosulfapyridine, NTP TR 457 

between dosed and control mice (Table F5). 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. 

Dose Selection Rationale: Based on the absence of 

chemical-related mortality, body weight effects, and 

lesions, the high dose selected for the 13-week mouse 

study was 2,700 mg/kg. 

Survival and Body Weights of Mice in the 16-Day Gavage Study of Salicylazosulfapyridine 

Mean Body Weightb(g) Final Weight 

. Dose ' Initial Survivala

(mgkg) 

.. 

Male 

Vehicle Control 515 

675 515 

1,350 515 

2,700 515 

Female 

Vehicle Control 515 

675 415' ' 

1,350 515 

.2,700 415' 

-

* 

21.0 f 0.3 

20.8 f 0.4 . 

21.3 f 0.2 

21.3 f 0.3 

17.2 f 0.2 

17.2 5'0.2 

16.8 f 0.3 

16.7 f 0.3 

Final Changeto Controls Relative

23.6 f 0.4 2.6 f 0.4 

23.0 f 0.4 2.2 f 0.4 97 

-24.2 f 0.5 2.9 f 0.3 102 

24.1 f 0.4 2.9 f 0.4 102 

19.6 f 0.3 2.4 f 0.3 

20.8 f 0:2 3.5 f 0.4* 106 

20.1 f 0.3 3.3 f 0.3 103 

20.3 f 0.7 3.4 f 0.3 103 

Significantly different (PiO.05) from the control group by Dunnett's test . . 

a Number of animals surviving at 16 dayslnumber initially in group 

Weights and weight changes are given as mean f standard error. Subsequent calculations are based on animals surviving to the end of the 

study. ' Dayof death: 7 
SrPPicyn~QSmlh~y~~~~~~, N%P %R457 55 

nmmEK STUDY 

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 (Table 10). In 

1,350 mgkg male mice, the mean body weight gain 

was 86%that of controls, and in 2,700 mgkg male 

mice, the body weight gain was80%that of controls. 

No chemical-related clinical observations were noted 

in dosed male or female mice during the 13-week 

study. 

The hematology, clinical chemistry, and urinalysis 

data for mice in the 13-weekstudy are listed in 

Table G5. Reticulocytosis, evidenced byincreased 

reticulocyte numbers, occurred in all dosed groups of 

male and female mice. In general, reticulocytosis 

indicates an erythropoietic response to anemia. In this 

study, there was some evidence of anemia,characterized by decreased erythrocyte counts and hematocrit 

values of dosed groups of females. Methemoglobinemia occurred in 1,350 and 2,700 mg/kg female mice, 

evidenced by increased methemoglobin concentrations. Sulfonamides can cause methemoglobinemia, 

and this could explain the minimal responsive anemia. 

The urine specific gravity values were increased in all 

male and female dose groups, suggesting an increased 

need for conservation of body water. The cause of 

the increased urine concentration was unknown. 

Increased urine concentration could, however, be related to a mild dehydration which could have masked 

the severity of the anemia. Leukocytosis, evidenced 

by increased leukocyte numbers, occurred in 

2,700 mgkg male and female mice. The leukocytosis 

was characterized by increases in mature neutrophil 

and lymphocyte numbers. There were no microscopic 

significance is unknown. While elevated peripheral 

neutrophil and lymphocyte numbers can be related to 

increased production, increases can also occur as a 

result of altered cell margination or homing, tissue 

migration, and recirculation. There were minimal 

increases in alanine aminotransferase and alkaline 

phosphatase activities in 1,350 and.2,700 mgkg male 

mice; these changes may be consistent with the 

centrilobular hypertrophy observed microscopically in 

the liver. Minimal decreases in thyroxine concentration occurred in all dosed male and female mice. 

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 significantlygreater 

than those of controls (Table F6). 

There were no chemical-related gross lesions. Microscopic evaluation of the liver revealed centrilobular 

hypertrophy in five 1,350 mgkg and all 2,700 mgkg 

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 (Table H2). 

DoseSelectionRationale: Based on the absence of 

biologically significant body weight differences and of 

potentiallylife-threateninghepatotoxicityin dosed 

mice, the high dose selected for the 2-year study was 

lesions that could account for the leukocytosis and the2,700 mgkg . 
56 Salicylazosulfapyridine,NTP TR 457 

TAIBLE10 

Survival and Body Weighis of Mice in the 13-Week Gavage Study of Salicylazosulfapyridine 

Mean Body Weightb (g) Final Weight 

Initial Survivala Dose Final Change Relative Controls to

Male 

Vehicle Control 10110 22.3 f 0.3 31.3 f 0.6 9.0 f 0.5 

675 10110 22.7 f 0.4 30.9 f 0.4 8.2 f 0.6 99 

1,350 10110 22.5 f 0.3 30.3 f 0.5 . 7.7 f 0.3* 97 

2,700 10110 23.1 f 0.3 30.3, f 0.4 7.2 f 0.3** 97 ,. 

Female 

Vehicle Control 10110 18.9 f 0.3 26.5 f 0.5 7.6 f 0.4 

675 10110 19.0 f 0.3 26.2 f 0.3 7.1 f 0.3 99 

1,350 10110 18.6 f 0.2 26.4 f 0.3 7.8 f 0.3 100 

2,700 10110 18.5 f 0.3 26.4 f 0.5 7.8 f 0.4 100 

* Significantly different (PsO.05) from the control group by Williams’ or Dunnett’s test 

** PsO.01 

a Number of animalssurvivingat 13 weekslnumber initially in group 

Weights and weight changes are given as mean f standard error. 
Salicylazosulhpyridine,. NTP TW057 57 

%YEAR STUJlCDY mice had mean body weights that were 6% to 18 % Suwival lower than those of controls; the finalmeanbody Estimates of 2-year survival probabilities for male andweight of 2,700 mgkg males was 12% less than that 

female mice are shown in Table 11 and in the Kaplan- of controls. From week 14 to the end of the study, Meier survival curves (Figure 6). Survival of all 2,700 mgkg female mice had mean body weights that dosed groups of male and female mice was similar to were 5 % to 19% lower than those of controls; the 

that of controls. final mean body weight of these dosed females was 

10% less than that of controls. No chemical-related 

Body Weights, ClinicalFindings, clinical observations were notedinmale or female 

and Clinical Chernistvy mice. There were no chemical-related differences in 

Mean body weightsof 675 and 1,350 mg/kg male and triiodothyronine, reverse triiodothyronine, thyroxine, 

female mice were similar to thoseof controls through- or thyroid-stimulating hormone concentrations beout the study (Figure 7 and Tables 12 and 13). From tween dosed and control mice at the 15-month evaluweek 12 to the end of the study, 2,700 mg/kg male ation (Table G6). 

TABLE11 

Survival of Mice in the %YearGavage Study of SalicynazosnlB~lpyriaine 

Vehicle Control 675 mglkg 1,350 mglkg 2,700 mglkg

Male 

Animals initially in study 60 6 0 ' 60 60 

15-Month interim evaluationa 10 10 10 10 

Accidental deathsa 2 0 0 0 

Moribund 5 4 3 4 

Natural deaths 3 5 6 0 

Animals surviving to study termination 40 41 41 46 

Percent probability of survival at end of studyb 84 83 82 93 

Mean survival (days).c 649 654 664 653 

Survival analysisd P=0.251N P=l.Ooo P=l.Ooo P=0.350N 

Female 

Animals initially in study 60 60 60 60 

15-Month interim evaluationa 10 10 10 10 

Accidental deathsa 2 0 1 2 

Moribund 5 6 8 6 

Natural deaths 2 3 4 4 

Animals surviving to study termination 41 41 37e 38 

Percent probability of survival at end of study 86 83 77 80 

Mean survival (days) 643 656 641 630 

Survival analysis P=O.484 P=O.839 P=O.327 P=O.594 

a Censored from survival analyses 

Kaplan-Meier estimated neoplasm incidence at the end of the study after adjustment for intercurrent mortality 

Mean of all deaths (uncensored, censored, and terminal sacrifice) 

The result of the life table trend test (Tarone, 1975) is in the vehicle control column, and the results of the life table painvise comparisons 

(Cox, 1972) with controls are in the dosed columns. A negative trend or lowkr mortality in a dose group is indicated by N. e Includes one animal that died the last week of the study 
Salicylaiosulfapyridine,NTP TR 457 

1.01 ................ . ....... -...... 

b 

1

4

-I 

1t 0.9- ................... ..............-. ;............... .................................. * ................ ....... L"......... I... 

3 

E 

0" 

v) 

4 

7 

0 

m 

CI: 0.8 _ ..............-.. ;................. t............... ................................. 

a. 

A 13% UO/*O 

... . . 0.7 

0 1s 30 I os 

WEEKS ON STUDY 

FIGURE6 

Kaplan-Meier SurvivalCurves for Male and Female Mice 

Administered Salicylazosulfapyridine by Gavage for 2 Years 
59 

80 . . 

SI .................. i.................... ........... .... ..... .............. 

................... 1...........-....... i............... ......... ....... 90 

as ................... 1............. ..... ......... DQ. 

t o+ ..... "............ <:........ f..;..@D..o..o..* ......................................... ..................... ;............._.... 40 

ss ....................,...............-....,....................b ................... 0...................

.................... ........................................... .................... <................... sa 

..... .................... e................... .................. 29 0 0 MO/KO 

0 074MG/RG 

aa ................... i 

0 8700 %/KO 

13 1 I I , I 

19 so 45 80 79 BO 19 

WEEKS ON STUDY 

60 

9s . ............................................................ -................... ^.. ......... 

iC 

A

90 ................... ......................................... '................. .........-......... i fin Ai i a-0' 

A A 4s ................... ................... ...... .... ........................... 1 

".......... -................... 0 

3 o o ~ ~ o ~ 

40 ................... o,..O.).. .................. .......................... ............. 

SI ................... ................... .................... .)....................I. ................... 3................... 

90 .................... ,.................... t.................... 5.................... 1 ........ . .......... + ............._...

25 .................................................. e................... .. ................ FEMALE MICE ..... 0 OMO/KO 

0 075 MO/KG 

ao ..................................... c :.................... c ................... ,".. ................ A lS30 M/KO ..... 0 8700 %/KO 

14 I I I i I I 

19 so 45 80 79 eo 14 

WEEKS O N STUDY 

FIGURE 7 

Growth Curves ffor Male rand Female Mice 

Administered Salicylazosulfapyridine by Gavage for 2 Years 

~ ~ o 
60 Salicylazosulfapyridine,NTP TR 457 

TAIBLE 12 

Mean Body Weights and Survival of Male Mice inthe 2-Year Gavage Study of Salicylazosulfapyridine 

.Weeks Vehicle Control 675 mdkg 1.350 mdke

011 Av. Wt. NO.of Av. Wt: Wt. (% of NO.of Av. Wt.Wt. (% of NO.of .Av.Wt. Wt. (% of NO.of 

Stedy . (g) Survivors . (g) controls) Survivors (g) controls) Survivors (9) controls) Survivors 

1 23.5 60 23.2 99 60 23.3 99 " 60 23.2 99 60 

2 25.3 60 25.O 99 60 24.8 98 60 25.0 99 58 

3 25.9 60 26.3 102 60 26.4 102 60 26.6 103 58 

.4 27.7 60 27.6 100 60 27.4 99 60 27.7 100 58 

5 28.4 60 28.5 100 60 28.1 99 60 28.1 99 58 

6 29.1 60 28.7 99 60 28.7 99 60 28.3 97 58 

30.0 60 29.9 100 60 30.0 100 60 29.5 98 58 7. 

8 31.4 60 30.6 98 60 30.5 97 60 30.4 97 58 

9 31.6 60 31.2 99 60 30.5 97 60 30.7 97 58 

1.0 32.0 60 31.3 98 60 31.5 98 60 30.8 96 58 

11 32.9 60 32.0 97 60 32.2 98 60 31.7 96 58 

12 33.5 60 32.5 97 59 32.1 96 60 31.4 94 58 

13 34.6 60 33.8 98 59 33.3 96 '60 32.5 94 58 

14 35.2 60 34.0 97 59 33.4 95 60 32.5 92 58 

18 38.2 60 37.1 97 59 36.3 95 60 34.2 90 58 

22 40.7 60 40.1 99 59 38.9 96 60 37.0 91 58 

26 44.7 59 42.3 95 59 40.7 91 60 37.9 85 58 

30 45.2 59 44.1 98 59 42.7 95 60 39.2 87 58 

34 47.7 59 45.6 96 59 44.0 92 60 39.4 83 58 

38 48.7 59 47.0 97 59 44.6 92 60 40.0 82 58 

42 49.2 59 47.9 97 59 46.2 94 ' 0 6
 40.1 82 58

46 49.7 59 48.8 98 58 47.5 96 60 41.0 83 58 

50 50.2 59 50.3 100 58 47.8 95 60 41.8 83 58 

54 51.7 58 52.1 101 58 50.0 97 60 43.1 83 58 

58 52.6 58 53.4 102 58 51.7 98 59 44.7 85 58 

ti2 52.8 56 54.0 102 58 53.0 100 59 45.9 87 58 

66a 5313 46 55.2 104 48 52.2 98 49 46.2 87 48 

70 54.0 46 55.5 103 48 52.4 97 49 45.2 84 48 

74 53.6 46 56.6 106 48 54.7 102 47 48.2 90 47 

78 54.4 46 56.1 103 48 54.0 99 47 45.8 84 47 

82 54.3 46 56.5 104 47 53.3 98 47 47.2 87 47 

$16 52.7 46 55.8 106 46 53.1 101 47 46.2 88 47 

(90 52.9 44 56.6 107 45 53.7 102 46 46.8 89 47 

94 '53.4 44 56.4 106 45 54.3 102 45 47.2 88 46 

98 52.3 41 54.5 104 42 50.1 96 44 44.8 86 46 

102 52.0 40 56.7 109 41 52.1 100 41 45.7 88 46 

Mean for weeks 

1-13 29.7 29.3 99 29.1 98 28.9 97 

14-52 45 .O 43.7 97 42.2 94 38.3 85 

53-102 53.1 55.3 104 52.7 99 45.9 86 

-

a Interim evaluationoccurredduring week 66. 
P8 Z'ZP 66 6'6P 

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65 501 6'92 65 POI 5'9Z 9'5Z 09 65 ZOI 1'9Z 9 

65901 50165 9'SZ 8'52 E'PZ 09 . 6S EO1 6'PZ 5 

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awu OOL'Z axlaw OSE'I awm EL9 iWUO3 W!WA waAA 
62 Salicylazosulfapyridine, NTP TR 457 

Pathology and Statistical Analysis 

This section describes the statistically significant and 

biologically noteworthy changes in the incidences of 

neoplasms and/or nonneoplastic lesions of the liver, 

spleen, forestomach, and other organs. Summaries of 

the incidences of neoplasms and nonneoplastic lesions, 

individual animal tumor diagnoses, statistical analyses 

of primary neoplasms that occurred withan incidence 

of ;It least 5 % in at least one dose group, and historical incidences for the neoplasms mentioned in this 

section are presented in Appendix C for male mice 

and Appendix D for female mice. 

Liver: The absolute and relative liver weights of 

1,350 mg/kg male mice were significantly greater 

than those of controls at the 15-month interim evaluation (Table F7). Incidences of hepatocellular adenoma (single and multiple or multiple) in all dosed 

groups of male and female mice were significantly 

greater than those in controls at 2 years (Tables 14, 

C3!, and D3) and occurred with a positive trend. Furthermore, the incidences of hepatocellular adenoma 

(single and multiple) in 675 and 2,700 mg/kg male 

mice and all dosed groups of female mice exceeded 

the ranges in historical controls fromNTP 2-year corn 

oil gavage studies (Tables 14, C4a, and D4a). The 

incidences of hepatocellular carcinoma (single and 

multiple) in 1,350 mg/kg male miceandalldosed 

groups of female mice were significantly greater than 

those in controls. The incidences of hepatocellular 

carcinoma (single and multiple)invehicle control, 

675, and 1,350 mg/kg male mice and all dosed groups 

of female mice exceeded the ranges in historical contro1.s from NTP 2-year corn oil gavage studies 

(Tables 14, C4a, and D4a). Additionally, the combined incidences of hepatocellular adenoma or carcinoma were greater in dosed mice than those in controls, and in males, the incidences increased with 

increasing dose. 

The incidences of nonneoplastic cytologic alterations 

ofthe liver in 1,350 and 2,700 mg/kg maleand 

female mice at the 15-month interim evaluation were 

significantly greater than those in controls (Tables 14, 

C5, and D5). At the end of the 2-year study, the 

incidences of eosinophilic foci in all dosed groups of 

male and female mice were significantly greater than 

those in controls. The incidences of clear cell foci in 

675 and 2,700 mg/kg male .mice were significantly 

greater than that in controls. 

Hepatocellular adenomas were nodular and occasionally multinodular masses of hepatocyte proliferation 

that varied in their tinctorial properties. Characteristic 

features included well-defined borders, variable compression of the surrounding parenchyma, alteration of 

the hepatic architecture with the ,absence of portal 

areas and central veins, and hepatic cords intersecting 

at an angle to the surrounding hepatic parenchyma. 

Carcinomas were generally larger and more irregular 

than adenomas. Compression and invasion of thesurrounding parenchyma were typical, as were abnormal 

growth patterns with normal appearingand/or atypical 

hepatocytes arranged in solid sheets and trabeculae 

three or more cells thick. Eosinophilic foci were typically nodular lesions with generally discrete borders 

and normal lobular architecture with hepaticcords that 

blended with those of the surrounding normal parenchyma. The hepatocytes of eosinophilic foci were 

often larger and more eosinophilic than histologically 

normal hepatocytes ofthe adjacent parenchyma. 

Clear cell foci were discrete nodular lesions that consisted of enlarged vacuolated hepatocytes. 

Cytologic alteration was observed at the 15-month 

interim evaluation and was most prominent in male 

mice receiving 2,700 mg/kg. It was characterized primarily by loss of cytoplasmic vacuolization, presumably due to the loss of glycogen from centrilobular 

hepatocyteswith concomitant accentuation of the 

lobular hepatic architecture and an apparent increase 

inthe size of the hepatocytes. Lesions of minimal 

severity involved hepatocytes immediately surrounding the central veins. With increasing severity, more 

hepatocytes were involved and cytoplasmic vacuolization was essentially absent from centrilobular hepatocytes and decreased in hepatocytes located more 

peripherally in the hepatic lobules. The loss of cytoplasmic vacuolization was most likely physiologic in 

nature. Mean body weightsof 2,700 mg/kg males 

and females at 15 months and the end of the 2-year 

study were lower than those of the control groups. 

The degree of hepatocellular vacuolization is known 

to change with the nutritional status of the animal; it 

is often decreased or absent in early death or moribund animals and in animals fasted prior to sacrifice. 
Hepatocellular cytoplasmic vacuolization may also 2-year evaluation is not clear. The severity of the 

vary with the timeof day depending upon whether the lesion was essentially minimal at 15 months, and at 

animal is in a pre- or postprandial status. The signifi- 2 years it may have been masked by spontaneous 

cance of the apparent absence of this change at the changes that occur in the liver with advancing age. 

Control Vehicle

Male 

15-Month Interim Evaluation 

NumberExaminedMicroscopically 10 

Alterationsa Cytologic 0

Hepatocellular Adenoma 2

Hepatocellular Carcinoma 0

%Year Study 

NumberExaminedMicroscopically 50 

Clear Cell Foci 2 

Eosinophilic Foci 6 

Hepatocellular Adenoma (Multiple) 

Overall rate 1/50 (2 %) 

Hepatocellular Adenoma (Single and Multiple)b 

rate' Overall 13/50 (26%) 

rated Adjusted 32.5% 

ratee Trminal 13/40 (33%) 

First (days) incidencg 728 (T) 

Logistic testf regression P<O.001 

Hepatocellular Carcinoma (Multiple) 

Overall rate 1/50 (2%) 

Hepatocellular Carcinoma (Single and Multiple)g 

Overall rate 13/50 (26%) 

Adjusted rate 29.2% 

Terminal rate 9/40 (23 %) 

First incidence (days) 420 

Logistic regression test P=0.154N 

Hepatocellular Adenomaor Carcinomah 

Overall rate 24/50 (48%) 

Adjusted rate 54.3% 

Terminal rate 20/40 (50%) . 

First incidence (days) 420 

Logistic regression test P<O.001 

(continued) 

675 mglkg 1,350 mglkg 2,700 mglkg 

10 10 10 

2 8** lo** 

2 0 2 

0 1 0 

50 50 50 

14** 5 11* 

19** 20** 22** 

21/50 (42%)** 15/50 (30%)** 27/50 (54%)** 

32/50 (64%) 28/50 (56%) 42/50 (84%) 

76.2% 66.6% 87.5% 

666 

31/41 (76%) 

678 

27/41 (66%) 

497 

40/46 (87%) 

P<O.O01 P=O.O02 P <0.001 

4/50 (8%) 5/50 (10%) 0/50 (0%) 

15/50 (30%) 23/50 (46%) 8/50(16%) 

33.8% 51.0% 17.4% 

12/41 (29%) 19/41 (46%) 8/46 (17 %) 

567 61 1 728 (T) 

P=O.415 P=O.O35 P=0.159N 

84.4% 

38/50 (76%) 

82.6% 

38/50 (76%) 

91.7 % 

4/50 (88%) 

567 

34/41 (83%) 

61 1 

33/41 (80%) 

497 

42/46 (91 %) 

P=O.O04 P=O.O05 P <0.001 

* Significantly different(PsO.05) from the vehicle control group by the Fisher exact test (interim evaluation)or the logistic regression test 

(2-year study)

** PsO.01 

(T)Terminal sacrifice 
64 Salicylazosulfapyridine, NTP TR 457 

TAIBLE14 

Incidences of Neoplasms and,Nonneoplastic Lesionsof theLiverinMiceinthe2-YearGavageStudy . of Salicylazosulfapyridine(continued) 

mg/kg675Vehicle Control 1,350 mg/kg 2,700 mg/kg 

Female 

15-Month Interim Evaluation 

Number Examined Microscopically 10 10 10 10 

Cytologic Alterations 0 3 6** 8** 

Hepatocellular Adenoma 1 0 1 2 

2-Year Study 

Number Examined Microscopically 50 50 50 49 

Eosinophilic Foci 5 17** 15* 19** 

Hepatocellular Adenoma (Multiple) 

Overall rate 3/50 (6%) 12/50 (24%)** 12/50 (24%)** 13/49 (27%)** 

Hr:patocellular Adenoma (Single and Multiple)' 

Overall rate 12/50 (24%) 28/50 (56%) 25/50 (50%) 28/49 (57%) 

Adjusted rate 29.3% 63.6% 65.7% 71.7% 

Terminal rate 12/41 (29%) 25/41 (61X) 24/37 (65%) 27/38 (71%) 

First incidence (days) (T) 735 539 618 655

Logistic regression test P=O.002 P=O.001 P=O.002 P<O.001 

Hepatocellular Carcinoma (Multiple) 

Overall rate 0150 (0%) 2/50 (4 %) 1/50 (2%) 1/49 (2%) 

Hepatocellular Carcinoma (Single and Multiple)' 

Overall rate 2/50 (4%) 10150 (20%) 10/50 (20%) 9/49 (18%) 

Adjusted rate 4.9% 23.7% 27.0% 23.7% 

Terminal rate 2/41 (5%) 9/41 (22%) 10/37 (27%) 9/38 (24%) 

First incidence (days) 735 (T) 551 735 (T) 735 (T) 

Logistic regression test P=O.O59 P=O.O17 P=O.009 P=O.O19 

Hepatocellular Adenoma or Carcinomak 

Overall rate 14/50 (28%) 32/50 (64%) 28/50 (56%) 29/49 (59%) 

Adjusted rate 34.1% 71.1% 73.6% 74.3% 

Terminal rate 14/41 (34%) 28/41 (68%) 27/37 (73%) 28/38 (74%) 

First incidence (days) 735 (T) 539 618 655 

Logistic regression test . P=O.005 P<O.001 P=O.001 P <0.001 

a Number of animals with lesion 

Historical incidence for 2-year NTP corn oil gavage studies with vehicle control groups (mean f standard deviation): 254/763 (33.3% f 

13.4%). range 14%-58% 

Number of animals with neoplasm per number of animals with liver examined microscopically 

Kaplan-Meier estimated neoplasm incidence at the end of the study after adjustment for intercurrent mortality 

e Observed incidence at terminal kill 

Reneath the vehicle control incidence are the P values associated with the trend test. Beneath the dosed group incidence are the P values 

corresponding to pairwise comparisons between the vehicle controls and that dosed group. The logistic regression test regards neoplasms 

in animals dying prior to terminal kill as nonfatal. A negative trend or a lower incidence in a dose group is indicated by N. 

Historical incidence: 127/763 (16.6% f 4.5%). range 8%-24% 

h Historical incidence: 340/763 (44.6% f 14.6%).range 25%-72% 

j Historical incidence: 99/759 (13.0% f 8.4%).range 2%-28% 

Historical incidence: 38/759 (5.0% f 3.7%).range 0%-14% 

Historical incidence: 131/759 (17.3% f 9.8%),range 4%-37% 
Spleen: The absolute and relative spleen weights of groups of female mice were significantly greater than 

females receiving 1,350 and 2,700 mgkg were signif- that in controls. 

icantly greater than those of controls at the 15-month 

interimevaluation(TableF7).Theincidences of 

hematopoietic cell proliferation and hemosiderin pigment in all dosed groups ofmaleandfemalemice 

were significantly greaterthan those in controls at the 

15-month interim evaluation (Tables 15, C5, and D5). 

Theincidences of hemosiderin pigmentindosed 

groups of maleandfemalemiceattheendofthe 

2-year study were significantly greater than those in 

controls. At theend of the 2-yearstudy,theincidences of hematopoietic cellproliferationindosed 

Hematopoieticcellproliferationwasaminimalto 

moderate change consisting ofa diffuse increase in the 

number of hematopoietic cells within the splenic red 

pulp. Hemosiderin pigment was generally a minimal 

to mild change characterizedby increased numbers of 

macrophages containing numerous prominent, irregular, brown-black, intracytoplasmic granules scattered 

withintheredpulp.Increasedsplenichemosiderin 

pigmentissuggestive of increasederythrocytedestruction (hemolysis). 

Vehicle Control 675 mgkg 1,350 mgkg 2,7OQ mgkg 

IS-Month !Interim Evaluation 

NumberExaminedMicroscopically10 

HematopoieticCellProliferationa 2 

Hemosiderin Pigment 0 

%Year Study 

NumberExaminedMicroscopically 50 

Hematopoietic Cell Proliferation 11 

Hemosiderin Pigment 2 

Female 

15-Month !Interim Evaluation 

NumberExaminedMicroscopically10 

HematopoieticProliferation Cell 2 

Hemosiderin Pigment 0 

%Year Study 

NumberExaminedMicroscopically 50 

Hematopoietic Cell Proliferation 7 

Hemosiderin Pigment 14 

* 

(1 .5)b 

4* 

8* 

10 

(1.0) 

(1.5) 

lo** (1.5) 

lo** (1.6) 

10 

lo** (2.0) 

lo** (2.6) 

10 

(2.0) 

(2.0) 25** 

16(2.4) 

50 

(1.2) 32** (1.4) 

20* (2.5) 

49 

47**(1.6) 

13(1.9) 

50 

(2.5) 

10 

lo** (1.7) 

7* (1.6) 

lo** (2.0) 

lo** (2.4) 

10 

lo** (2.3) 

10 

lo** (2.0) 

(2.4) 

(1.3) 37**(1.6) 

21**(2.5) 

50 

39** (1.5) 

19** (2.5) 

50 

46** (1.8) 

23**(2.2) 

49 

Significantly different (PgO.05) from the vehicle control groupby the Fisher exact test (interim evaluation) or the logistic regression test 

(2-year study)

** PiO.01 

a Numberof animals with lesion 

Average severity grade of lesions in affected animals: 1 =minimal, 2=mild, 3=moderate, 4=marked 
66 Salicylazosulfapyridine,NTP TR 457 

Forestomach: The incidence ofsquamous cell papil- age-related changes encountered in chronic studies, 

loma wassignificantly lower in 2,700 mg/kg females 2)the increased or decreased incidences were marthan in controls (vehicle control, 5/50; 675 mg/kg, ginal and did not have a clear dose-related trend, or 

1/50; 1,350 mg/kg, 1/50;2,700 mg/kg, 0/50; 3)therewasusuallyno similar effect in animals of the 

Table: D3). The incidences of hyperplasia were 

significantly lower in 2,700 mg/kg males (18/50, 

11/50,11/49, 10150; Table C5) and in 1,350 and 

2,700 mg/kg females (12150, 6/49, 4\50,4/49; 

Table DS). -Epithelial (squamous) hyperplasia and 

hyperkeratosis with or without the occurrence of 

squamous cell neoplasms are sometimes encountered 

in gavage studies. Such lesions may be associated 

with irritation or trauma inflicted during the gavage 

procedure. The biological of significancethe

chemical-related lower incidences of forestomach 

lesions in dosed male and female mice is not certain, 

but may be related to the antiinflammatory properties 

of salicylazosulfapyridine. 

Other Organs: The incidences of hemangiosarcoma 

(0150, 2/50, 4/50, 1/49; Table D3) were marginally 

greater in the spleen of dosedfemalemice than in 

controls. The incidences of mineralization of the 

glandular stomach (1/50, 11/50, 9/48, 7/50; 

Table C5) were greater in hosed males than in controls. The incidences of thyroid gland follicle degeneration (1/50, 5/50, 6/50, 10/50) and dilatation in the 

uterus (4/50, 17/50, 11/50, 11/49) in dosed groups of 

females were greater than in controls (Table D5). 

The incidences of dilatation in the seminal vesicle 

(14/50, 2/50, 4/50, 0/50)and thyroid gland follicular 

cell hyperplasia (9/50, 4/50, 3/50, 2/50) were lower 

in dosed males than in controls (TableC5). The incidences of renal tubule casts (13/50, 11/50, 12/50, 

3/50) and regeneration (38/50,40/50, 40/50, 24/50) 

were lower in males administered 2,700 mg/kg than 

in controls (Table C5). The incidence of ductal cysts 

in the preputial gland (45/49, 40/49, 31/49, 38/50; 

Table C5) was lower males in administered 

1,350 mg/kg than in controls. The incidences of 

thyroid gland follicular cell hyperplasia (20/50, 5/50, 

2/50, 1/50; Table D5) were lower in dosed females 

than in controls. The incidence of cysts within 

thyroid gland follicles (6/50, 4/50, 3/50, 0/50; 

Table DS),was lower in femalesadministered 

2,700 mg/kg than in controls. 

The biological significance of theseincidencesis 

uncertain. In general, 1) the lesions represent typical 

other sex. 

GENETICTOXICOLOGY 

Salicylazosulfapyridine (33-6,666 pg/plate) was not 

mutagenic in Salmonella typhimurium strains TA97, 

TA98, TA100, or TA1535 when tested in a preincubation protocol with and without induced rat or 

hamster liver S9 metabolic activation enzymes 

(Table El). Itdidnot induce sister chromatid exchanges (Table E2) or chromosomal aberrations 

(Table E3) in cultured Chinese hamster ovary cells, 

with or without induced rat liver S9 (Bishop et al., 

1990). In contrast to the negative results obtained 

in vitro, results from in vivo genotoxicity tests with 

salicylazosulfapyridine yielded generally positive 

results. 

Salicylazosulfapyridine gave a strongly positive 

response in a micronucleus assay conducted with 

peripheral blood smears obtained from mice used in 

the 13-week toxicity study (Table E4; Bishop et al., 

1990). Significant increases in the frequencies of 

micronucleated polychromatic erythrocytes were 

observed in maleand female mice administered 

2,700 mg salicylazosulfapyridine/kg body weight. 

Significantly elevated micronucleus frequencies were 

also observed in normochromaticerythrocytes in male 

and female mice administered 1,350, 675, or

2,700 mg/kg. A dose-related increase in the percentage of polychromatic erythrocytes was also observed 

in all dosed groups of males and females, indicating 

that erythropoiesis was stimulated in animals dosed 

with salicylazosulfapyridine. 

Salicylazosulfapyridine was subsequently tested for 

induction of chromosomal aberrationsin bone marrow 

cells of male mice following single gavage administrations of 250 to 1,000 mg/kg (Table E5; Bishopet al., 

1990). These doses, selected on the basis of results 

from a preliminary range-finding study, were less than 

all but the lowest dose tested in the 13-week study. 

No significant increases in chromosomal aberrations 

were noted in either of the two trials conducted. 
67 

Subsequent micronucleus tests withsalicylazosulfa- aberrations in nonstandard protocols that were depyridine confirmed the positive response observed in signed to measure the influence of treatment schedthe initial 13-week peripheral blood test. A small but ules. Table E8 presents results from a series of bone 

significant increase in polychromatic erythrocytes wasmarrow micronucleus tests performed with male and 

observed in the bone marrow of male mice administered 250 to 2,OOO mg salicylazosulfapyridine/kgbody 

weight in corn oil by gavage, twice at 24-hour intervals; sampling was performed 24 hours after administration of the second dose (Table E6; Bishop et al., 

1990). 

A specialized micronucleus test was performed with 

salicylazosulfapyridine to investigate the aneuploidyinducing potential of the compound, implied by the 

positive results from micronuclei assays (which detect 

both numerical and structural chromosomal aberrations) and the negative results from the in vitro and 

in vivo chromosomal aberrations assays (which detect 

only structural damage). In this micronucleus/ 

kinetochore-stainingassay (Table E7; Witt et al., 

1992b), salicylazosulfapyridine induced dose-related 

increases in the number oftotalmicronucleated 

polychromatic erythrocytes; in addition, the micronuclei that were induced were predominantly kinetochore-positive (contained a fluorescent signal indicating the presence of a kinetochore and presumably, 

therefore, an entire chromosome rather than a fragment). Salicylazosulfapyridine also induced a significant increase in kinetochore-negative micronuclei, but 

only at the highest dose tested in the second trial; the 

majority of induced micronuclei stained positive for 

the presence of a kinetochore. Theseresultsimply 

that salicylazosulfapyridine produces aneuploidogenic 

rather than clastogenic chromosomal effects. 

Becausepositiveresultswereobservedinmicronucleus experiments that employed multiple dosing 

protocols (standard for a micronucleus assay), and 

because the single-dosechromosomal aberrations 

protocol was negative, salicylazosulfapyridine was 

tested for induction of micronuclei and chromosomal 

female mice. Significant increases in micronucleated 

polychromatic erythrocytes were observed in each of 

two trials in which femalemice were dosed with 

salicylazosulfapyridine three times by gavage. Male 

mice were administered salicylazosulfapyridine either 

by single (Trial 1) or triple (Trial 2) gavage protocols. 

Results of the first experiment were negative, whereas 

results obtained in the second trial using the tripletreatment protocol were positive, thus underscoring 

the relationship between dosing schedule and detection 

of a genotoxic response. 

Salicylazosulfapyridine was further tested for induction of chromosomal aberrations in a nonstandard, 

triple-treatment protocol that used three gavage 

administrations separated by 24-hour intervals in an 

attempt to mimic the same exposure environment that 

produced the positive micronucleus test results. The 

highest dose used in this experiment was

4,000 mg/kg. Again, no induction of chromosomal 

aberrations by salicylazosulfapyridine was notedin 

mouse bone marrow cells (Table E9). These results 

further support the hypothesis that salicylazosulfapyridine is aneuploidogenic. 

Because the carcinogenicity of salicylazosulfapyridine 

wasbeingevaluatedbythe NTP in both rats and 

mice, a micronucleus test was also performed in male 

rats using the triple-treatment protocol (Table E10). 

The overall results were considered to be equivocal 

because the weakly positive response obtained in the 

first trial with doses as great as 2,700 mgkg did not 

repeat in thesecond trial when doses as great as 

3,000 mgkg were tested. Thus, there appears to be 

a species difference in response to bone marrow 

erythrocyte micronucleus induction by salicylazosulfapyridine. 
(arrows). H&E; 6.5 x 

papillomas (*) and variable mucosal (transitional) epithelial hyperplasia 

salicylazosulfapyridine in corn oil by gavage for 2 years. Multiple epithelium forming papillary fronds supported by fibrous stroma. H&E; 13X 

Uriniry bladder from a male F344/N rat administered 337.5 mglkg Higher magnification of Plate 1. Papillomas consist of hyperplastic mucosal 

mATE 1 mATE 2 

base of the papilloma (arrows). H&E; 52 x 

fibrovascular stroma. Note hyperplastic mucosal epithelium adjacent to the 

papilloma showing hyperplastic epithelium within the neoplasm supported by hyperplasia; note folding of the mucosa. H&E; 52 X 

salicylazosulfapyridine in corn oil by gavage for 2 years. Details of a salicylazosulfapyridine in corn oil by gavage for 2 years. Moderate mucosal 

Urinary bladder from a male F344/N rat administered 337.5 mglkg Urinary bladder from a male F344/N rat administered 337.5 mgkg 

mATE 3 mTE 4 
into the lumen. H&E; 25 X 

hyperplasia; the hyperplastic epithelium forms thick complex folds that project F344lN rat administered 337.5 mglkg salicylazosulfapyridine in corn oil by 

salicylazosulfapyridine in corn oil by gavage for 2 years. Marked mucosal Multiple concretions (arrows) occlude the lumen of the urethra of a male 

Urinary bladder from a male F344lN rat administered 337.5 mglkg Example of concretions observed in the urinary bladder, ureter and urethra. 

mATE 5 EATE 6 

gavage for 2 years. H&E; 26 x 

transitional epithelium (arrows). H&E; 13 X 

with concretions'(C) in the lumen and incorporated within hyperplastic papillary thickening of the transitional epithelium of the renal pelvis. 

salicylazosulfapyridine in corn oil by gavage for 2 years. Renal pelvis (P) salicylazosulfapyridine in corn oil by gavage for 2 years. Nodular and 

Kidney from a male F344/N rat administered 337.5 mglkg Kidney from a male F344/N rat administered 337.5 mglkg 

UTE 7 mATE 8 

H&E; 104x 
projecting into the renal pelvis. H&E; 52 x 

transitional hyperplasia consist of a nodule of polyhedral to spindle cells necrosis; note lack of cellular detail, pyknotic nuclei and cellular debris. 

salicylazosulfapyridine in corn oil by gavage for 2 years. Focal atypical salicylazosulfapyridine in corn oil by gavage for 2 years. Renal papillary 

Kidney from a male F344/N rat administered 337.5 mg/kg Kidney from a male F344/N rat administered 337.5 mglkg 

PLATE 9 KATE 10 

H&E; 20X 
Salicylazosulfapyridine, a drug widely usedinthe werealsoevidentin 337.5 mg/kg male rats in the 

treatment of ulcerative colitis, has complex biological 13-week study. There were minor decreases in thyactivities when it is cleaved into 5-aminosalicylic acid roxine concentration in all groups of dosed'male and 

(anti-inflammatory) and sulfapyridine (antimicrobial). female mice ip the13-week study. However, there 

Salicylazosulfapyridine was nominated for toxicity andwere no differences in triiodothyronine and thyroidcarcinogenicity study by the National Cancer Institute stimulatinghormone concentrations between dosed 

based on its widespread use and because it is a repre- and control animals, suggesting the thyroxinechanges 

sentative of the aryl sulfonamide class. werenotbiologically relevant. Sulfonamides are 

known goitrogens which act by inhibiting thyroid 

The 16-day and 13-week studies were conducted to peroxidase, an enzyme required for the iodination of 

characterize the toxic effects of salicylazosulfapyridinethyroglobulin during thyroid hormone biosynthesis 

in F344/N rats and B6C3F, mice, to identify potential 

target organs, and to estimate dose ranges that would 

be compatible with growth and survival in longer term 

studies. In the 16-day studies, salicylazosulfapyridine 

was administered (0, 675, 1,350, and 2,700 mg 

salicylazosulfapyridine/kg)in corn oil by gavage to 

groups of five male and five female F344/N rats and 

B6C3F, mice. Final mean body weights of dosed rats 

ranged from 5 % to 32% less than those of controls 

while final mean body weights and weight gains of 

dosed mice were generally similar to those of controls. Chemical-related gross lesions included enlarged, discolored thyroid glands in male and female 

rats and enlarged cecae/large intestines in male and 

female rats and inmale mice. Microscopiclesions 

were present in the thymus in male and female rats 

given 2,700 mg/kg and in the thyroid and pituitary 

glands in male and female rats receiving 1,350 or 

2,700 mgkg. Rat serum triiodothyronine and thyroxine concentrations were decreased ina dosedependent 

manner and this wasmost pronounced in males. 

(Capen et al., 1991). Rats appear to be especially 

susceptible to this effect but the mouse also is sensitive to the goitrogenic effects of sulfonamides (Capen 

et al., 1991). Thyroid sensitivity in F344 rats but not 

B6C3F, mice fed sulfamethazine, an analogue of salicylazosulfapyridine, has been reported (Heath and 

Littlefield, 1984). 

In the 13-weekmouse study, there was marginal 

evidence of a responsive anemia. The anemia was 

probably related to a methemoglobinemia as exhibited 

infemalemice given 1,350 or2,700 mg/kg. Increased erythrocyte methemoglobin concentrations 

cause increased redcell turnover. The anemia that 

results then stimulates an erythropoietic response 

resultinginreticulocytosis. Sulfonamides probably 

cause methemoglobinemia indirectly, through metabolites, since they do not cause increased methemoglobin formation in vitro (Nelson, 1979). Activation 

of salicylazosulfapyridine to the sulfapyridine metabolite by liver microsomes has been shownto cause metConsidered together, the clinical chemistry and micro-hemoglobinemia in vitro (Pirmohamed et al., 1991). 

scopic evaluations revealed evidence indicative of Interestingly, pharmacokinetic studies indicated much 

chemical-inducedincreasedactivity of the thyroid higher maximum plasma concentrations of sulfapyrigland. dine in mice than in rats following oral administration 

of salicylazosulfapyridine (NTP, 1994a; Appendix L). 

Subsequent investigations were conducted to charac- The maximum plasma concentrations of sulfapyridine 

terize the toxic effects of repeated exposures of in female mice were equal to, or greater than, those 

salicylazosulfapyridine in rats and mice with emphasisfor male mice at the same dose. The area under the 

on morphologic and functional alterations of the concentration-time curve was greater for female mice 

pituitary-thyroid axis. In rats, the decreased concen- compared to males for all doses. Thus, female mice 

trations of triiodothyronine and thyroxine evident in were exposed to higher concentrations of sulfapyri2,700 mgkg male and female rats in the 16-day study dine for a longer period of time than male mice, 
70 Salicylazosulfapyridine,NTP TR 457 

possibly explaining the observed gender difference in 

methemoglobin. 

There were minimal increases in alanine aminotransferase and alkaline phosphatase activities in 1,350 and 

2,700 mg/kg male mice, and these changes may be 

There were several reasons thyroid gland neoplasms 

were expected to occur in the 2-year rat study. These 

included: 1) the perturbation of serum hormone concentrations indicative of pituitary gland disturbances 

(thyroid-stimulating hormone, triiodothyronine, and 

thyroxine); 2) histologic evidence of thyroid gland 

consistent with the centrilobular hypertrophy observedfollicular cell hyperplasia; and 3) the .reported goitromicroscopically in the liver. 

The significance of the ,chemical-related decreases 

in !;perm motility in all dosed groups of male rats in 

the 13-week study is unclear. Nevertheless,, these 

results are consistent with those of Pholpramool and 

Srikhao (1983) who noted slightly decreased sperm 

motility in Fischer rats receiving up to 600 mg salicylazosulfapyridine/kg body weight by gavage. Other 

investigators have shown that when male SpragueDawley rats were fed salicylazosulfapyridine at doses 

up to 617 mg/kg per day for 8 weeks and then mated 

to virgin females, there were dose-dependent declines 

in fertility accompanied by significant reductions in 

litter sizes. Male rats fed the metabolite sulfapyridine 

also had reduced litter sizes when mated to untreated 

virgins, while those fed the metabolite 5-aminosalicylic acid sired litters of comparable size to those of 

control rats (O’Moriin et al., 1984). Similarly, NTPsponsored reproductive assessment of salicylazosulfapyridine using continuous breeding protocols found 

decreased male fertility in the absence of changes in 

spe:rm motility at doses up to 400 mg/kg (corn oil 

gavage) in Sprague-Dawley rats (EHRT, 1992). The 

different outcomes in these studies may be the result 

of strain differences. An evaluation of sperm quality 

(density, motility, and morphology) in time-course 

studies indicated that the metabolite sulfapyridinemay 

be directly toxic to developing spermatozoa in humans 

(Toovey et al., 1981). 

Doses selected for the 2-year study in male and 

female rats (0, 84, 168, and 337.5 mg/kg by corn oil 

gavage) were based on the results of the13-week 

study. It was anticipated that the high dose 

(337.5 mg/kg) would cause marginal toxicity as 

suggested by the lower body weightgainsand the 

thyroid gland effects observed in the 16-day and 

13-week studies, and that 84 and 168 mg/kg would 

probably encompass no-adverse-effect-levels with 

respect to toxicity. 

genic and carcinogenic activities of sulfonamides in 

general, and of the structural analogues of salicylazosulfapyridine in particular. Consequently, male rats 

that appeared to be the most sensitive in the 13-week 

study were given 337.5 mg salicylazosulfapyridine/kg 

body weight for 6 months, after which animals 

received only the corn oil vehicle for the remainder of 

the 2-year study. In this way the progression or 

regression of thyroid gland lesions in the absence of 

a sustained exposure could be evaluated. 

The thyroid gland hyperplasia seen in the 13-week 

study was not observed in the 2-year study, and there 

wasno evidence of chemical-related thyroid gland 

follicular cell adenomas or carcinomas. While follicular cell neoplasms of the thyroid gland are rare in 

untreated F344 rats, they are readily induced by 

chemicals with goitrogenic activity; this is seen as a 

secondary result of an increase in thyroid-stimulating 

hormone by goitrogens (Capen, 1997). In the present 

studies, hypothyroidism (decreased triiodothyronine 

and thyroxine concentrations) was evidentin the male 

and female rats receiving 2,700 mg/kg in the 16-day 

study but was only evident in the 337.5 mg/kg male 

rats in the .13-week study. At the 1 5-month evaluation, .there was no biochemical evidence of hypothyroidism indicating that the thyroid gland effects in 

rats were transient; the rats may have adapted to the 

mildly goitrogenic doses used in this study. Carcinogenic activities of structurally related compounds have 

proved to be inconsistent. A related para-amino aryl 

sulfonamide, sulfamethoxazole, produced thyroid 

gland tumors in rats (Swarm et al., 1973). In contrast, another structural analogue, sulfisoxazole, was 

not carcinogenic in F344/N rats or in B6C3F, mice 

(NCI, 1979). When sulfamethoxazole was fed to rats 

for 52 or 60 weeks, it induced thyroid gland adenomas with evidence of lung metastasis. However, 

withdrawal of the drug from the diet after 6 or 

13 weeks yielded normal thyroid gland follicle structure (Swarm et al., 1973). 
S;aliCyp~QSMPh~yyrridine,N p TR 457 71 

The principal adverse effect of salicylazosulfapyridine 

in rats in the 2-year study was transitional epithelial 

papilloma of the urinary tract, which wasaccompanied by increased incidences of concretions (diagnosed as calculi at necropsy). There was a significant 

(P <0.01) positive association between the presence of 

grossly diagnosed concretions (calculi) and the development of transitional epithelial papillomas of the 

urinary bladder in male rats. The observed concretions were considered to be precipitated salicylazosulfapyridine or a metabolite, and they were present in 

all animals with urinary tract papillomas as well.as in 

some rats that did not develop papillomas. However, 

the animals without papillomas invariably had mild to 

moderate mucosa (transitional) hyperplasia, which 

may be a precursor to epithelial neoplasms of the urinary tract. The association between calculi and papillomas of the urinary bladder has been observed in 

other studies (Okumura et al., 1992; NTP, 1983b, 

1986) and supports the contention that urinary calculi/ 

concretions observed in the present studies may have 

influenced the development of papillomas. The 

mechanical irritation caused by calculikoncretions 

may have stimulated epithelial cell proliferation and 

subsequent neoplasm development. 

Core-study male rats showed a significant, doserelated increase in the incidence of urinary bladder 

transitional epithelial papillomas as well as a corresponding increase in the incidence of mucosa hyperplasia. The increased neoplasm incidence was considered to be chemical related. The stop-exposure male 

rats had no urinary bladder neoplasms or concretions/ 

calculi, which further suggests that these lesions may 

be correlated. Transitional epithelial papillomas of 

the kidney were not observed in male rats, but there 

were increased incidences of transitional epithelial 

hyperplasia and atypical transitional epithelial hyperplasia that were dose related. 

In female rats, two 168 mg/kg animals had urinary 

bladder transitional epithelial papillomas, and the increased incidence of mucosa hyperplasia was significant and dose related. Two 337.5 mgkg female rats 

had transitional cell papillomas of the kidney, and the 

increased incidences of transitional epithelial hyperplasia were significant and increased with increasing 

dose. Transitional epithelial papillomas of the kidney 

are rare in control female rats and hadnotbeen 

observed in 920 corn oil gavage controls in previous 

NTP studies. Moreover, these neoplasms were not 

found in 1,977 untreated or in 1,944 corn oil gavage 

control female F344/N rats (Haseman et al., 1990). 

Because of the rarity of these neoplasms, these two 

neoplasms (and the two urinary bladder papillomas 

with a similar morphology) were considered to be 

chemical related. 

The incidence of mononuclear cell leukemia in male 

rats receiving 337.5 mgkg was significantly lower 

than that in controls. There were also concurrent 

increases in the incidences of splenic hematopoietic 

cell proliferation and hemosiderin pigmentation in 

337.5 mgkg male rats. The biological significance of 

these effects is uncertain. However, in a number of 

NTP studies, chemicals that have resulted in a significant reduction in the incidence of mononuclear cell 

leukemiahave frequently been associated with 

changes suggestive ofhematopoietictoxicity at 

13 weeks, including increased incidences of splenic 

hematopoietic cell proliferation, hemosiderin pigmentation, and fibrosis (NTP, 1982, 1989, 1994b, 1996). 

This association has not been observed in all studies 

in which the incidence of mononuclear cell leukemia 

in treated groups is lower -than in controls (NTP, 

1987, 1992). 

The doses selected for the 2-year study in male and 

female mice were 0, 675, 1,350, or2,700 mg/kg 

salicylazosulfapyridine, based on the absence. ,of 

chemical-related mortality, body weight effects, and 

lesionsinthe13-week study. Inthe 2-year study, 

salicylazosulfapyridineinduced liver neoplasms in 

mice. The incidences of hepatocellular adenoma in all 

dosed groups of male and female mice were at least 

doublethosein controls, significantly greater than 

those in controls by logistic regression analysis, and 

occurred with positive trends., Multiple hepatocellular 

adenomas occurred in dosed males and females more 

frequently than in controls. The incidences of hepatocellular adenoma inmalemice receiving 675 and 

2,700 mgkg and in all groups of dosed female mice 

exceededthehistorical control ranges from NTP 

2-year corn oil gavage studies. The incidences of 

hepatocellular carcinoma in 1,350 mg/kg male mice 

and all dosed groups of female mice were significantly 

greater than those in controls, and multiple hepatocellular carcinomas were also evident in dosed male 

andfemalemice. The incidences of hepatocellular 

carcinoma in 675 and 1,350 mg/kg male mice and all 
72 Salicylazosulfapyridine, NTP TR 457 

dosed groups of female mice exceeded the ranges in study ranged from 90 to 260 pM (23 to 65 pg/mL) in 

historical controls from NTP 2-year corn oil gavage malesand 110 to 484 p M '(28 to 120 pg/mL) in 

studies. Collectively,' the combinedincidences of females. Again, the plasma concentrations attained in 

ade.noma or carcinoma in both male and female dosed the mouse study. are similar to human therapeutic 

groups were statistically greater than those in controls.plasma concentrations. 

Considered together, these data provide clear evidence 

that the increased incidences of liver neoplasms were Toxicokinetic studies in male F344/N rats indicated 

related to the administration of salicylazosulfapyri- that salicylazosulfapyridine was less well absorbed by 

dine At the end of the 2-year study, incidences of rats than by mice: At an equivalent dose(675 mg/kg) 

eosinophilic foci in dosed groups of males and femalesthe highest plasma concentrations of salicylazosulfawere greater than those in controls. pyridine and sulfapyridine in rats were approximately 

20% and 30% of those in mice, respectively (NTP, 

The incidences of hemosiderin pigment and hemato- 1994a). Pharmacokinetic measurements following 

poietic cell proliferation in,all dosed groups of male intravenous administration .indicated, that rats would 

and female mice were greater than those in the con- clear salicylazosulfapyridine more slowly than mice 

trols at the 15-month interim evaluation and at the end(Appendix L). Thus, peak exposure may be less in 

of the 2-year study, and the increases were generally 

significant. 

In 2,700 mg/kg female mice, the incidence of squamous cell papilloma of the forestomach was significantly lower than that in controls. The incidences of 

forestomach hyperplasia were significantly lower than 

-those in controls in 2,700 mg/kg malesand in 

1,350 and 2,700 mg/kg females. The biological significance of the lower incidences of the forestomach 

lesions in dosed male and female mice is not certain, 

but may be related to the anti-inflammatory properties 

of salicylazosulfapyridine. 

Results from the toxicokinetic studies in F344/N rats 

and B6C3Fl mice allow a comparison of the plasma 

concentrations of salicylazosulfapyridine and its 

metaboliteswith those attained in humans at therapeutic dose concentrations. Following a 4 g dose of 

sa1:icylazosulfapyridine to human male volunteers 

(approximately 50 mg/kg), the medianmaximum 

serum concentration of salicylazosulfapyridine was 

21 pg/mL (53 pM) andthemaximummean serum 

concentration of sulfapyridine plus its metabolites was 

33 A- 9 pg/mL (Schroder and Campbell, 1972). In a 

B6C3Fl mouse toxicokinetic study (NTP, 1994a; 

Appendix L), maximum plasma concentrations of 

salicylazosulfapyridine ranged from 16 to 42 p M in 

males and 40 to 212 pM infemales given doses 

ranging from 67.5 to 2,700 mg/kg. Thus, except for 

the highest doses in females, plasma concentrations of 

salicylazosulfapyridine were comparable to those 

observed in humans. The maximum plasma concentration of sulfapyridine in themousetoxicokinetic 

rats, but the duration of exposure may be longer than 

in mice. This observation may explain in part why 

mice did not accumulate salicylazosulfapyridine precipitate in the urinary tract even though mice were 

given an 8-fold greater dose than rats. 

There have been a limited number, ofin vitro studies 

conductedwith salicylazosulfapyridine and/or its 

metabolites, and in general, the results have indicated 

that none of these compounds are mutagenic in vitro 

(Voogd et al., 1980; Zeiger et al., 1988; Bishop 

et al., 1990; Witt et al., 1992b). The exception was 

an in vitro study with human lymphocytes that showed 

increased frequencies of micronuclei, an indication of 

chromosomal damage, after treatment with salicylazosulfapyridine (Mackay et al., 1989). Results from 

in vivo studies further suggest that salicylazosulfapyridine can induce chromosomal damage, perhaps in the 

form of aneuploidy, in mammalian cells. Chronic 

exposure to therapeutic concentrations of salicylazosulfapyridine was associated with increases in micronuclei and sister chromatid exchanges in peripheral 

blood lymphocytes of ulcerative colitis patients (Fox 

et al., 1987). Several studies have shown induction of 

micronuclei in mouse bone marrow following multiple 

treatments with high doses of salicylazosulfapyridine 

(Bishop et al., 1990; Witt et al., 1992a). A bone 

marrow micronucleus test using the same protocol in 

rats and micegave negative results in both cases 

(Appendix E). The plasma concentrations of salicylazosulfapyridine and sulfapyridine in F344 rats were 

shown to be 20% to 30% of that attained in mice 

following administration of comparable doses of salicylazosulfapyridine (NTP, 1994a). Although multiple 
73 

dosing withsalicylazosulfapyridine, compared with a related to the mutagenicity observed in miceand 

single dose administration, did not lead to increased humans (Zheng et al., 1993). 

plasma concentrations of salicylazosulfapyridinein 

B6C3Fi mice, the peak (CmJ and total (AUC) concentrations of sulfapyridine were increased by multiple dosing (Zheng et al., 1993). In vivo and in vitro 

test results indicated that the genotoxic moiety of the 

salicylazosulfapyridine molecule is primarily the 

sulfapyridine subunit; 5-aminosalicylic acid has been 

shown not to induce genetic damage. Therefore, the 

data from mouse and rat in vivo studies show that the 

mouse is more susceptible to salicylazosulfapyridineinduced genetic damage, and the results are consistent 

with the observation that mice have greater systemic 

exposures to sulfapyridine. 

The evidence from clinical studies. indicates that the 

increases in the frequency of sister chromatid exchanges and micronuclei in patientsmaintained on 

salicylazosulfapyridine therapy for treatment of 

inflammatory bowel disease are caused by the drug 

per se andnotbythe disease (Fox et al., 1987; 

Mackay et al., 1989). In these clinical studies, the 

dose of salicylazosulfapyridine and the length of treatment to a large extent determined the magnitudeof the 

observed genetic damage. The high sulfapyridine 

plasma concentrations resulting from'salicylazosulfapyridine administration in mice supports the assumption that sulfapyridine and/or its metabolites(s) may be 

C'oNJeLusn'oNs 

Under the conditions of these 2-year gavage studies, 

there was some evidence of carcinogenic activity* of 

salicylazosulfapyridine in maleand female F344/N 

rats based on increased incidences of neoplasms in the 

urinary tract. There was an increased incidence of 

transitional epithelial papilloma of theurinary bladder 

in males and a low incidenceof rare transitional 

epithelial papillomas of the kidney and of the urinary 

bladder in females. There was clearevidence of 

carcinogenicactivity of salicylazosulfapyridine in 

maleandfemale B6C3F, mice based on increased 

incidences of hepatocellular neoplasms. 

Increased incidences of nonneoplastic lesions of the 

urinary bladder and kidney in male and female rats 

andofthespleeninmale rats were observed. Increasedincidences of nonneoplastic lesions of the 

liver 'and spleen in maleand 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. 

* Explanation of Levels of Evidence of Carcinogenic Activity is on page 11. A summary of the Technical Reports Review Subcommittee 

comments and the public discussion on this Technical Report appears on page 13. 

~ ~~ .. ..L ~ . -~.. .
74 Salicylazosulfapyridine, NTP TR 457 
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Vender, R.J., and Spiro, H.M. (1982). Inflammatory 

bowel disease and pregnancy. J. Clin. Gastroenterol. 

4, 231-249. 

Vilaseca, J., Tor, J., Puig Costa, M., Ribera, A., 

Martin, C., and Guardia, J. ,( 1980). Chronic ulcerative colitis and autoimmune hemolytic anemia. Med. 

Clin. (Barc.) 75, 126-128. 

Voogd, C.E., Van der Stel, J.J., and 

Jacobs, J.J.J.A.A. (1980). On the mutagenic action 

someof enzyme immunoassay substrates. J. 

Immunol. Methoh 36,55-61. 
83 

r 

Wilkieson, C.A., Madhok, R., Hunter, J.A., and Witt, K.L., Gudi, R., and Bishop, J.B. (1992b). 

Capell, H.A. (1993). Toleration, side-effects and Induction of kinetochore positive and negative microefficacy of sulphasalazine in rheumatoid arthritis pa- nucleiinmousebone marrow cells by salicylazotients of different ages. Quart. J. Med. 86, 501-505. sulfapyridine and sulfapyridine. Mutat. Res. 283, 

53-57. 

Williams, D.A. (1971). A Zeiger, E., Anderson, B., Haworth, S., Lawlor, T., test for differences 

and Mortelmans, K. (1988). Salmonella mutagenicity between treatment means when several dose levels are 

compared with a zero dose control. Biometrics 27, 

103-1 17. 

Williams, D.A. (1972). The comparison of several 

dose levels with a zero dose control. Biometrics 28, 

5 19-53 1. 

Witt, K.L., Bishop, J.B., McFee, A.F., and 

Kumaroo, V. (1992a). Induction of chromosomal 

damage in mammalian cells in vitro and in vivo by 

sulfapyridine or 5-aminosalicylic acid. Mutat. Res. 

283,59-64. 

tests: IV. Results from the testing of 300 chemicals. 

Environ. Mol. Mutagen. Ill (Suppl. 12), 1-158. 

Zeiger, E., Haseman, J.K., Shelby, M.D., 

Margolin, B.H., and Tennant, R.W. (1990). 

Evaluation of four in vitro genetic toxicity tests for 

predicting rodent carcinogenicity: Confirmation of 

earlier results with 41 additional chemicals. Environ. 

Mol. Mutagen. I6(Suppl. 18), 1-14. 

Zheng, W., Winter, S.M., Mayersohn, M., 

Bishop,J.B.,andSipes, I.G. (1993). Toxicokinetics 

ofsulfasalazine (salicylazosulfapyridine) and its 

metabolitesin B6C3FI mice. Drug Metab. Dispos. 

21, 1091-1097. 
Salicylazosulfapyridine, NTP TR 457 
OF 
86 Salicylazosulfapyridine, NTP TR 457 

Disposition Summary 

Animals initially in study 70 60 60 

6-Monthinterim evaluation 10 

IS-Month interim evaluation 10 10 10 

Early deaths 

Accidental deaths 1 4 1 

Moribund 13 11 16 

Natural deaths 1 2 2 

Survivors 

Died last week of study 1 

Terminal sacrifice 35 33 30 

Animalsmicroscopically exanlined 70 60 60

Systems Examined at 6 Months With No Neoplasms Observed 

Alimentary System 

Cardiovascular System 

Endocrine System 

General Body System 

Genital System 

Hematopoietic System 

Integumentary System 

Musculoskeletal System 

Nervous System 

Respiratory System 

Special Senses System 

Urinary System 

15-Month Interim Evaluation 

Alimentary System 

Intestine large:, colon (10) (9) (10)

Carcinoma 1 (10%) 

Liver (10) (10) (10)

Hepatocellular adenoma 1 (10%) 

Endocrine System 

Adrenal medulla (10)

Pheochromocytoma benign 

Islets, pancreatic (10)

Adenoma 

Pituitary gland (10) (10) (10)

Pars distalis, adenoma 1 (10%) 

Genital System 

Preputial gland 

Adenoma 

3 (30%) 

60 

10 

2 

15 

10 

23 

60 

(10) 

(10) 

(10) 

70 

10 

10 

3 

13 

4 

30 

70 

I . 

(10) 

1 (10%) 

(10) 

1 (10%) 

(9)

2 (22%) 
Vehicle Control 84 m@!z 168 mgkg 337.5 mdkg 

(StoplExposure) 

337.5 mgkg 

~~~~ 

8s-MonEh hE@dm&~U&~OPS (continued) 

Genital System(continued) 

Testes 

Bilateral, interstitial cell, adenoma 

Interstitial cell, adenoma 

(10) 

6 (60%) 

3 (30%) 

(10) 

6 (60%) 

1 (10%) 

(10) 

6 (60%) 

4 (40%) 

(10) 

9 (90%) 

1 (10%) 

(10) 

3 (30%) 

6 (60%) 

Respiratory System 

Lung 

Alveolar/bronchiolar carcinoma 

(10)

1 (10%) 

(10) (10) (10) (10) 

Cardiovascular System 

Systems Examined With No Neophms Observed .. 

General Body System 

Hematopoietic System 

Integumentary System 

Musculoskeletal System 

Nervous System 

Special Senses System 

Urinary System 

2-Y@ar$E” 

Alimentary System 

Intestine large, cecum 

Intestine small, duodenum 

Carcinoma 

Intestine small, jejunum 

Intestine small, ileum 

Leiomyosarcoma 

Liver 

Fibrous histiocytoma, metastatic, ski 

Hepatocellular carcinoma 

, Hepatocellular adenoma 

Histiocytic sarcoma 

Leiomyosarcoma, metastatic, stomach, 

forestomach 

Osteosarcoma, metastatic, bone 

Osteosarcoma; metastatic, tissue NOS 

Mesentery 

Fibrous histiocytoma 

Leiomyosarcoma, metastatic, stomach, 

forestomach 

Osteosarcoma, metastatic, tissue NOS 

Pancreas 

Fibrous histiocytoma 

Leiomyosarcoma, metastatic, stomach, 

forestomach 

Mixed tumor benign 

Acinar cell, carcinoma, multiple 

Acinar cell, adenoma, multiple 

Acinar cell, adenoma 

2 (4%) 

2 (4%) 
88 Salicylazosulfapyridine,NTP TR 457 

2-Year Study (continued) 

Alimentary System(continued) 

Pharynx 

Squamous cell carcinoma 

Squamous cell papilloma 

Salivary glands 

Adenoma 

Schwannorna malignant, metastatic, 

skin 

Stomach, forestomach 

Leiomyosarcoma 

Squamous cell carcinoma 

Squamous cell papilloma 

Stomach, glandular 

Tongue 

Squamous cell carcinoma 

Cardiovascular System 

Blood vessel 

Vena cava, chordoma, metastatic, 

spinal cord 

Heart 

Schwannorna benign 

Schwannorna malignant 

. Endocrine System 

Adrenal cortcx 

Adenoma 

Histiocytic sarcoma 

Adrenal medulla 

Pheochromocytoma malignant 

Pheochromocytoma complex 

Pheochromocytoma benign 

Bilateral, pheochromocytoma benign 

Islets, pancre:atic 

Adenoma 

Carcinoma 

Pituitary gland 

Ganglioneuroma 

Pars distalis, adenoma 

Pars internledia, adenoma 

Thyroid gland 

Bilateral, C-cell, carcinoma 

C-cell, adenoma 

C-cell, carcinoma 

Follicular cell, adenoma 

Follicular cell, carcinoma 

(50) 

(50) 

1 (2%) 

1 (2%) 

(49) 

(50) 

(50) 

3 (6%) 

2 (4%) 

4 '(8%) 

1 (2%) 

(50) 

(50) 

(50) 

(50) 

1 (2%) 

6 (12%) 

I (2%) 

(50) 

3 (6%) 

1 (2%) 

(50) 

16 (32%) 

(50) 

1 (2%) 

9 (18%) 

1(2%) 

(50) 

1 (2%) 

(50) 

1 (2%) 

(50) 

1 (2%) 

(50) 

1(2%) 

5 (10%) 

1(2%) 

(50) 

6 (12%) 

(49). 

18 (37%) 

1 (2%) 

(50) 

5 (10%) 

2 (4%) 

3 (6%) 

1 (2%) 

(50) 

(50) 

2 (4%) 

(49) 

7 (14%) 

1 (2%) 

(50) 

5 (10%) 

(49) 

18 (37%) 

(50) 

6 (12%) 

1 (2%) 

1 (2%) 

.I,' , . I 

(50) 

1 (2%) 

(50) 

2 (4%) 

(50) 

2 (4%) 

3 (6%) 

1 (2%) 

(49) 

(49) 

18 (37%) 

(50) 

4 (8%) 

2 (4%) 

2 (4%) 
2-y&ZP %Udy (continued) 

General Body System 

Tissue NOS 

Osteosarcoma 

Genital §ystem 

Epididymis 

Preputial gland 

Adenoma 

Carcinoma 

Prostate 

Adenoma 

Seminal vesicle 

Testes 

Bilateral, interstitial cell, adenoma 

Interstitial cell, adenoma 

Hematopoietic §ystem 

Bone marrow 

Histiocytic sarcoma 

Lymph node 

Inguinal, fibrous histiocytoma,

metastatic, skin 

Mediastinal, osteosarcoma, metastatic, 

tissue NOS 

Pancreatic, histiocytic sarcoma 

Lymph node, mandibular 

Histiocytic sarcoma 

Lymph node, mesenteric 

Spleen 

Hemangiosarcoma 

Histiocytic sarcoma 

Leiomyosarcoma, metastatic, stomach, 

forestomach 

Osteosarcoma, metastatic, tissue NOS 

Thymus 

Thymoma benign 

Integumentary System 

Mammary gland

Carcinoma 

Fibroadenoma 

Fibroadenoma, multiple 

(1) (1) 

1 (100%) 

(49) (49) 

148) (49) (47) (49) (49) 

1 (2%) 

3 (6%) 4 (8%) 4 (9%) 3 (6%) 4 (8%) 

1 (2%) 
90 Salicylazosulfapyridine,NTP TR 457 

2-Ye'ar Sttudy (continued) 

htegumentary System (continued)

Skin 

Basal cell adenoma 

Basal cell carcinoma 

Keratoacanthoma 

Squamous cell papilloma 

Trichoepithelioma 

Subcutaneous tissue, fibroma 

Subcutaneous tissue, fibrosarcoma 

Subcutaneous tissue, fibrous histiocytoma 

Subcutaneous tissue, lipoma 

Subcutaneous tissue, liposarcoya 

Subcutaneous tissue, schwannoma benign 

Subcutaneous tissue, 

schwannoma malignant 

Musculoskeletal System 

Bone 

Hamartoma 

Osteosarcoma 

Skeletal muscle 

Fibrous histiocytoma 

Hemangio.;6 arcoma 

Histiocytic sarcoma 

Osteosarcoma, metastatic, tissue NOS 

Nervous System 

Brain 

Astrocytoma malignant 

Glioma malignant 

Meningioma benign 

Spinal cord 

Respiratory System 

Lung 

Alveo1arlb:ronchiolar adenoma 

Alveo1arlb:ronchiolar carcinoma 

Chordoma, metastatic, spinal cord 

Histiocytic sarcoma 

Osteosarcoma, metastatic, bone 

Osteosarcoma, metastatic, tissue NOS 

Nose 

Squamous cell carcinoma 

Special Senses System 

Zymbal's gland 

Carcinoma 

(50) 

2 (4%) 

3 (6%) 3 (6%) 

1 (2%) 1 (2%) 

2 (4%) 6 (12%) 

1 (2%) 

1 (2%) 

1 (2%) 

1 (2%) 2 (4%) 

(50) (50) 

1 (2%) 

1 (2%) 

1 (2%) 

(1) (1) 

1 (2%) 

(50) 

(1) 

1 (100%) 
Leukemia mononuclear 

Neoplasm Summary 

Total animals with primary neoplasms' 

15-Month interim evaluation 9 10 10 10 10 

2-Year study 49 46 49 48 46 

Total primary neoplasms 

15-Month interim evaluation 12 11 11 10 13 

2-Year study 

Total animals with benign neoplasms 

144 132 141 124 120 

15-Month interim evaluation 9 10 10 10 10 

2-Year study 49 44 48 47 42 

Total benign neoplasms 

15-Month interim evaluation 10 11 11 10 13 

2-Year study 114 103 113 112 93 

Total animals with malignant neoplasms 

15-Month interim evaluation 2 

2-Year study 23 24 23 9 21 

Total malignant neoplasms 

15-Month interim evaluation 2 

2-Year study 

Total animals with metastatic neoplasms 

30 29 28 12 27 

2-Year study 2 4 5 4 

Total metastatic neoplasms 

2-Year study 5 12 13 I 

a Number of animals examined microscopically at the site and the number of animals with neoplasm 

Number of animals with any tissue examined microscopically 

Primary neoplasms: all neoplasms except metastatic neoplasms 

Vehicle Cont~ol mag 168 mgkg 337.5.mglkg 337.9 rnglkg

(StoplExposure) 

Urinary System 

Kidney (50) (50) (50) (50) (50)

Lipoma 1 (2%) 

Osteosarcoma, metastatic, bone 1 (2%) 

Renal tubule, adenoma 1 (2%) 

Urinary bladder (50) (49) (50) (50) (47)

Papilloma 2 (4%) 4 (8%) 

Papilloma, multiple 2 (4%) 

Systemic Lesions 

Multiple organsb (50) (50) (50) (50) (50)

Histiocytic sarcoma 1(2%) 

18 (36%) 10 (20%) 

Mesothelioma malignant (4%) 2

13 (26%) 3 (6%) 12 (24%) 

(6%) (2%) 13 2 (4%) 1 (2%) 
Salicylazosulfapyridine; N"TR 457 

TABLEA2! 

Individual Animal Tumor Pathologyof Male Rats in the 2-Year Gavage Studyof Salicylazosulfapyridine: 

Vehicle Control 

1445556666677777777777777 

Number of Days on Study 577025035.7801122222222.22'2. 

0370315236487769999999999 

0000000000000000000000000 

Carcass ID Number 1453253045440520012233345 

1272916970815882651504964 

Alimentary System 

Esophagus ......................... Intestine large, colon ......................... Intestine large, rectum ......................... Intestine large, cecum .......................... Intestine small, duodenum .......................... Intestine small, jejunum ......................... Intestine small, ileum ......................... Leiomyosarcoma X 

Liver ......................... Hepatocellular adenoma X 

Leiomyosarcoma, metastatic, stomach, 

forestomach X 

Mesentery + + + + ++ + + 

Fibrous histiocytoma X 

Leiomyosarcoma, metastatic, stomach, .. forestomach X 

Pancreas ......................... Fibrous histiocytoma X 

Leiomyosarcoma, metastatic, stomach, 

forestomach X 

Acinar cell, adenoma X xx x X 

Acinar cell, adenoma, multiple x 

Acinar cell, carcinoma, multiple X 

Salivary glands ......................... Stomach, forestomach ......................... Leiomyosarcoma X 

Squamous cell papilloma X 

Stomach, glandular ++++++++M++++++++++++++++ 

Cardiovascular System 

Blood vessel ......................... Heart .......................... 

Endocrine System 

Adrenal cortex ......................... Adrenal medulla ......................... Pheochromocytoma complex X 

Pheochromocytoma benign X X X 

Bilateral, pheochromocytoma benign X x x 

Islets, pancreatic ......................... Adenoma X 

Carcinoma 

Parathyroid gland +++++++++++++++++M++++++M 

Pituitary gland ......................... Ganglioneuroma 

Pars distalis, adenoma X X x x x xx x 

+: Tissue examined microscopically M: Missingtissue X: Lesionpresent 

A: Autoly!;is precludes examination I:Insufficienttissue Blank: Notexamined 
Tmm A2 

~[ndividuna~ ~nba~ TWUUOI~.nwhonoa ~ f f ale ~aus in uhe 2-~ear Gavage ~tudy ~f ~aa~cy~azcpsu~ffa~yIt.id~~e:

V&k!e @hT~~It.d (continued) 

'171111111177111111111711~ 

Number of Days on Study 3333333333333333333333333 

1111111111111111111111111 

0000000000000000000000000 Total 

Carcass IDNumber OOOO111111222223333444555~Tissuesl 

1348024619023411358035235 Tumors 

Alimentary System 

Esophagus ......................... 50 

Intestine large, colon +++++++++++++++M+++++++++ 49 

Intestine large, rectum ......................... 50 

Intestine large, cecum ......................... 50 

Intestine small, duodenum ......................... 50 

Intestine small,jejunum .......................... 50 

Intestine small, ileum ++M++++++++++++++++++++++ 49 

Leiomyosarcoma 1 

Liver ......................... 50 

Hepatocellular adenoma X 2 

Leiomyosarcoma, metastatic, stomach, 

forestomach 1 

Mesentery + + + + + + +++ 11 

Fibrous histiocytoma 1 

Leiomyosarcoma, metastatic, stomach, 

forestomach 1 

Pancreas ......................... 50 

Fibrous histiocytoma 1 

Leiomyosarcoma, metastatic, stomach, 

forestomach 1 

Acinar cell, adenoma X X X X 9 

Acinar cell, adenoma, multiple X X 3 

Acinar cell, carcinoma, multiple 1 

Salivary glands ......................... 50 

Stomach, forestomach ......................... 50 

Leiomyosarcoma 1 

Squamous cell papilloma 1 

Stomach, glandular ......................... 49 

Cardiovascular System 

Blood vessel ......................... 50

Heart ......................... 50 

Endocrine System 

Adrenal cortex ......................... 50 

Adrenal medulla ......................... 50 

Pheochromocytoma complex 1 

Pheochromocytoma benign X X xxxx X X X 12 

Bilateral, pheochromocytoma benign 3 

Islets, pancreatic ......................... 50 

Adenoma X 2 

Carcinoma X 1 

Parathyroid gland ........................ 46 

Pituitary gland ......................... 50 

Ganglioneuroma X 1 

Pars distalis, adenoma xxx X X X 14 
94 Salicylazosulfapyridine, NTP TR'457 

TABLEAl! 

1ndividua:lAnimal Tumor Pathology of Male Rats in the 2-Year Gavage Study of SalicyIazosu1fapyridine:i 

Vehicle Control (continued) 

144555666,6677777777777777 

Number of Days on Study 577025035.7801122222222222 

0370315236487769999999999 

0000000000000000000000000 

Carcass ID Number 1453253045440520012233345 ' 

1272916970815882651504964 

Endocrine System(continued) 

Thyroid gland ......................... C-cell, adenoma X 

C-cell, carcinoma X 

Follicular cell, adenoma 

Follicular cell, carcinoma X 

General Body System 

Tissue NOS + 

Genital System 

Epididymis ......................... Mesothelioma malignant, metastatic, mesentery 

Preputial gland ......................... Adenoma X 

Carcinoma X 

Prostate ......................... Seminal vesicle ......................... Testes ......................... Bilateral, interstitial cell, adenoma x x xxxx xxxxxxxxxxxxxx 

Interstitial cell, adenoma X 

Hematopoietic System 

Bone marrow ......................... Lymph node ++ +++ + 

Lymph node, mandibular ......................... Lymph node, mesenteric ......................... Spleen ......................... Leiomyosa.rcoma, metastatic, stomach, 

forestomach X 

Thymus +I ++++I ++++++++++++++++++ 

Thymoma benign 

Integumentary System 

Mammary gland ......................... Fibroadenoma x x 

Skin ......................... Basal cell adenoma X 

Keratoacanthoma X 

Squamous cell papilloma 

Subcutaneous tissue, fibroma X 

Musculoskelletal System 

Bone ......................... Skeletal muscl:e + 

Fibrous histiocytoma X . . . . 

Nervous System 

Brain ......................... Astrocytoma malignant X 
Number of Days on Study 

Carcass IUD Number 

Endocrine System (continued) 

Thyroid gland 

C-cell, adenoma 

C-cell, carcinoma 

Follicular cell, adenoma 

Follicular cell, carcinoma 

General Body System 

Tissue NOS 

Genital System

Epididymis 

Mesothelioma malignant, metastatic, mesentery 

Preputial gland 

Adenoma 

Carcinoma 

Prostate 

Seminal vesicle 

Testes 

Bilateral, interstitial cell, adenoma 

Interstitial cell, adenoma 

Hematopoietic System 

Bone marrow 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Leiomyosarcoma, metastatic, stomach, 

forestomach 

Thymus 

Thymoma benign 

Integumentary System

Mammary gland 

Fibroadenoma 

Skin 

Basal cell adenoma 

Keratoacanthoma 

Squamous cell papilloma 

Subcutaneous tissue, fibroma 

Musculoskeletal System

Bone 

Skeletal muscle 

Fibrous histiocytoma 

Nervous System

Brain 

Astrocytoma malignant 

1111771171111711111111717 

3333333333333333333333333' 

1111111111111111111111111 

0000000000000000000000000 Total 

0000111111222223333444555 Tissues/ 

1348024679023471358035235 Tumors 

.......................... 50 

x x 3 

x 2 

x xx x 4 

1 

1 

......................... 50 

x 1 

......................... 50 

1 

x 2 

......................... 50 

......................... 50 

......................... 50 

xxxxxxxxxxxxxxxxxxxxxxxx 44 

1 

......................... 50 

+ + + 9 

......................... 50 

......................... 50 

......................... 50 

1 

......................... 47 

x 1 

+++++++M+++M+++++++++++++ 48 

x 3 

.......................... 50 

x 2 

x x 3 

x 1 

x 2 

......................... 50 

1 

1 

......................... 50 

1 
96 Salicylazosulfapyridine, NTP TR 457 

TABLEA2 _. . II Individual AnimalTumor Pathology of Male Rats in the 2-Year GavageStudy of Salicylazosulfapyridine: I " 

' : ; VehicleControl (continued) . 

. , 14.4 5.5 5 6 6 6 6 6 7 7 7 7 7 7.7 7 7 7 7 7 7 7 

Number of Days on Study 5770250357801122222222222 ., I

0370315236487769999999999.. ~~ ~ ~~ ~~ ~~ ~~~~ ~~ 

0 0 0 0.0 0 0 0 0 0-0 0 0 0 0 0 0.0 0-0 0 0 0 0 0.' 

Carcass ID Number '1453253045440520012233345 

127291697081588265150496.4. ' 

Respiratory System .. . .. 

Lung

Alveolar/t'ronchiolar adenoma X 

......................... X 

Nose ........................... Squamous cell carcinoma X 

Trachea ......................... 

Zymbal's gland 

Special Senses System 

+ 

Carcinoma X ~ ~ ~~ 

UrinarySystem . 

Kidney .......................... Urinary bladder ......................... ~~ ~ ~ ~~~ ~~ 

Systemic Lesions 

Multiple organs 

Leukemia mononuclear 

......................... X X x x xx x x .. 1 

Mesothelioma malignant ,I . 
Number of Days on Study 

. . 

Carcass ID Number 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma 

Nose 

Squamous cell carcinoma 

Trachea 

Special Senses System 

Zymbal's gland 

Carcinoma 

Urinary System 

Kidney 

Urinary bladder 

Systemic ]Lesions 

Multiple organs 

Leukemia mononuclear 

Mesothelioma malignant 

1711111111111111111111711 

3333333333333333333333333 

'1 11 11 11-1 11 11 11 1.1 11 11 11.1-1 1 

0000000000000000000000000 Total 

0000111111222223333444555 Tissues/ 

13~48024619023471358035235Tumors 

......................... 50 

2 

......................... 50 

1 

......................... 50 

1 

1 

......................... 50 

.......................... 50 

......................... 50 

X xx xx 13 

X 1 
98 Salicylazosulfapyridine,NTP TR 457 

TABLE,421 

Individual1 Animal Tumor Pathologyof Male Rats in the 2-Year Gavage Study of Salicylaz&ulfapyridine: 84 mg/kg 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatocellular carcinoma 

Histiocytic. sarcoma 

Mesentery 

Pancreas 

Mixed hurlor benign 

Acinar cell, adenoma 

Salivary glands 

Stomach, fore:stomach 

Stomach, glandular 

Cardiovascular System 

Blood vessel 

Heart 

Schwannoma malignant 

Endocrine System

Adrenal cprtex 

Histiocytic sarcoma 

Adrenal medulla 

Pheochromocytoma benign 

Bilateral, pheochromocytoma benign 

Islets, pancreatic 

Adenoma 

Carcinoma 

Parathyroidgland . 

Pituitary gland 

Pars distalis, adenoma 

Thyroid gland 

Bilateral, C-cell, carcinoma 

C-cell, adenoma 

C-cell. carcinoma 

General Body System 

None 

Genital System 

Epididymis 

Preputial gland 

Adenoma 

Carcinoma 

0224445666666667777777777 

8341261025568990222222222 

5434405554920129699999999 

00010011000111110000,00111 

8780990098700001977899000 

1886495986338025729503147 . 

......................... 

......................... 

......................... 

......................... 

.......................... 

+I+++++++++++++++++++++++ 

+I+++++++++++++++++++++++ 

.......................... X 

X 

+ ++.+ + + ......................... 

X X X 

......................... 

......................... 

......................... 

......................... 

......................... 

......................... X 

++++M++++++++++++++++++++ 

......................... x x 

++++++M++++++++++++++++++ 

.......................... xxx x X 

......................... 

x x X X 

~~ 

......................... 

......................... 

X 
 

Number of Days on Study 

Carcass E D Number 

Alimentary System 

Esophagus 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatocellular carcinoma 

Histiocytic sarcoma 

Mesentery 

Pancreas 

Mixed tumor benign 

Acinar cell, adenoma 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Cardiovascular System 

Blood vessel 

Heart 

Schwannoma malignant 

Endocrine System 

Adrenal cortex 

Histiocytic sarcoma 

Adrenal medulla 

Pheochromocytoma benign 

Bilateral, pheochromocytoma benign 

Islets, pancreatic 

Adenoma 

Carcinoma 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Thyroid gland 

Bilateral, C-cell, carcinoma 

C-cell, adenoma 

C-cell, carcinoma 

General Body System 

None 

Genital System 

Epididymis 

Preputial gland 

Adenoma 

Carcinoma 

IIIIIIIIIIII'IIIIIIIIIIIII 

2233333333333333333333333 

9922222222222333333333333 

110000000000000001 1 1 1 1 1 1 1 Total 

117711,1888888999911111112 Tissues/ 

2114567023419125601346890 Tumors 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

......................... 49 

......................... 49 

......................... 50 

1 

1 

+ ++ + ++ + + 14 

......................... 50 

X 1 

xx X 6 

......................... 50 

......................... 50 

......................... 50 ~~~~~ ~~~~~~~~~~ 

.......................... 50 

......................... 50 

x, 1 

......................... 50 

1 

......................... 49 

X xxx 'X X 6 

X 1 

......................... 50 

X 3 

X 1 

......................... 49 

......................... 50 

x x x xxxx x xx X 16 

......................... 50 

X 1 

X X xx X 9 

X 1 

......................... 50 

......................... 50 

X 1 

1 
100 Salicylazosulfapyridine,NTP TR 457 

TABLEA2! 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Gavage Study of Salicyiazosulfapyridine: 84 mg/kg 

(continued) 

Number of Days on Study 

Carcass ID Number 

Genital System(continued) 

Prostate 

Adenoma 

Seminal vesicle 

Testes 

Bilateral, interstitial cell, adenoma 

Interstitial cell, adenoma 

Hematopoietic System 

Bone marrow 

Histiocytic sarcoma 

Lymph node 

Pancreatic, histiocytic sarcoma 

Lymph node, mandibular 

Histiocytic sarcoma 

Lymph node, mesenteric 

Spleen 

Hemangiosarcoma 

Histiocytic: sarcoma 

Thymus 

Integumentary System 

Mammary gland 

Fibroadenoma 

Skin 

Basal cell adenoma 

Basal cell carcinoma 

Keratoacanthoma 

Squamous cell papilloma 

Subcutaneous tissue, fibroma 

Subcutaneoustissue, liposarcoma 

Musculoskeletal System 

Bone 

Hamartoma 

Skeletal muscle 

Histiocytic sarcoma 

Nervous System 

Brain 

Peripheral nerve 

Spinal cord 

Respiratory System 

Lung 

Histiocytic sarcoma 

Nose 

Trachea 

022444566666666.7777777777 

83412610255689'90222222222 

54344055549201296999.99999 

0001001100011111000000111 

8780990098700001977~899000 

1886495986338025729503147 

......................... 

......................... 

+++++M+++++++++++++++++++ 

xxxxxxxxxxxxxxxx 

X 

......................... X 

+ ++ + ++ 

X ......................... X 

......................... 

......................... X 

X ......................... 

++++++++++M++++++++++++++ 

X ......................... X X 

X 

x 

X 

xx X 

X 

.......................... X 

+ 

X 

......................... + 

+ 

......................... X ......................... 

......................... 
.,- ~ 

BaPiCyB~QSolfa~y~i8ine, Nm TR 457 non

TABLE A2 

I[ndMdanaUAnnimaU ,TUIIMIQ~ Pathdogy offMale RaUs in the %Year Gavage Study off SaPcyU~os,snnnffa~y~~d~n@: 80 mgkg

(continued) 

Number of Days on Study 

Carcass ID Number 

Genital System (continued) 

Prostate 

Adenoma 

Seminal vesicle 

Testes 

Bilateral, interstitial cell, adenoma 

Interstitial cell, adenoma 

Hematopoietic System 

Bone marrow 

Histiocytic sarcoma 

Lymph node 

Pancreatic, histiocytic sarcoma 

Lymph node, mandibular 

Histiocytic sarcoma 

Lymph node, mesenteric 

Spleen I 

Hemangiosarcoma 

Histiocytic sarcoma 

Thymus 

Integumentary System 

Mammary gland 

Fibroadenoma 

Skin 

Basal cell adenoma 

Basal cell carcinoma 

Keratoacanthoma 

Squamous cell papilloma 

Subcutaneous tissue, fibroma 

Subcutaneous tissue, liposarcoma 

Musculoskeletal System 

Bone 

Hamartoma 

Skeletal muscle 

Histiocytic sarcoma 

Nervous System 

Brain 

Peripheral nerve ,

Spinal cord 

Respiratory System 

Lung 

Histiocytic sarcoma 

Nose 

Trachea 

7777777777777777777777777 

2233333333333333333333333 

9922222222222333333333333 

1100000000000000011111111 Total 

117777788888899991 11 11 11.2 Tissues/ 

2714567023479125601346890 Tumors 

......................... 50 

X 1 

......................... 50 

......................... 49 

xxxxxxxx xx x xxxxxxxxxx 37 

X x x 4 

......................... 50 

1 

++ ++ ++++ + 15 

1

+++++++++++++++++++++++++I 50 

1 

......................... 50 

......................... 50 

1 

1 

......................... 50 

......................... 49 

X X X 4 

......................... 50 

X 3 

1 

X X 3 

X 2 

X X 5 

1 

......................... 50 

1 

1 

1 

......................... 50 

1 

1 

......................... 50 

1 

......................... 50 

......................... 50 
102 Salicylazosulfapyridine,NTP TR 457 

TABLEA;! 

Individual Animal Tumor Pathologyof Male Rats in the 2-Year Gavage Studyof Salicylazosulfapyridine: 84 mg/kg 

(continued) 

Number of Days on Study 

Carcass ID Number 

Special Sen.sesSystem 

EY e 

Urinary System 

Kidney 

Urinary bladder 

Systemic Lesions 

Multiple 0rga.m 

Histiocytic sarcoma 

Leukemia mononuclear 

Mesothelioma malignant 

0224445666666667777777777 

8341261025568990222222222 

5434405554920129699999999 

0001001100011111000000111 

8780990098700001977899000 

1886495986338025729503147 

+ 

......................... 

+++++A+++++++++++++++++++ 

......................... X 

X xxxxxx x x x 
 

7777777777777777777777717 

Number of Days on Study 2233333333333333333333333 

9922222222222333333333333 

110000000000000001 1 1 1 1 1 1 1 Total 

Carcass ID Number 1177777888888999911111112 Tissues/ 

2714567023479125601346890 Tumors ~ 

Special Senses System 

Eye + 2 

Urinary System 

Kidney ......................... 50 

U~~IMQJ bladder ......................... 49 

Systemic Lesions 

Multiple organs ......................... 50 

Histiocytic sarcoma 1 

Leukemia mononuclear xxx X xx xx 18 

Mesothelioma malignant X 1 
104 Salicylazosulfapyridine, NTP TR 457 

TABLEA2 

Individual Animal Tumor Pathologyof Male Rats in the 2-Year Gavage Studyof Salicylazosulfapyridine: 168 mg/kg 

2344455555666'666777777777 

Number of Days on Study 3913301347'113899000222222 

364572569811905624499.9999 

1111111111111111111~111111 

Carcass ID1 Number 3348477543375464655334456 

9644718383755096218483940 __ __ ~~~~~~ _____ __ ~ 

Alimentary System 

Esophagus +++++++++M++++++++++++++'+ 

Intestine large, colon .......................... Intestine large, rectum .......................... Intestine large, cecum 

' 

......................... Intestine small, duodenum ......................... Intestine smaii, jejunum ......................... Intestine small, ileum ............................ . Liver ......................... Hepatocellular carcinoma 

Osteosarcoma, metastatic, tissue NOS X 

Mesentery ++ + ++ + ++ + 

Osteosarcoma, metasiatic, tissue NOS X 

Pancreas ......................... Acinar cell, adenoma X X 

Acinar cell, adenoma, multiple X 

Pharynx + + 

Squamous cell carcinoma X 

Squamous cell papilloma X 

Salivary glands ......................... Stomach, forestomach .......................... Stomach, glandular ......................... 

Cardiovascular System 

Blood vessel ......................... Vena cava, chordoma, metastatic, 

spinal cord 

Heart ......................... Schwannclma malignant 

Endocrine System , . 

Adrenal cortex ......................... Adenoma X 

Adrenal medulla ......................... . Pheochromocytoma malignant 

Pheochromocytoma benign 

Bilateral, pheochromocytoma benign X 

Islets, pancreatic ......................... Adenoma X X 

Parathyroid gland ++++++++++++++++++++++++M 

Pituitary gland +++++++++++M+++++++++++++ 

Pars distalis, adenoma X x x xxx xx X 

Pars intermedia, adenoma 

Thyroid gland ......................... C-cell, adenoma x x X 

C-cell, carcinoma 

Follicular cell, adenoma X 

Follicular cell, carcinoma X 

.. i 
no5 

Number of Days on Study 

Carcass E D Number 

Alimentary System 

Esophagus 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatocellular carcinoma 

Osteosarcoma, metastatic, tissue NOS 

Mesentery 

Osteosarcoma, metastatic, tissue NOS 

Pancreas 

Acinar cell, adenoma 

Acinar cell, adenoma, multiple 

Pharynx 

Squamous cell carcinoma 

Squamous cell papilloma 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Cardiovascular System 

Blood vessel 

Vena cava, chordoma, metastatic, 

spinal cord 

Heart 

Schwannoma malignant 

Endocrine System 

Adrenal cortex 

Adenoma 

Adrenal medulla 

Pheochromocytoma malignant 

Pheochromocytoma benign 

Bilateral, pheochromocytoma benign 

Isleis, pancreatic 

Adenoma 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Pars intermedia, adenoma 

Thyroid gland 

C-cell, adenoma 

Ccell, carcinoma 

Follicular cell, adenoma 

.Follicular cell, carcinoma 

~ ~~ ~~~ ~ ~~ 

1111111111111111111111111 

2222333333333333333333333 

9999222222222222222222222 

1111111111111111111111111 Total 

6611333444555556666611111 Tissues/ 

1849125125026793456102367 Tumors 

......................... 49 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

X 1 

1 

+ + + + + + + ++ 18 

1 

......................... 50 

X X X 5 

X 2 

2 

1 

1 ......................... 50 

......................... 50 

......................... 50 

......................... 50 

X 1 

......................... 50 

X 1 

......................... 50 

1 

......................... 50 

X 1 

X xxx X 5 

1 ......................... 50 

X X X X 6 

......................... 49 

......................... 49 

xxx xx xxx X 1 8

X 1 

......................... 50 

X X 5 

X X 2 

xx 3 

1 
106 Salicylazosulfapyridne, NTP TR 457 

TABLEA2 

Individual Animal Tumor Pathology of Male Rats in the2-YearGavage Study of Salicylazosulfapyridine: 168 mg/kg 

(continuea) 

Number of Days on Study 

Carcass 11) Number 

General Body System 

Tissue NOS 

Osteosarcoma 

Genital System 

Epididymis 

Mesothelioma malignant, metastatic, mesentery 

Preputial gland 

Adenoma 

Carcinoma 

Prostate 

Seminal vesicle 

Testes 

Mesothelioma malignant, metastatic, mesentery 

Bilateral, interstitial cell, adenoma 

Interstitial cell, adenoma 

Hematopoietic System 

Bone marrow 

Lymph node 

Mediastinal, osteosarcoma, metastatic, 

tissue NOS 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Mesothelioma malignant, metastatic, mesentery 

Osteosarc.oma, metastatic, tissue NOS 

Thymus 

Integumentary System 

Mammary gland 

Carcinoma 

Fibroadenoma 

Fibroadenoma, multiple 

Skin 

Keratoacanthoma 

Squamous cell papilloma 

Trichoepithelioma 

Subcutancous tissue, fibroma 

Subcutaneous tissue, schwannoma malignant ~ ~~ 

Musculoskeletal System 

Bone 

Skeletal muscle 

Osteosarcoma. metastatic. tissue NOS 

Nervous System 

Brain 

Glioma malignant 

Spinal cord 

2344455555666666777777777 

3913301347113899000222222 

3645725698119056244999999 

1111111111111111111111111 

3348477543375464655334456 

96447'18383755096218483940 

+ 

X 

......................... X ......................... 

X X X 

......................... 

......................... 

......................... 

x x x x x xx x x xx x x x xx 

x x x X X 

......................... +++++ + + ++ + 

X 

......................... 

......................... 

......................... X 

X 

++++++I++++++++++++++++++ 

I 

+++++++++++++++++++M++MM+ 

X 

X 

.......................... x xx 

X 

X 

X 

......................... + 

X 

......................... X 

+ 
Salicylrazosulh~yridine,NTB TR 4159 

Number of Days on Study 

Carcass ID Number 

General Body System 

Tissue NOS 

Osteosarcoma 

Genital System 

Epididymis 

Mesothelioma malignant, metastatic, mesentery 

Preputial gland 

Adenoma 

Carcinoma 

Prostate 

Seminal vesicle 

Testes 

Mesothelioma malignant, metastatic, mesentery 

Bilateral, interstitial cell, adenoma 

Interstitial cell, adenoma 

Hematopoietic System 

Bone marrow 

Lymph node 

Mediastinal, osteosarcoma, metastatic, 

tissue NOS 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Mesothelioma malignant, metastatic, mesentery 

Osteosarcoma, metastatic, tissue NOS 

Thymus 

Integumentary System 

Mammary gland 

Carcinoma 

Fibroadenoma 

Fibroadenoma, multiple 

Skin 

Keratoacanthoma 

Squamous cell papilloma 

Trichoepithelioma 

Subcutaneous tissue, fibroma 

Subcutaneous tissue, schwannoma malignant 

Musculoskeletal System 

Bone 

Skeletal muscle 

Osteosarcoma, metastatic, tissue NOS 

Nervous System 

Brain 

Glioma malignant 

Spinal cord 

7777177777777777777777777 

2222333333333333333333333 

9999222222222222222222222 

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Total 

6677333444555556666671777 Tissues1 

1849125125026793456702367 Tumors 

1 

1 

......................... 50 

X 2 

......................... 50 

x x 2 

3 

......................... 50 

......................... 50 

......................... 50 

X 1 

x x x x x x x x x x x x x x x x x x x x x x 39 

X X 7 

......................... 50 

++ + + 14 

1 

......................... 50 

......................... 50 

......................... 50 

1 

1 

......................... 49 

......................... 47 

1 

x x X 4 

X 1 

......................... 50 

3 

X X X 3 

1 

1 

1 

......................... 50 

1 

'1 

......................... 50 

1 

1 
10s Salicylazosulfapyridine,NTP TR 457 

TABLEA2 

Individual Animal Tumor Pathology of Male Rats in the 2-Year Gavage Study of Salicylazosulfapyridine: 168 mg/kg 

(continued) 

Number of Days on Study 

Carcass ID Number 

~~ 

Respiratory System 

Lung 

Chordoma, metastatic, spinal cord 

Osteosarcoma, metastatic, tissue NOS 

Nose 

Trachea 

Special Senses System 

Eye 

Urinary System 

Kidney 

Lipoma 

Renal tubule, adenoma 

Ureter 

Urethra 

Urinary bladder 

Papilloma 

Systemic Lesions 

Multiple organs 

Leukemia :mononuclear 

Mesothelioma malignant 

2344455555666666777777777 

3913301347113899000222222 

3645725698119056244999999 

1111111111111111111111111 

3348477543375464655334456 

9644118383755096218483940 ~_____ ~~ ~~ ~~ ~~ 

......................... .. 

X 

......................... 

......................... 

+ + 

......................... 

+ 

+ + ......................... 

......................... X xxx x xx x .x 

X 
TABLE A2 

hdividunaU Auni~maUTumor Pathk~g~~ OP MaUe Rats in the %YearGavage Study off SaPicyUsszosulff~pyr~~i~e: 168 mgkg 

(continued) 

7777777777777777777777777 

Number of Days on Study 2222333333333333333333333 

9999222222222222222222222 

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Total 

Carcass I D Number 6677333444555556666677777 Tissues/ 

1849125125026793456702367 Tumors 

Respiratory System 

Lung ......................... 50 

Chordoma, metastatic, spinal cord X 1 

Osteosarcoma, metastatic, tissue NOS I 

Nose ......................... 50 

Trachea ......................... 50 

Special Senses System 

Eye + 3 

Urinary System 

Kidney ......................... 50 

Lipoma X 1 

Renal tubule, adenoma X 1 

Ureter 1 

Urethra 2 

Urinary bladder ......................... 50 

Papilloma X X 2 

Systemic Lesions 

Multiple organs ......................... 50 

Leukemia mononuclear X xx 12 

Mesothelioma malignant X 2 
110 Salicyiazosulfapyridine,NTP TR 457 

TABLEA2 

Individuad Animal Tumor Pathology of Male Rats in the 2-YearGavage Study of Salicylazosulfapyridine: 337.5 mg/kg 

Number of Days on Study 

Carcass Ill Number 

Alimentary System 

Esophagus 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Fibrous histiocytoma, metastatic, skin 

Osteosarcoma, metastatic, bone 

Mesentery 

Pancreas 

Mesothelioma malignant, metastatic, mesentery 

Mixed turnor benign 

Acinar cell, adenoma 

Salivary glands 

Adenoma 

Stomach, forestomach 

Squamous cell carcinoma 

Stomach, glandular 

Tongue 

Sauamous cell carcinoma 

Cardiovasaular System 

Blood vessel 

Heart 

Schwannoma benign 

Endocrine !System 

Adrenal cortex 

Adrenal medulla 

Pheochrornocytoma benign 

Bilateral, pheochromocytoma benign 

Islets, pancreatic 

Adenoma 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Thyroid gland 

C-cell, adenoma 

C-cell, carcinoma 

Follicular cell, adenoma 

General Body System 

None 

1244455566666666666777777 

2423768813455557889000001 

2086.351535538992476444588 

2222222122222222222111222 

230020093203223112099930'0 

7343,601774942854852235056 

. , 

......................... 

......................... 

.......................... 

......................... 

......................... 

+++++++++++++++++++++++M+ 

......................... 

......................... X 

X 

++ + + + ......................... Ji 

x xx x .......................... 

......................... 

......................... 

.......................... 

......................... X 

......................... 

......................... 

X 'X 

X ......................... X X 

++++++++M++++M+++++M+++++ 

......................... X xxxx x x X x x ......................... X X 

X 
1111171117777177777771717 

Number of Days on Study 2222222222223333333333333 

36999999-99991111222222222 

22112222222211122,22222222 Total 

Carcass ID Number 1199012223339990111112334 Tissues/ 

1969850392691487023671180 Tumors 

Alimentary System 

Esophagus ++++++++++++++M++++++++++ 49 

Intestine large, colon +M+++++++++M+++++++++++++ 48 

Intestine large, rectum ++++++++++++++++++++++++I 49 

Intestine large, cecum ......................... 50 

Intestine small, duodenum ......................... 50 

Intestine small, jejunum ......................... 49 

Intestine small, ileum ......................... 50 

Liver ......................... 50 

Fibrous histiocytoma, metastatic, skin 1 

Osteosarcoma, metastatic. bone 1 

Mesentery + + + + ++ + 12 

Pancreas ......................... 50 

Mesothelioma malignant, metastatic, mesentery 1 

Mixed tumor benign X 1 

i Acinar cell, adenoma xxx X x x 10 I Salivary glands ......................... 50 - I

-i Adenoma X 1

! Stomach, forestomach ......................... 50 I/ 

Squamous cell carcinoma X 1 

Stomach, glandular ......................... 50 

Tongue + 1 

Squamous cell carcinoma X 1 

Cardiovascular System 

Blood vessel ......................... 50 

Heart ......................... 50 

Schwannoma benign X 2 

Endocrine System 

Adrenal cortex ......................... 50 

Adrenal medulla ......................... 49 

Pheochromocytoma benign X X x x X I 

Bilateral, pheochromocytoma benign 1 

Islets, pancreatic ......................... 50 

Adenoma X X X 5 

Parathyroid gland ........................ 45 

Pituitary gland +++++++++++++++++++M+++++ 49 

Pars distalis, adenoma X xx X xxx X 18 

Thyroid gland ......................... 50 

Ccell, adenoma xxxx 6 

C-cell, carcinoma 1 

Follicular cell, adenoma X 1 

General Body System 

None 
112 Salicylazosulfapyridine,NTP TR 457 

Number of Days on Study _. ' 

Carcass ID IVumber 

Genital Syst'em 

Epididymis 

Mesothe1io:mamalignant, metastatic, mesentery 

Preputial gland 

Prostate 

Seminal vesicle 

Mesothelioma malignant, metastatic, mesentery 

Testes 

Mesothelioma malignant, metastatic, mesentery 

Bilateral, interstitial cell, adenoma 

Interstitial cell, adenoma 

Hematopoietic System 

Bone marrow 

Lymph node 

Inguinal, fibrous histiocytoma, metastatic, skin 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Thymus 

Integumentary System 

Mammary gland 

Fibroadenoma 

Skin 

Keratoacanthoma 

Squamous cell papilloma 

Subcutaneoustissue, fibroma 

Subcutaneoustissue, fibrosarcoma 

Subcutaneoustissue, fibrous histiocytoma 

Musculoskeletal System 

Bone 

Osteosarcoma 

Skeletal muscl'e 

Mesothelioma malignant, metastatic, mesentery 

Nervous System 

Brain 

Respiratory System 

Lung 

Osteosarcoma, metastatic, bone 

Nose . . Trachea 

~1244455566666666666777777-

2423768813455557889000001 

208.,63515355.3899247644458.8 

2222222122222222222111222 

2300200932032231120999300 

7343.6017749,42854852235056 

......................... X 

......................... 

......................... 

........................... X 

........................... X 

x x x x xx xx x x x x x x x xx x 

X . . X 

......................... ++ + + + . . 

X ' 

++++++++++++++++++++++++I-

++++++M++++++++++++++++++ 

......................... 

.......................... 

......................... X 

......................... , 

X X 

X 

X 

X 

......................... X 

+ 

X 

.......................... 

......................... X 

......................... 

......................... 
183 

. .. ." .- ppI_~ 

TABLE A2 

II~C"U ~nima~ llhnwn~r P~~MO~JJ OITMane R~US in the 2-yem Gavage ~tunc~~y OIT~a~~cy~~~~~ffa~pgrridliane: 337.5 mg/kg

(continued) 

Number of Days on Study 

Carcass ID Number 

Genital System 

Epididymis 

Mesothelioma malignant, metastatic, mesentery 

Preputial gland 

Prostate 

Seminal vesicle 

Mesothelioma malignant, metastatic, mesentery 

Testes 

Mesothelioma malignant, metastatic, mesentery 

Bilateral, interstitial cell, adenoma 

Interstitial cell, adenoma. 

Hematopoietic System 

Bone marrow 

Lymph node 

Inguinal, fibrous histiocytoma, metastatic, skin 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Thymus ' 

Integumentary System 

Mammary gland 

Fibroadenoma 

Skin 

Keratoacanthoma 

Squamous cell papilloma 

Subcutaneous tissue, fibroma 

Subcutaneous tissue, fibrosarcoma 

Subcutaneous tissue, fibrous histiocytoma 

Musculoskeletal System 

Bone 

Osteosarcoma 

Skeletal muscle 

Mesothelioma malignant, metastatic, mesentery 

Nervous System 

Brain 

Respiratory System 

Lung 

Osteosarcoma, metastatic, bone 

Nose 

Trachea 

Special Senses System 

None 

77771177777~77771177777177 

2222222222223333333333333 

3699999999991111222222222 

2211222.2222211122222'22222 

1199012223339990111112334 

1969850392691487023671180 

......................... 

X 

......................... 

......................... 

......................... 

......................... 

X X 

xxxxxxxxxxxxxxxxxxxxxxxxx 

......................... 

+++ + + 

......................... 

......................... 

......................... 

......................... 

+I+++++++++++++++++++++++ 

X X ......................... 

X xx 

......................... 

......................... 

......................... 

......................... 

......................... 

Total 

Tissues/ 

Tumors 

50 

2 

50 

50 

49 

1 

'50 

3 

43 

2 

50 

10 

1 

49 

49 

50 

49 

49 ' 

3 

50 

2 

1 

3 

1 

1 

50 

1 

1 

1 

50 

50 

1 

50 

50 
114 Salicylazosulfapyridine,NTP TR 457 

TABLE A2 

Individual. Animal Tumor Pathologyof Male Rats in the 2-Year Gavage Studyof Salicylazosulfapyridine: 337.5 mg/kg 

(continued) 

Number of Days on Study 

Carcass ID Number 

Urinary System 

Kidney 

Ureter 

Urethra 

Urinary bladder 

Mesothelioma malignant, metastatic, mesentery 

Papilloma 

Papilloma, multiple ~~ ~ ~~~ ~~ 

Systemic Lesions 

Multiple organs 

Leukemia mononuclear 

Mesothelioma malignant 

12444555,66666666666777777 

2423768813455557889000001 

2086351535538992476444588 

2222222122222222222111222 

2300200932032231120999300 

7343601774942854852235056 

......................... + +M + 

+ ......................... X 

X 

......................... X X X 

X ’ 
Salicylazosulhgyridine, NTP TR 457 1n5 

Number of Days on Study 

Carcass IDNumber 

Urinary System 

Kidney 

Ureter 

Urethra 

Urinary bladder -

Mesothelioma malignant, metastatic, mesentery 

Papilloma 

Papilloma, multiple 

Systemic Lesions 

Multiple organs 

Leukemia mononuclear 

Mesothelioma malignant 

1111111111117111111111111 

2222222222223333333333333 

3699999999991111222222222 

2211222222221112222222222 Total 

1199012223339990111112334 Tissues/ 

Tumors 1969850392691481023611180 

......................... 50 

++ + + + + + +++ 13 

++ + + 5 

......................... 50 

1 

X X X X 4 

X 2 

......................... 50 

3 

X x 3 
116 Salicylazosulfapyridine,NTP TR 457 

TABLEA;! 

Individual Animal Tumor Pathology of Male Rats in the %Year Gavage Study of Salicylazosulfapyridine: 

337.5 mgJkg (Stop-Exposure) 

Number of Days on Study 

Carcass I D 1 Number

Alimentary System 

Esophagus 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Carcinoma 

Intestine small, jejunum . 

Intestine small, ileum 

Liver 

Mesentery 

Pancreas 

Acinar cell, adenoma 

Acinar cell, adenoma, multiple 

Pharynx 

Squamous cell papilloma 

Salivary glands 

Schwannoma malignant, metastatic, skin 

Stomach, forestomach 

Stomach, glandular 

Cardiovascular System 

Blood vessel 

Heart 

Schwannoma benign 

Endocrine System 

Adrenal cort~:x 

Adenoma 

Adrenal medulla 

Pheochromocytoma malignant 

Pheochro~nocytoma benign 

Bilateral, pheochromocytoma benign 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Thyroid gland 

C-cell, adenoma 

C-cell, carcinoma 

Follicular cell, carcinoma 

General Body System 

None 

0112334445555566666777777 

8234682360446844689022222 

5000133066278548246499999 

2222222232232223222222222 

6975988909708560598755667 

8601394748455391708948265 

......................... 

......................... 

......................... 

......................... 

+A+++++++++++++++++++++++ 

+A+++++++++++++++++++++++ 

.+A+++++++++++++++++++++++ 

......................... + + + ++ 

+A++++++++++A++++++++++++ 

X 

+ 

X 

......................... 

......................... 

.......................... 

......................... 

......................... 

~~ ~~ 

.......................... 

........................... X 

X 

+A+++++++++++++++++++++++ 

+M+M+++++++++++++++++++++ 

......................... X x xxxxx x x x ......................... X X 
TABLE & 

Individual Aunhal Tumor Pathdog off Male Rats in the %YearGavage Study off SallcynazosoPhpyricine: 

337.5 mgkg (StQp-lEXpOSure) (continued) 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Carcinoma 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Mesentery 

Pancreas 

Acinar cell, adenoma 

Acinar cell, adenoma, multiple 

Pharynx 

Squamous cell papilloma 

Salivary glands 

Schwannoma malignant, metastatic, skin 

Stomach, forestomach 

Stomach, glandular 

Cardiovascular System 

Blood vessel 

Heart 

Schwannoma benign 

Endocrine System 

Adrenal cortex 

Adenoma 

Adrenal medulla 

Pheochromocytoma malignant 

Pheochromocytoma benign 

Bilateral, pheochromocytoma benign 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Thyroid gland 

C-cell, adenoma 

C-cell, carcinoma 

Follicular cell, carcinoma 

General Body System 

None 

7777777777777777777777777 

2222233333333333333333333 

9999900000000000000000000 

2223322222222222222222333 Total 

8890055666666777788999000 Tissues/ 

16107690134.57237837259369 Tumors 

......................... 50 

......................... 50 

++++++++++++++++++++++I++ 49 

......................... 50 

......................... 49 

1 

......................... 49 

......................... 49 

......................... 50 

+ + +++ +++ +++ 16 

......................... 48 

X 2 

X X 2 

1 

1 

......................... 50 

X 1 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

X 1 

......................... 50 

X X 2 

.......................... 50 

X 2 

X X 3 

X 1 

......................... 49 

......................... 48 

......................... 49 

xx x x X xx X 18 

......................... 50 

X X 4 

X X 2 

X X 2 
118 Salicylazosulfapyridine, NTP TR 457 

TABLEA2 

Individual Animal Tumor Pathologyof Male Rats in the 2-Year Gavage Studyof Salicylazosulfapyridine: 

337.5 mg/kg (Stop-Exposure)(continued) 

Number of Days on Study 

Carcass ID Number 

~ ~~ ~~ ~~ ~~ ~~ 

Genital Sy,stem 

Epididymis 

Mesothelioma malignant, metastatic, 

mesente:ry 

Preputial gland 

Adenoma 

Carcinoma 

Prostate 

Seminal vesicle 

Testes 

Mesothelioma malignant, metastatic, 

mesentery 

Bilateral, interstitial cell, adenoma 

Interstitial cell, adenoma 

Hematopoietic System 

Bone marrow 

Lymph node 

Lymph node, mandibular 

Lymph node., mesenteric 

Spleen 

Thymus 

Integumentary System 

Mammary gland 

Fibroadenoma 

Skin 

Keratoacanthoma 

Squamous cell papilloma 

Subcutaneous tissue, fibroma 

Subcutaneous tissue, lipoma 

Subcutanr:ous tissue, schwannoma benign 

Subcutaneous tissue, schwannoma malignant 

Musculoskeletal System 

Bone 

Osteosarcoma 

Skeletal muscle 

Hemangiosarcoma 

Nervous System 

Brain 

Meningioma benign 

Peripheral nerve 

Spinal cord 

0112334445555566666777777 

8234682360446844689022222 

5000133066278548246499999 

-_ _ ~ ~ ~~ ~~ ~~ ~~ 

2222222232232223222222222 

6975988909708560598755667 

8601394748455391708948265 

......................... 

.......................... X 

X ......................... 

........................... 

......................... 

x x X xx.xxx xx 

xx 

......................... + ++ + + ......................... 

......................... 

......................... 

+++++++++M+++++++++++++++ 

......................... X X 

......................... X 

X 

X 

X 

......................... X 

+ 

X 

......................... X 

+ 3. 

+ 
Carcass ID Number 

Genital System 

Epididymis 

Mesothelioma malignant, metastatic, 

mesentery 

Preputial gland 

Adenoma 

Carcinoma 

Prostate 

Seminal vesicle 

Testes 

Mesothelioma malignant, metastatic, 

mesentery 

Bilateral, interstitial cell, adenoma 

Interstitial cell, adenoma 

Hematopoietic System 

Bone marrow 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Thymus 

Integumentary System 

Mammary gland 

Fibroadenoma 

Skin 

Keratoacanthoma 

Squamous cell papilloma 

Subcutaneous tissue, fibroma 

Subcutaneous tissue, lipoma 

Subcutaneous tissue, schwannoma benign 

Subcutaneous tissue, schwannoma malignant 

Musculoskeletal System 

Bone 

Osteosarcoma 

Skeletal muscle 

Hemangiosarcoma 

Nervous System 

Brain 

Meningioma benign 

Peripheral nerve 

Spinal cord 

2223322222222222222222333 Total 

8890055666666777788999000 Tissues1 

1610769013457237837259369 Tumors 

......................... 50 

X X 2 

......................... 50 

X x xx 5 

X 2 

......................... 50 

......................... 50 

......................... 50 

X X 2

xx'xxxxxxxxxxxxxxxx xxxxxx 34 

X 3 

......................... 50 

+ 6 

......................... 50 

++++++++++++++M++++++++++ 49 

......................... 50 

......................... 49 

+++M+++++++++++++++++++++ 49 

x . x 4 

......................... 50 

X X 3 

X 1 

x x X X X 6 

X 1 

1 

X 2 

........................... 50 

1 

1 

1 

......................... 50 

1 

2 

1 
120 Salicylazosulfapyridine, NTP TR 457 

TABLEA2 

Individual Animal Tumor Pathologyof Male Rats in the 2-Year Gavage Studyof Salicylazosulfapyridine: 

337.5 mg/kg (Stop-Exposure)(continued) 

0112334445555566666777777 

Number of Days on Study 8234682360446844689022222 

500013306627854824649'9999 

-22.222222322-32223222222222 

Carcass ID Number 6975988909708560598755667 

8601394748455391708948265 

Respiratory System 

Lung ......................... Alveolar/bronchiolar carcinoma 

Osteosarcoma, metastatic, bone X 

Nose ......................... Trachea .......................... ~~ ~ ~ 

Eye 

Special Senses System + 

Urinary System 

Kidney 

Osteosarcoma, metastatic, bone 

......................... X 

Urinary bladder +A+++++A++++A++++++++++++ 

Systemic Lesions 

Multiple organs 

Leukemiamononuclear X 

......................... x x 

Mesothelioma malignant 
~~ ~ 

Number of Days on Study 

Carcass ID Number 

Respiratory System 

Lug

Alveolarlbronchiolar carcinoma 

Osteosarcoma, metastatic, bone 

Nose 

Trachea 

Special Senses System 

E Ye 

Urinary System 

Kidney 

Osteosarcoma, metastatic, bone 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Leukemia mononuclear 

Mesothelioma malignant 

~~ ~~ ~ ~~ ~ 

7771777777777777777777717 

2222233333333333333333333 

9999900000000000000000000 

2223322222222222222222333 Total 

8890055666666777788999000 Tissues/ 

1610769013457237837259369 Tumors 

......................... 50 

X X 2 

1 

......................... 50 

......................... 50 

1 

......................... 50 

1 

......................... 47 

......................... 50 

x xx x x x X 10 

X X 2 
122 Salicylazosulfapyridine, NTP TR 457 

TABLEA3a 

Statisticall Analysis of Primary Neoplasms in Male Rats in the 2-Year Gavage Study of Salicylazosulfapyridine 

Vehicle Control 

AdrenalMedulla:BenignPheochromocytoma 

Overall ratea 15/50 (30%) 

Adjusted rateb 38.2% 

Terminal ratec 11/35 (31X )

First incidence (days) 653 

Life table testd P=0.303N 

Logistic regression testd P=O. 147N 

Cochran-Arnlita e testd P=O.@lN I Fisher exact test 

Adrenal Medulla: Benign, Complex, or Malignant Pheochromocytoma 

Overall rate 16/50 (32%) 

Adjusted rate 39.8% 

Terminal rate 11/35 (31%) 

First incidence (days) ' 653 

Life table test P=0.262N 

Logistic regression test ' P=0.114N 

Cochran-Annitage test P=0.068N 

Fisher exact test 

MammaryGland:Fibroadenoma 

Overall rate ' 3/50 (6%) 

Adjusted rate 8.6% 

Terminal rate 3/35 (9%) 

Firstincidence(days) , 729 (T) 

Life table test P=O.378 

Logistic regression test P=O.493 

Cochran-Amitage test P=0.554N 

Fisher exact test 

Mammary Gland Fibroadenoma or Carcinoma 

Overall rate 3/50 (6%) 

Adjusted rate 8.6% 

Terminal rate 3/35 (9%) 

First incidence (days) 729 (T)

Life table test P=O.357 

Logistic regression test P=O.477 

Cochran-Armitage test P=O.569N 

Fisher exact test 

Pancreas:Adenoma 

Overall rate 12/50 (24% 

Adjusted rate 31.4% 

Terminal rate 9/35 (26%) 

First incidencme (days) 684 

Life table test P=O.282 

Logistic regression test P=O.482 

Cochran-Armitage test P=0.488N 

Fisher exact tiest 

84 mg/kg 

7/49 (14%) 

21.2% 

7/33 (21%) 

729 (T) 

P=0.064N 

P=0.057N 

P=0.050N 

7/49 (14%) 

21.2% 

7/33 (21%) 

729 (T) 

P=0.045N 

P=0.036N 

P=0.031N 

4/50 (8 X) 

11.5% 

3/33 (9%) 

69 1 

P=O.468 

P=O.466 

P=O.500 

4/50 (8%) 

11.5% 

3/33 (9%) 

691 

P=O.468 

P=O.466 

P=O.500 

6/50 (12%) 

17.3% 

5/33 (15%) 

659 

P=0.127N 

P=0.116N 

P=0.096N 

168 mg/kg 

6/50 (12%) 

18.7% 

5/31 (16%) 

704 

P=0.054N 

P=0.041N 

P=0.024N 

7/50 (14%) 

21.8% 

6/31 (19%) 

704 

P=0.067N 

P=0.048N 

P=0.028N 

5/50 (10%) 

15.4% 

4/31 (13%) 

696 

P=O.292 

P=O.286 

P=O.357 

6/50 (12%) 

18.0% 

4/31 (13%) 

696 

P=O.187 ' 

P=O.185 

P=O.243 

7/50 (14%) 

20.7% 

5/31 (16%) 

61 1 

P=0.243N 

P=O.214N 

P=0.154N 

337.5 mg/kg 

8/49 (16%) 

27.5% 

4/23 (17%) 

635 

P=0.324N 

P=0.127N 

P=0.084N 

8/49 (16%) 

27.5% 

4/23 (17%) 

635 

P=0.265N 

P=0.087N 

P=0.056N 

3/50 (6%) 

11.4% 

2/23 (9%) 

684 

P=O.480 

P=O.577 

P=0.661N 

3/50 (6%) 

11.4% 

2/23 (9%) 

684 

P=O.480 

P=O.577 

P=0.661N 

10150 (20%) 

35.9% 

6/23 (26%) 

704 

P=O.394 

P=O.590 

P=0.405N 
Pancreas:AdenomaorCarcinoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

Pancreatic!Islets:Adenoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

Pancreatic !Islets: Adenoma or Carcinoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

Pituitary Gland (Pars Distalis): Adenoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

PreputialGland:Carcinoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

VehicleControl 

13/50 (26%) 

34.1 X 

10135 (29%) 

684 

P=O.353 

P=0.537N 

P=0.403N 

2/50 (4%) 

5.3% 

1/35 (3%) 

684 

P=O.O56 

P=O.127 

P=O.146 

3/50 (6%) 

8.1% 

2/35 (6%) 

684 

P=O.124 

P=O.245 

P=O.280 

14/50 (28%) 

34.3% 

9/35 (26%) 

473 

P=O.O37 

P=O.162 

P=O.198 

2/50 (4%) 

5.3% 

1/35 (3%) 

708 

P=0.291N 

P=0.226N 

P=0.241N 

84 mglkg 

6/50 (12,%) 

17.3% 

5/33 (15 %) 

659 

P=O.O87N 

P=0.077N 

P=0.062N 

3/50 (6%) 

9.1% 

3/33 (9%) 

729 (T) 

P=O.472 

P=O.474 

P=O.500 

4/50 (8 %) 

12.1% 

4/33 (12 %) 

729 (T) 

P=O.466 

P=O.467 

P=O.500 

16/50 (32%) 

43.1% 

12/33 (36%) 

680 

P=O.345 

P=O.355 

P=O.414 

1/50 (2%) 

2.2% 

0/33 (0%) 

424 

P=0.527N 

P=O.47ON 

P=O.5OON 

168 mg/kg 

7/50 (14%) 

20.7% 

5/31 (16%) 

61 1 

P=0.180N 

P=0.154N 

P=O.l05N 

6/50 (12%) 

17.1% 

4/31 (13%) 

502 

P=O.109 

P=O.125 

P=O.134 

6/50 (12 X) 

17.1% 

4/31 (13%) 

502 

P=O.199 

P=O.224 

P=O.243 

18/49 (37%) 

45.4% 

10131 (32%) 

396 

P=O.174 

P=O.216 

P=O.238 

3/50 (6%) 

7.4% 

0/31 (0%) 

502 

P=O.451 

P=O.518 

P=O.500 

337.5 mg/kg 

10150 (20%) 

35.9% 

6/23 (26%) 

704 

P=O.469 

P=O.526N 

P=0.318N 

5/50 (10%) 

18.3% 

3/23 (13%) 

613 

P=O.113 

P=O.190 

P=O.218 

5/50 (10%) 

18.3% 

3/23 (13%) 

613 

P=O.198 

P=O.314 

P=O.357 

18/49 (37%) 

50.5% 

7/22 (32%) 

428 

P=O.O64 

P=O.216 

P=O.238 

0150 (0%) 

0.0% 

0/23 (0%) -e 

P=0.330N 

P=0.269N 

P=0.247N 
124 Salicylazosulfapyridine,NTP TR 457 

TABLEA?la 

Statistical Analysisof Primary Neoplasmsin Male Ratsin the 2-Year Gavage Study of SalicylazosuJfapyridine(continued) .. . 

VehicleControl 84 mg/kgmg/kg 168 337.5 mg/kg 

Preputial Gland: Adenoma or Carcinoma 

Adjusted rate 

Overall rate 

7.7% 

3/50 (6%) 

5.1% 

2/50 (4%) 

13.4% 

5/50 (10%) 

0.0% 

0150 (0%) 

First incidence (days) 

Terminal rate 

684 

1/35 (3%) 

424 

1/33(3%) 

502 

2/31 (6%) 0123 (0%) -

Life table test P=0.238N P=0.533N P=O.303 P=0.185N 

Logistic regression test P=0.156N P=0.486N P=O.357 P=0.133N 

Cochran-Armitage test P=0.161N 

Fisher exact test P=0.500N P=O.357 P=0.121N 

Skin: Squamous CellPapilloma 

Adjusted rate 

Overall rate 

2.9% 

1/50 (2%) 

6.1% 

2/50 (4%) 

9.7% 

3/50 (6%) 

3.1% 

1/50 (2%) 

Life table test 

First incidence (days) 

Terminal rate 

P=O.485 

729 (T) 

1/35 (3%) 

P=O.479 

729 (T) 

2/33 (6%) 

P=O.262 

729 (T) 

3/31 (10%) 

P=O.691 

696 

0123 (0%) 

' 

Logistic regression&st P=O.547 P=O.479 P=O.262 P=O.741 

Cochran-Armitage test P=0.577N 

Fisher exact test P=O.500 P=O.309 P=0.753N 

Skin: Keratoacanthoma , .. 

Adjusted rate 

Overall rate 

8.6% 

3/50 (6%) 

8.8% 

3/50 (6%) 

8.0% 

3/50 (6%) 

6.8% 

2/50 (4%) 

Terminal rate 3/35 (9%) 2/33 (6%) 0/31 (0%) 0123 (0%) 

First incidence (days) 687 729 (T) 726 639

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

Skin: Squamous Cell Papilloma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidenc'e (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

Skin: Basall CellAdenoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

or Keratoacanthoma 

P=0.545N 

P=0.438N 

P=0.397N 

4/50 (8X) 

11.4% 

4/35 (11%) 

729 (T) 

P=O.522 

P=0.473N 

P=0.401N 

2/50 (4%) 

5.4% 

1/35(3X)

717 

P=0.120N 

P=0.092N 

P=0.073N 

P=O.609 

P=O.637 

P=O.635 

P=O.629 

P=0.661N 

5/50 (10%) 

14.7% 

4/33 (12%) 

726 

P=O.463 

P=O.453 

P=O.500 

3/50 (6%) 

8.7% 

2/33 (6%) 

709 

P=O.467 

P=O.473 

P=O.500 

P=0.661N 

6/50 (12X) 

16.9% 

3/31 (10%) 

639 

P=O.304 

P=O.314 

P=O.370 

0150 (0%) 

0.0% 

0131 (0%) -

P=0.276N 

P=0.263N 

P=0.247N 

P=0.655N 

P=0.569N 

P=0.500N 

3/50 (6%) 

9.7% 

0123 (0%) 

687 

P=O.627 

P=0.574N 

P=O.500N 

0/50 (0%) 

0.0% 

0/23 (0%) -

P=0.331N 

P=0.282N 

P=0.247N 

126 'Salicylazosulfapyridine,NTP TR 457 

VehicleControl 84 mg/kg 168 mg/kg 337.5 mg/kg 

~ 

Testes:Adenoma 

First incidence (days) 

Terminal rate 

Adjusted raes 

Overall rate 

. . 

477 

34/35 (97A ) 

97.8% 

45\50 (90%) 

515 

30/33 (91X) 

93.2% 

41/49 (84%) 

414 

31/31 (100%) 

100.0% 

46/50 (92%) 

473 

23/23 (100%) 

100.0% 

45/50 (90%) 

Life table test P=O.003 P=0.462N P=O.157 P=O.o09 

Logistic regression test P=O.208 P-0.434N P=O.253- P=O.405 

Cochran-Annitage test P-0.405 

Fisher exact test P=0.264N P=O.500 P=0.630N . 

ThyroidGland(C-cell):Adenoma 

Adjusted rate 

Overall rate 

8.6% 

3/50 (6%) 

25.4% 

9/50 (1896) 

14.6% 

5/50 (10%) 

22.9% 

6/50 (12%) 

Life table test 

First incidence (days) 

Terminal rate. 

P=O.172 

729 (T) 

3/35 (9%) 

P=O.O50 

680 

7/33 (2146) 

P=O.298 

680 

3/31 (10%) 

P=O.O93 

696 

4/23 (17%) 

Logistic regression test P20.293 P=O.O48 P=O.299 P=O. 145 

Cochran-Arnnitage test P=O.397 

Fisher exact test P=O.o61 P=O.357 P=O.243 

Adjusted rate 

Overall rate 

Thyroid Gl.and (C-cell): Adenoma or Carcinoma 

14.3% 

5/50 (105%) 

31.2% 

11/50 (22%) 

20.7 % 

7/50 (14%) 

24.9% 

7/50 (14%) 

Life table test 

First incidence (days) 

Terminal rate 

P=O.235 

729 (T) 

5/35 (14%). 

Pe0.066 

680 

9/33 (27%) 

P=O.301 

680 

5/31 (16%) 

P=O. 162 

653 

4/23 (17%) 

Logistic regression test P=O.404 P=O.o63 P=O.298 P=O.278 

Cochran-Amitage test P=O.527 

Fisher exact test ' P=O.O86 P=O.380 P=O.380 

Adjusted rate 

Overall rate 

Thyroid Gland (Follicular Cell): Adenoma 

11.4% 

4/50 (8 46) 

0.0% 

0150 (0%) 

8.5% 

3/50 (6%) 

4.3 x

1/50 (2%) 

Life table test 

First incidence (days) 

Terminal rate 

P=0.353N 

729 (T) 

4/35 (1 1 96) 0133 (0%) 

P=O.O7ON 

- P=0.558N 

502 

2/31 (6%) 

P=0.324N 

729 (T) 

1/23 (4%) 

Logistic regmsion test P=O.25ON P=O.O70N P=O.524N P=0.324N 

Cochran-Annitage test P=0.232N 

Fisher exact (est P=O.O59N P=O.500N P=0.181N 

Adjusted rate 

Overall rate 

Thyroid Gl,md (Follicular Cell): Adenoma or Carcinoma 

14.3% 

5/50 (10%) 

0.0% 

0/50 (0%) 

11.2% 

4/50 (8 X) 

4.3% 

1/50 (2%) 

Life table test 

First incidence (days) 

Terminalate . 

P=0.273N 

729 (T) 

5/35 (14%) 0/33 (0%) 

P=0.038N 

-

P=0.569N 

502 

2/31 (6%) 

P=0.221N 

729 (T) 

1/23 (4%) 

Logistic regression test P=O.l8ON P=0.038N P=0.534N P=0.221N 

Fisher exact test 

Cochran-Amitage test P=O.l62N 

P=0.028N P=O.5OON P=O.l02N 
I 

i i

j I

i 

I 

i 

j 

Urinary Bladder: Papilloma

i

I Overall rate . 

i Adjusted rate 1 Terminal rate 

First incidence (days)

I

i Life table test 

Logistic regression test I Cochran-Armitage test 

Fisher exact test

iI

I AI1 Organs: MalignantMesothelioma 

I Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) i Life table test i 

Logistic regression test j Cochran-Armitage test 

Fisher exact test

II

! All Orgians: Mononuclear Cell Leukemia 

Overall rate I Adjusted rate 1 Terminal rate 1 First incidence (days) 

i Life table test j Logistic regression test i

i Cochran-Armitage test 

i Fisher exact test 

All Organs: Benign Neoplasms 

Overall rate I Adjusted rate 

Terminal rate I First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

I

i All Organs: Malignant Neoplsasm I 

I 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days)

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

0/50 (0%) 

0.0% 

0135 (0%) -

P<O.001 

-p<o.001 P<0.001 

1/50 (2%) 

2.9% 

1/35 (3%) 

729 (T)

P=O.094 

P=O.149 

P=O.165 

13/50 (26%) 

32.0% 

8/35 (23 %) 

477 

P=0.021N 

P=0.002N 

P=0.002N 

49/50 (98%) 

100.0% 

35/35 (100%) 

473 

P=O.007 

P=O.518 

P=O.5OON 

23/50 (46%) 

49.9% 

12/35 (34%) 

477 

P=0.045N 

P<0.001N 

P=O.OOlN 

0/49 (0%) 

0.0% 

0133 (0%) ----

1/50 (2%) 

3.0% 

1/33 (3%) 

729 (T) 

P=O.749 

P=O.749 

P=0.753N 

18/50 (36%) 

44.3% 

11/33 (33%) 

414 

P=O.167 

P=O.165 

P=O.194 

4/50 (88%) 

100.0% 

33/33 (100%) 

515 

P=0.401N 

P=0.086N 

P=0.056N 

24/50 (48%) 

55.1% 

14/33 (42%) 

414 

P=O.412 

P=O.385 

P=O.500 

2/50 (4%) 

6.5% 

2/31 (6%) 

729 (T) 

P=O.212 

P=O.212 

P=O.247 

2/50 (4%) 

5.9% 

1/31 (3%) 

695 

P=O.463 

P=O.465 

P=O.500 

12/50 (24%) 

29.5% 

4/31 (13%) 

435 

P=O.545 

P=0.501N 

P=0.500N 

48/50 (96%) 

100.0% 

31/31 (100%) 

396 

P=O.264 

P=0.886N 

P=0.500N 

24/50 (48 %) 

51.9% 

9/31 (29%) 

414 

P=O.340 

P=O.580 

P=O.500 

6/50 (12%) 

22.1% 

3/23 (13%) 

653 

P=O.o06 

P=O.O11 

P=O.O13 

3/50 (6%) 

10.3% 

1/23 (4%) 

585 

P=O.207 

P=O.292 

P=O.309 

3/50 (6%) 

6.8% 

0123 (0%) 

428 

P=0.040N 

P=O.OWN 

P=O.WN 

47/50 (94%) 

100.0% 

23/23 (100%) 

428 

P=O.025 

P=0.998N 

P=0.309N 

9/50 (1 

8%) 

22.8% 

1/23 (4%) 

428 

P=0.049N 

P=O.002N 

P=0.002N 
128 Salicylazosulfapyridine, NTP TR 457 

TABLEA321 

Statistical .Analysis of Primary Neoplasms inMale Rats in the 2-Year Gavage Study of Salicylazosulfapyridine(continued)

Vehicle Control 84 mglkg 168 mglkg 337.5 mglkg

All Organs: BenignorMalignantNeoplasms 

Overall rate 49/50 (98%) 46/50 (92%) 49/50 (98%) . 48/50 (96%) 

Adjusted rate 100.0% 100.0%100.0% 100.0% 

33/33 (100%)

414 

Life table test P=O.007 P=0.561N P=O.211 P=O.O18 

Logistic regression test P=O.512 P=0.722N -f -

Cochran-Armitage test P=O.597 

Fisher exact test P=0.500N P=0.181N P=0.753N 

(T)Terminal sacrifice 

a Number of neoplasm-bearing animalslnumber of animals examined. Denominator is number of animals examined microscopically for adrenal gland, 

pancreas, pancreatic islets, pituitary gland, preputial gland, testes, thyroid gland, and urinary bladder; for other tissues, denominator is number,of animals 

necropsied. 

Kaplan-Meier estimated neoplasm incidence at the end of the study after adjustment for intercurrent mortality 

Observed incidence at terminal kill 

Beneath the vehicle control incidence are theP values associated with the trend test. Beneath the dosed group incidence are theP values corresponding to 

pairwise comparisons between the vehicle controls and that dosed group. The life table test regards neoplasms in animals dying prior to terminal kill as 

being (directly or indirectly) the cause of death. The logistic regression test regards these lesions as nonfatal. The Cochran-Armitage and Fisher exact tests 

compare directly the overall incidence rates. For all tests, a negative trend or a lower incidence in a dose group is indicatedby N. e Not applicable; no neoplasms in animal group 

Value of statistic cannot be computed. 

Terminal rate 

First incidence. (days) 428 396 

35/35 (100%)

473 

31/31 (100%) 23/23 (100%) 
129 Salicylazosul~aa9yricine,NTP TR 4§7 

337.5 mglkg 

4/50 (8%) 

13.3% 

4/30 (13%) 

729 (T) 

P=0.016N 

P=0.015N 

P=0.005N 

6/50 (12 %) 

19.4% 

5/30 (17 %) 

704 

P=0.048N 

P=0.039N 

P=0.014N 

4/50 (8 %) 

12.1% 

2/30 (7%) 

644 

P=O.414 

P=O.412 

P=O.500 

4/48 (8%) 

12.8% 

3/30 (10%) 

696 

P=0.064N 

P=0.062N 

P=0.033N 

4/48 (8%) 

12.8% 

3/30 (10%) 

696 

P=0.041N 

P=0.040N 

P=0.019N 

0149 (0%) 

0.0% 

0130 (0%) -e

P=0.154N 

P=O.l5ON 

P=0.125N 

Adrenal Medulla: Benign Pheochromocytoma 

Overall ratea 

Adjusted rateb 

Terminal rate' 

First incidence (days) 

Life table testd 

Logistic regression testd 

Fisher exact testd 

Adrenal Medulla: Benign, Complex, or Malignant Pheochromocytoma 

Vehicle Control 

15/50 (30%) 

38.2% 

11/35 (31%) 

653 . 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Fisher exact test 

MammaryGland:Fibroadenoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Fisher exact test 

Pancreas:Adenoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Fisher exact test 

Pancreas:Adenoma or Carcinoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Fisher exact test 

Pancreatic Islets: Adenoma or Carcinoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Fisher exact test 

16/50 (32%) 

39.8% 

11/35 (31%) 

653 

3/50 (6%) 

8.6% 

3/35 (9%) 

729 (T) 

12/50 (24%) 

31.4% 

9/35 (26%) 

684 

13/50 (26%) 

34.1 % 

10135 (29%) 

684 

3/50 (6%) 

8.1I 

2/35 (6%) 

684 
130 Salicylazosulfapyridine, NTP TR 457 

Vehicle Control 

PituitaryGlland (Pars Distalis): Adenoma 

Overall rate 14/50 (28X)

Adjusted rate 34.3% 

Terminal rate 9/35 (26%) 

First incidence (days) 473 

Life table test 

Logistic regression test 

Fisher exact test 

PreputialGland:Adenoma 

Overall rate 1/50 (2%) 

Adjusted rate 2.5% 

Terminal rate: 0/35 (0%) 

First incidence (days) 684 

Life table test 

Logistic regrcssion test 

Fisher exact last 

PreputialGland: Adenoma or Carcinoma 

Overall rate 3/50 (6%) 

Adjusted rate 7.7% 

Terminal rate 1/35 (3%) 

First incidence (days) 684 

Life table test 

Logistic regression test 

Fisher exact test 

Skin: Keratoacanthoma 

Overall rate 3/50 (6%) 

Adjusted rate 8.6% 

Terminal rate 3/35. (9%) 

First incidence (days) 729 (T)

Life table test 

Logistic regression test 

Fisher exact test 

Skin: Squamous Cell Papilloma or Keratoacanthoma 

Overall rate 4/50 (8X) 

Adjusted rate 11.4% 

Terminal rate 4/35 (1 1%) 

First incidence (days) 729 (T)

Life table test 

Logistic regression test 

Fisher exact test 

Skin: Squamous Cell Papilloma, Keratoacanthoma, or Basal Cell Adenoma 

Overall rate 6/50 (12I) 

Adjusted rate 16.5% 

Terminal rate 5/35 (14%) 

First incidence (days) 717 

Life table test 

Logistic regression test 

Fisher exact test 

~ 

337.5 mgkg 

18/49 (37%) 

46.4% 

10/30 (33%) 

423 

P=O.140 

P=O.177 

P=O.238 

5/50 (10%) 

16.7% 

5/30 (17%) 

729 (T) 

P=O.o72 

P=O.O64 

P=O.102 

7/50 (14%) 

21.8% 

6/30 (20%) 

383 

P=O.106 

P=O.122 

P=O.159 

3/50 (6%)

10.0% 

3/30 (10%) 

729 (T) 

P=O.591 

P=O.591 

P=0.661N 

4/50 (8%) 

13.3% 

4/30 (13A) 

729 (T)

P=O.557 

P=O.557 

P=0.643N 

4/50 (8%) 

13.3% 

4/30 (13X) 

729 (T) 

P=0.474N 

P=0.502N 

P=O.37ON 
Sran~cynn;eQSuBfra~yg.PiBine, NTp 457 131 

TABLE &fib 

StatbticaU AnnaUysb off Wman-y NeopUmms hn “le Iwsnas in Uhe St~p-lE~p~re ~Ev~UU~U~UHI off SaPcyUarzosoBffapy~~~~nne

(continued) 

Vehicle Control 337.5 mg/kg 

Skin (SubcutaneousTissue):Fibroma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Fisher exact test 

Skin (Subcutaneous Tissue): Benignor MalignantSchwannomra 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Fisher exact test 

Testes:Adenoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Fisher exact test 

ThyroidGland(C-cell):Adenoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Fisher exact test 

Thyroid Gland (C-cell): Adenoma or Carcinoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Fisher exact test 

Thyroid Gland (Follicular Cell):Adenoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Fisher exact test 

2/50 (4%) 

5.7% 

2/35 (6%) 

729 (T) 

Ob0 (0%) 

0.0% 

0135 (0%) -

45/50 (90%) 

97.8% 

34/35 (97%) 

477 

3/50 (6%) 

8.6% 

3/35 (9%) 

729 (T) 

5/50 (10%) 

14.3% 

5/35 (14%) 

729 (T) 

4/50 (8%) 

11.4% 

4/35 (11%) 

729 (T) 

6/50 (12%) 

19.3% 

5/30 (17 %) 

696 

P=O.O89 

P=O.O77 

P=O.134 

3/50 (6%) 

7.9% 

1/30 (3%) 

423 

P=O.103 

P=O.156 

P=O.121 

37/50 (74%) 

97.3% 

29/30 (97%) 

423 

P=0.466N 

P=0.308N 

P=0.033N 

4/50 (8%) 

12.3% 

2\30 (7 X)

648 

P=O.409 

P=O.410 

P=O.500 

6/50 (12%) 

18.5% 

4/30 (13 %) 

648 

P=O.387 

P=O.373 

P=O.500 

0150 (0%) 

0.0% 

0130 (0%) -

P=0.083N 

P=0.083N 

P=0.059N 
132 Salicylazosulfapyridine, NTP TR 457 

TABLEA:3b 

Statistical Analysis of Primary Neoplasms in Male Rats in the Stop-Exposure Evaluationof Salicylazosulfapyridine 

(continued) 

Thyroid Gland (Follicular Cell): Adenoma or Carcinoma 

Overall rate 

Adjusted ratt: 

Terminal rata 

First incidence (days) 

Life table test 

Logistic regression test 

Fisher exact test 

All Organs: Mononuclear Cell Leukemia 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Fisher exact test 

AllOrgans: Benign Neoplasms 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Fisher exact test 

All Organs: Malignant Neoplasms 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Fisher exact test 

All Organs: Benign or Malignant Neoplasms 

Overall rate 

Adjusted rate 

Terminal rate: 

First incidence (days) 

Life table test 

Logistic regression test 

Fisher exact test 

(T)Terminal :sacrifice 

Vehicle Control 337.5 mglkg 

5/50 (10%) 2/50 (4%) 

14.3X 6.7% 

5/35 (14%) 2/30 (7 %) 

729 (T) 729 (T) 

P=0.280N 

P=0.280N 

P=0.218N 

13/50 (26%) 10150 (20%) 

32.0% 30.9% 

8/35 (23 %) 8/30 (27 %) 

477 648 

P=0.492N 

P=0.468N 

P=0.318N 

49/50 (98 %) 42/50 (84%) 

100.0% 100.0% 

35/35 (100%) 30/30 (100%) 

473 423 

P=O.552 

P=0.329N 

P=0.015N 

23/50 (46%) 21/50 (42%) 

49.9% 55.7% 

12/35 (34%) 14/30 (47%) 

477 361 

P=O.459 

P=0.525N 

P=0.420N 

49/50 (98%) 46/50 (92 %) 

100.0% 100.0% 

. 35/35 (100%) 30130 (100%) 

473 361 

P=O.259 

P=O.814 

P=0.181N 

a Number of neoplasm-bearing animalslnumber of animals examined. Denominator is number of animals examined microscopically for adrenal gland, 

pancreas, pancreatic islets, pituitary gland, preputial gland, testes, and thyroid gland; for other tissues, denominator is number of animals necropsied. 

Kaplan-Meier estimated neoplasm incidence at the end of the study after adjustment for intercurrent mortality 

Observed incidence at terminal kill 

Beneath the dosed group incidence are the P values corresponding to painvise comparisons between the vehicle controls and the dosed group. The life table 

test regards neoplasms in animals dying prior to terminal kill as being (directly or indirectly) the cause of death. The logistic regression test regards these 

lesions as nonfatal. The Fisher exact test compares directly the overall incidence rates. For all tests, a lower incidence in the dosed group is indicated by 

N. 

e Not app1ic:able;no neoplasms in animal group 

.. 
TABLE A0 

Historical Incidence d UJrinnary Bladder Papilloma in MaleFX4/41/NRats Receiving Corn Oil by Gavagea 

Incidence in Controls 

Overall Historical Incidence 

Total 1” (0.1%) 

Standard deviation 0.5% 

Range 0%-2% 

a Data as of 17 June 1994 
134 Salicylazosulfapyridine,NTP TR 457 

TABLEA!! 

Summary of the Incidence of Nonneoplastic Lesions in Male Ratsin the 2-Year Gavage Study 

of Salicyl:azosulfapyridinea 

Disposition Summary 

Animals initially in study 

6-Month interim evalu&n 

15-Month interim evaluation 

Early deaths 

Accidental deaths 

Moribund 

NaNral deaths 

Survivors 

Died last week of.study 

Terminal sacrifice 

Animals examined microscopically 

6-Month Interim Evaluation 

Alimentary System 

Intestine large, colon 

Parasite metazoan 

Intestine large, rectum 

Parasite metazoan 

Liver 

Clear cell focus 

Granuloma 

Inflammation, chronic 

Metaplasia, osseous 

Pancreas 

Atrophy 

Tongue 

Hemorrhage 

Cardiovascular System 

Heart 

Cardiomyopathy 

Endocrine System 

Adrenal cortex 

Accessory adrenal cortical nodule 

Pituitary gland ’

Pars distalis, cyst 

Pars distalis, hyperplasia, focal 

Pars intermedia, cyst 

Thyroid gland 

Follicular c,ell, hypertrophy 

VehicleControl 84 mg/kgmg/kgmg/kg 337.5337.5 mglkg 168

70 60 

10 

10 10 

1 4 

13 11 

1 2 

35 33 

70 60 

(10) 

7 (70%) 

(10) 

1 (10%) 

(10) . . 

2 (20%) 

1(10%) 

(10)

2 (20%) 

60 

10 

1 

16 

2 

1 .. 

30 

60 

60 

10 

2 

15 

10 

23 

60 

(Stop-Exposure) 

70 

10 

10 

3 

13 

4 

30 

70 

1 (10%) 

1 (10%) 

(10) 

1 (10%) 

a Number of animals examined microscopicallyat the site and the number of animals with lesion 
6-ikfonEhInEerim ~VQ~ldQEbR(continued) 

Genital System 

Preputial gland (10) 

Intlamrnation, chronic 7 (70%) 

Inflammation, suppurative 2 (20%) 

Prostate (10) 

Corpora amylacea 1 (10%) 

Inflammation, suppurative 

Hematopoietic System 

Lymph node, mandibular 

Hyperplasia, lymphoid 

Pigmentation 

Lymph node, mesenteric 

Hemorrhage 

Spleen 

Hematopoietic cell proliferation 

Pigmentation, hemosiderin 

Thymus 

Hemorrhage 

Integumentary System 

Skin 

Cyst epithelial inclusion 

~ 

Respiratory System 

Lung (10) 

Hemorrhage 1 (10%) 

Inflammation, subacute 2 (20%) 

Alveolar epithelium, liyperplasia 1 (10%) 

Nose (10) 

Exudate 

Foreign body 

Fungus 

Mucosa, hyperplasia 

Mucosa, metaplasia, squamous 

Urinary §ystem 

Kidney (10) 

Mineralization 1 (10%) 

Nephropathy 8 (80%) 

Renal tubule, dilatation 

Systems Examined With No Lesions Observed 

General Body §ystem 

Musculoskeletal System 

Nervous System 

Special Senses System 

(10) 

7 (70%) 

1 (10%) 

(10)

1 (10%) 

1 (10%) 
136 Salicylazosulfapyridine, NTP TR 457 

TABLEA5 

SummarJrof the Incidenceof NonneoplasticLesions in MaleRats in the %Year Gavage Study 

of Salicy1,azosulfapyridine(continued) 

Vehicle Control 84 mug 

15-Month Interim Evaluation 

Alimentary System 

Intestine large, colon 

Parasite metazoan 

Intestine large, rectum 

Parasite metazoan 

Intestine small, ileum 

Hyperplasia, lymphoid 

Liver 

Basophilic focus 

Clear cell focus 

Granuloma 

Hepatodiaphragmatic nodule 

Inflammation, chronic 

Mixed cell focus 

Bile duct, hyperplasia 

Hepatocyte, vacuolization cytoplasmic 

Lobules, necrosis 

Mesentery 

Fat, hemorrhage 

Fat, necrosis 

Pancreas 

Atrophy 

Acinar cell, basophilic focus 

Acinar cell, hyperplasia, focal 

Stomach, forestomach 

Edema 

Mineralization 

Cardiovascular System 

Heart (10) (10)

Cardiomyopathy 6 (60%) 4 (40%) 

Inflammation, subacute 1 (10%) 

Pericardium, fibrosis 

Endocrine System 

Adrenal cortex 

Accessory adrenal cortical nodule 

Degeneration, fatty 

Hyperplasia, focal 

Pituitary gland 

Pars distalis, angiectasis 

.Pars distalis, cyst 

Pars distalis, hyperplasia, focal 

Thyroid gland 

Ultimobranchial cyst 

C-cell, hyperplasia 

Follicle, cyst 1(10%) 

Follicular dl, hypertrophy 

/.l,. . . .,., 

168 mg/kg . 337.5 mglkgmg/kg 337.5 

(Stop-Exposure) 

6 (60%) 

6 (60%) 

2 (20%) 

1 (10%) 

(1) 

1(100%) 

(10) 

5 (50%) 

1 (10%) 

3 (30%) 

(10) 

1(10%) 

(10) (10) (10)

6 (60%) 3 (30%) 5 (50%) 

1 (10%) 
I

i

I 

! 

Is-MOnEh hE6?&?8 &&Wih. 

Genital System 

Epididymis 

Atypia cellular 

Hypospermia 

Preputial gland 

Inflammation, chronic 

Inflammation, suppurative 

Prostate 

Corpora amylacea 

Edema 

Inflammation, suppurative 

Testes 

Interstitial cell, hyperplasia 

Seminiferous tubule, atrophy 

I 

Hematopoietic System 

Lymph node 

Mediastinal, hemarrhage 

Lymph node, mandibular 

Ectasia 

Hemorrhage 

Pigmentation 

Lymph node, mesenteric 

Hemorrhage 

Hyperplasia, lymphoid 

Spleen 

Fibrosis 

Hematopoietic cell proliferation 

Hemorrhage 

Pigmentation, hemosiderin 

Thymus 

cyst 

Hemorrhage 

htegumentmny System 

Mammary gland 

Hyperplasia, cystic 

Nervous System 

Brain 

Compression 

VehicleControl . . $4 mgkg 168 mglkg 337.5 mglkg 337.5 mg/kg 

(Stop-Exposure) 

(continued) 

(10) (10) 

2 (20%) 

2 (20%) 

(10) (10)

8 (80%) 8 (80%) 

2 (20%) 2 (20%) 

(10) (10) ' 

2 (20%) 

6 (60%) 7 (70%) 

(10) (10) 

4 (40%) 5 (50%) 

1(10%) 

(9) (9) (10) (10) 

1 (11%) 1 (11%) 2 (20%) 
138 Salicylazosulfapyridine, NTP TR 457 

TABLEA5 

Summar:yof the Incidenceof NonneoplasticLesions in MaleRats in the2-YearGavage Study 

of Salicyllazosulfapyridine(continued) 

VehicleControl 84mg/kg 168 mg/kg mg/kgmg/kg 337.5337.5

(Stop-Exposure) 

15-Month Interim Evaluation (continued) 

Respiratory System 

Lung (10)

Hemorrhage 

Infiltration cellular, histiocyte 1(10%) 

Inflammation, subacute 1 (10%) 

Alveolar epithelium, hyperplasia 1 (10%) 

Nose (10) (10) 

Exudate 1(10%) 

Foreign body 1 (10%) 

Fungus 1(10%) 

Mucosa, hyperplasia 1(10%) 

Mucosa, metaplasia, squamous 1 (10%) 

Urinary System 

Kidney (10) (10) (10) (10) (10) 

Mineralization 2 (20%) 

Nephropathy 10 (100%) 10 (100%) 10 (100%) 10 (100%) 10 (100%) 

Renal tubule, dilatation (10%) 1 2 (20%) 

Transitional epithelium, hyperplasia 1 (10%) 

Bulbourethral gland, ectasia 

Urethra (1) 

1(100%) 

Systems Ekamined With No Lesions Observed 

General Body System 

Musculoskeletal System 

Special Senses System 

2-Year Study 

Alimentary System 

Esophagus 

Inflammation, suppurative 

(49) (49) 

Perforation 

Intestine large, colon 

Inflammation, subacute 

Parasite metazoan 

Intestine large, rectum 

Parasite metazoan 

Ulcer 

Intestine large, cecum 

Dilatation 

Inflammation, subacute 

Parasite metazoan 

Intestine small, duodenum 

Erosion 

Ulcer 

Erosion 

Intestine small, jejunum (49) (50) 

1 (2%) 
n39 

VehicleControl mgM! ns mg/Q 337.5 mgkg 

(Stop-Exposure) 

337.5 mgkg 

Intestine small, ileum

Alimentary System (continued) 

&y@W&%6dy (continued) 

Inflammation, chronic active 

Lobules, necrosis 

Hepatocyte, vacuolization cytoplasmic 

Centrilobular, necrosis 

Centrilobular. atrophy 

Bile duct, hyperplasia 

Mixed cell focus 

Inflammation, subacute 

Inflammation, chronic 

Hepatodiaphragmatic nodule 

Hematopoietic cell proliferation 

Granuloma

Eosinophilic focus 

Developmental malformation

Degeneration, cystic 

Congestion 

Clear cell focus 

Basophilic focus 

Angiectasis 

Liver 

Fat, necrosis 

Fat, hemorrhage 

Fibrosis 

Accessory spleen 

Mesentery 

Acinar cell, hyperplasia, focal 

Acinar cell, cytoplasmic alteration 

Acinar cell, basophilic focus 

Pigmentation 

Atrophy 

Inflammation, chronic 

Atrophy 

Mucosa, hyperplasia 

Ulcer 

Perforation 

Mineralization 

Inflammation, chronic 

Erosion 

Edema 

Pancreas 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Ulcer 

Mineralization 

Erosion 

cyst 

(49) 

(50) 

23 (46%) 

33 (66%) 

2 (4%) 

3 (6%) 

10 (20%) 

1 (2%) 

2 (4%) 

2 (4%) 

1 (2%) 

1 (2%) 

4 (8%) 

39 (78%) 

3 (6%) 

1 (7%) 

1 (7%) 

1 (2%) 

26(52%) 

13 (93%) 

11 (22%) 

1 (2%) 

(14) 

(50) 

(50) 

(50) 

1 (2%) 

1 (2%) 

(50) 

1 (2%) 

(50) 

(50) 

24(48%) 

26 (52%) 

3 (6%) 

1 (2%) 

7 (14%) 

4 (8%) 

38 (76%) 

7 (14%) 

2 (4%) 

2 (4%) 

(18) 

14 (78%) 

1 (6%) 

1(6%) 

14 (28%) 

3 (6%) 

1 (2%) 

1(2%) 

18 (36%) 

1(2%) 

(50) 

(50) 

(50) 

1 (2%) 

1 (2%) 

1 (2%) 

(50) 

1(2%) 

1 (2%) 

20 (40%) 

(50) 

(50) 

16 (32%) 

2 (4%) 

1 (2%) 

9 (18%) 

1 (2%) 

21(42%) 

2 (4%) 

2 (4%) 

1 (8%) 

1 (2%) 

2 (4%) 

(12) 

19 (38%) 

9 (75%) 

(50) 

1 (2%) 

8 (16%) 

15 (30%) 

(50) 

(50) 

9 (18%) 

7 (14%) 

1 (2%) 

1(2%) 

1 (2%) 

2 (4%) 

4 (8%) 

1 (2%) 

2 (4%) 

1 (2%) 

(50) 

(49) 

(50) 

18 (36%) 

34 (68%) 

1 (2%) 

6 (12%) 

1 (2%) 

1 (2%) 

3 (6%) 

1 (2%) 

2 (4%) 

6 (12%) 

2 (4%) 

2 (4%) 

1(2%) 

3 (6%) 

37 (74%) 

(16) 

14 (88%) 

(48) 

21 (44%) 

14 (29%) 

3 (6%) 

3 (6%) 

(50) 

1 (2%) 
140 Salicylazosulfapyridine,NTP TR 457 

TABLEA5: 

Summary of the Incidenceof Nonneoplastic Lesions in Male Rats in the 2-Year Gavage Study 

of Salicylalzosulfapyridine(continued) 

Vehicle Control &I mg/kg 168 mg/kg mg/kgmglkg 337.5337.5 

(Stop-Exposure) 

2-Year Study (continued) 

Cardiovascular System 

Blood vessel (50) (50) Embolus 1 (2%) 

HyperWhy

Inflammation, chronic active 

Heart (50) (50)

Cardiomyopathy 23 (46%) 27 (54%) 

Foreign body 

Inflammation, subacute 

Mineralization 

Thrombosis 

Pericardium; fibrosis 1(2%) 

Adrenal cortex 

Endocrine System 

Basophilic focus 

Accessory adrenal cortical nodule 

(50) 

19 (38%) 

(50) 

19 (38%) 

(50) 

13 (26%) 

(50) 

12 (24%) 

Hemorrhage 

Hematopoietic cell proliferation 

Degeneration, fatty 

cyst 

Congestion 

8 (16%) 

1 (2%) 

1 (2%) 

2 (4%) 

1(2%) 

1 (2%) 

6 (12%) 9 (18%) 

1 (2%) 

2(4%) 

8 (16%) 

1 (2%) 

1 (2%) 

6 (12%) 

Necrosis 

Hypertrophy, focal 

Hyperplasia, focal 

Hyperplasia 

Parathyroid gland 

Hyperplasia 

Islets, pancreatic 

Hyperplasia 

Adrenal medulla 

Congestion 

Pituitary gland 

5 (10%) 

4 (8%) 

1 (2%) 

2 (4%) 

8 (16%) 

(50) 

(49) 

(50) 

(49) 

1 (2%) 

18 (37%) 

3 (6%) 

6 (12%) 

(50) 

(50) 

16 (32%) 

(49) 

(49) 

1 (2%) 

3 (6%) 

2 (4%) 

3 (6%) 

3 (6%) 

(50) 

9(18%) 

(49) 

1 (2%) 

(49) 

Pars distalis, hyperplasia, focal 

Pars distalis, cyst 

Pars distalis, angiectasis 

11 (22%) 

2(4%) 

1 (2%) 

13 (27%) 

3 (6%) 

2(4%) 

12 (24%) 

5 (10%) 

2 (4%) 4 (8%) 

5 (10%) 

5 (10%) 

Pars intermedia, angiectasis 2 (4%) 1 (2%) 1 (2%) 2 (4%) 

Pars intermedia, cyst 1 (2%) 1 (2%) 2(4%) 1 (2%) 

Pars intermedia, hyperplasia 1(2%) 

Pars nervosa, gliosis 1(2%) 

Ultimobranchial cyst 

Thyroid gland (50) 

1(2%) 4 (8%) 

(50) (50) 

2(4%) 

(50) 

1 (2%) 

(50) 

4 (8%) 

Follicular cell, hypertrophy 

Follicular cell, hyperplasia 

Follicle, cyst 

C-cell, hyperplasia 

1(2%) 

1 (2%) 

1(2%) 

14 (28%) 

3 (6%) 

9 (18%) 

2(4%) 

1 (2%) 

10 (20%) 

4 (8%) 

1 (2%) 

11 (22%) 

2 (4%) 

2 (4%) 

3 (6%) 

12 (24%) . 

General Body System 

None 
TABLEA5 

Smmary off the Incidence off Nonneoplastic Lesions ian Male Rats in the %Year Gavage Study 

QfSmBiCynazQSnnnffalpgrffidlirme(continued) 

VehicleControl M mglkg 161 mglkg 337.5 mglkg 337.5 mglkg 

(Stop-Exposure) 

$&" (continued) 

Genital System 

Hypospermia 

Atypia cellular 

Epididymis (50) 

40 (80%) 

25 (50%) 

(50)

33 (66%) 

34 (68%) 

(50) 

37 (74%) 

36 (72%) 

(50) 

36 (72%) 

31 (62%) 

(50)

25 (50%) 

32 (64%) 

Ectasia 

Preputial gland 

20 (40%) 

(50) 

17 (34%) 

(50) (50)

17 (34%) 

(50)

14 (28%) 

(50)

14 (28%) 

Inflammation, chronic 

Hyperplasia 

24 (48%) 

1 (2%) 

18 (36%) 16 (32%) 

1 (2%) 

9 (18%) 

2 (4%) 

25 (50%) 

1 (2%) 

Inflammation, suppurative 

Ectasia 

Corpora amylacea 

Prostate 

19 (38%) 

28 (56%) 

(50) 

28 (56%) 

15 (30%) 

(50) 

10 (20%) 

19 (38%) 

1 (2%) 

(50) 

17 (34%) 

(50)

11 (22%) 25 (50%) 

18 (36%) 

(50) 

1 (2%) 

Hemorrhage 

Edema 

1 (2%) 3 (6%) 

2 (4%) 

Epithelium, hyperplasia 

Inflammation, suppurative 

Hyperplasia, lymphoid 

Hemorrhage 

Dilatation 

Seminal vesicle 

8 (16%) 

17 (34%) 

1 (2%) 

1 (2%) 

(50) 

1 (2%) 

8 (16%) 

19 (38%) 

(50) 

21 (42%) 

13 (26%) 

1 (2%) 

1 (2%) 

(50) 

3 (6%) 

21 (42%) 

2 (4%) 

4 (8%)

(49) 

4 (8%) 

24 (48%) 

2 (4%) 

1 (2%) 

(50) 

Inflammation, suppurative 2 (4%) 4 (8%) 

Seminiferous tubule, atrophy 

Interstitial cell, hyperplasia 

Testes (50)

1 (2%) 

(49) 

2 (4%) 

5 (10%) 

1 (2%) 

8 (16%)

(50) 

2 (4%) 

1 (2%) 

(50) (50) 

3 (6%) 

5 (10%) 

Hematopoietic System 

Bone marrow 

Myelofibrosis 

Hypercellularity 

(50) 

3 (6%) 

(50) 

3 (6%) 

(50) 

5 (10%) 

(50) 

2 (4%) 

2 (4%) 

Mediastinal, pigmentation 

Mediastinal, hyperplasia, lymphoid 

Mediastinal, hemorrhage 

Mediastinal, ectasia 

Mediastinal, congestion 

Inguinal, hyperplasia, lymphoid 

Pigmentation 

Hyperplasia, plasma cell 

Hyperplasia, lymphoid 

Hemorrhage 

Ectasia 

Congestion 

Pigmentation 

Hyperplasia, lymphoid 

Hemorrhage 

Lymph node, mesenteric 

Lymph node 

Lymph node, mandibular 

(9) 

5 (56%) 

6 (67%) 

(50) 

1 (2%) 

5 (10%) 

4 (8%) 

5 (10%) 

(50) 

1 (2%) 

3 (6%) 

(14)

1 (7%) 

7 (50%) 

6(43%) 

(50) 

4 (8%) 

(10) 

5 (50%) 

1(10%) 

4 (40%) 

(49) 

7(14%) 

3 (6%) 

1 (2%) 

(49)

4 (8%) 

(6) 

3 (50%) 

1 (17%) 

3 (50%) 

1 (17%) 

1 (17%) 

(50) 

1 (2%) 

2 (4%) 

3 (6%) 

7(14%) 

4 (8%) 

1 (2%) 

1 (2%) 

(49) 

- - . 
142 Salicylkosulfapyridine, NTP TR 457 

TABLEA5 I (. .

Summar:yof the Incidence'of NonneoplasticLesions in Male Rats in the 2-Year Gavage Study 

of Salicyllazosulfapyridine(continued) ! . 

. . 

VehicleControl 84 mglkg 168 mg/kg 3373 mg/kg 337.5 mag 

(Stop-Exposure) 

&Year Study (continued) 

Hematopoietic System(continued) 

Spleen (50) (50) (50) (50)

Developinlental malformation 1(2%) 

8 (16%) (4 

Hematopoietic cell proliferation 14 (28%) 15 (30%) 17 (34%) 23 (46%) 14 (28%) " 

Hyperplasia, reticulum cell 1 (2%) 

Pigmentation,hemosiderin . 14 (28%) 15 (30%) 15 (30%) 30 (60%) 13 (26%) 

Lymphoid follicle, atrophy 5 (10%) 

Thymus (47) (50) (49) (49) (49) 

cyst 1 (2%) 

Hemorrhage 1 (2%) 1 (2%) 

Cortex, atrophy 2 (4%) 

Integumentary System 

Mammary gland (48) (49) (47) (49) 

Hyperplasia, cystic 11 (23%) 8 (16%) 12 (26%) 10 (20%) 

Fibrosis 8 (16%) 2 (4%) 2 2 (4%) 

Hyperplasia, lobular 2 (4%) 4 (8%) 3 (6%) 

Skin 

6 (12%) 

(50) (50) (50) (50) 

Acanthosis ' . 1 (2%)

Cyst epithelial inclusion 2 (4%) 3 (6%) 2 (4%) 1 (2%) 

Foreign body 1 (2%) 

Hemorrhage 1 (2%) 

Inflammation, chronic 1 (2%) 

Inflammation, suppurative 2 (4%) 

Subcutanenus tissue, edema 1 (2%) 

Musculoskeletal System 

Bone .. (50) (50) (50) (50) 

Calvarium, osteopetrosis 1 (2%) 

Nervous System 

Brain (50) (50) (50) (50) 

Compression 4 (8%) 4 (8%) 8 (16%) 4 (8%) 

Hemorrhage 1 (2%) 

3 (6%) (4%) 

Inflammation, chronic 1(2%) 

Mineralization 1 (2%) 

Necrosis 1 (2%) 1 (2%) 

Peripheral nerve (2) 

Atrophy 1 (50%) 

Hydrocephalus 1 (2%) 2 (4%) 2 
2-y&W &"y (continued) 

Respiratory system 

Lung

Congestion 

Edema 

Fibrosis 

Foreign body 

Hemorrhage 

Infiltration cellular, histiocyte 

Inflammation, granulomatous 

Inflammation, subacute 

Inflammation, suppurative 

Metaplasia, osseous 

Alveolar epithelium, hyperplasia 

Fat, mediastinum, necrosis 

Nose 

Exudate 

Foreign body 

Fungus 

Mucosa, hyperplasia 

Mucosa, metaplasia, squamous 

Trachea 

Hemorrhage 

Bpecial senses system 

Eye

Cataract 

Phthisis bulbi 

Retina, atrophy 

Urinary system 

Kidney 

Concretion 

cyst 

Hemorrhage 

Hydronephrosis

Infarct 

Inflammation, suppurative 

Mineralization 

Nephropathy 

Papilla, fibrosis 

Papilla, necrosis 

Renal tubule, cytoplasmic alteration 

Renal tubule, dilatation 

Renal tubule, hyperplasia

Renal tubule, hyperplasia, oncocytic 

(50) 

1 (2%) 

8 (16%) 

3 (6%) 

10 (20%) 

1 (2%) 

(50) 

15 (30%) 

4 (8%) 

6 (12%) 

6 (12%) 

1 (2%) 

(50) 

(50) 

4 (8%) 

3 (6%) 

5 (10%) 

1 (2%)

8 (16%) 

5 (10%) 

1 (2%) 

3 (6%) 

1 (2%) 

(50) 

12 (24%) 

7 (14%) 

4 (8%) 

1 (2%) 

2 (4%) 

(50) 

(2) 

2 (100%) 

2 (100%) 

1 (2%) 

1 (2%) 

(3) 

3 (100%) 

3 (100%) 

(50) 

3(6%) 

3 (6%) 

1 (2%) 

3 (6%) 

1 (2%) 

4 (8%) 

1(2%) 

2 (4%) 

1 (2%) 

3 (6%) 

(50) 

13 (26%) 

5 (10%) 

4 (8%) 

6 (12%) 

3 (6%) 

(1) 

1 (100%) 

(50) 

8 (16%) 

3 (6%) 

46 (92%) 

1(2%) 

3 (6%) 

Renal tubule, pigmentation 2 (4%) 4 (8%) 1 (2%) 

Transitional epithelium, hyperplasia 10 (20%) 10 (20%) 4 (8%)

Transitional epithelium, hyperplasia, 

atypical

Transitional epithelium, metaplasia 
144 Salicylazosulfapyridine, NTP TR 457 

TABLEA5 

Summary 'ofthe Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Gavage Study 

of Salicylazosulfapyridine(continued) 

Vehicle Control 84 mglkg ' 168 mg/kg 337.5 mg/kg , 337.5 mg/kg 

(Stop-Exposure) 

&Year Study (continued) 

Urinary System (continued) 

Ureter 

Concretion 

Dilatation 

Hemorrhage. 

Inflammation, subacute 1 (8%) 

Mucosa, hyperplasia 12 (92%) 

Urethra (2) 0) 

Concretion 1 (20%). 

Hemorrhage 1 (50%) 

Inflammation, subacute 1(20%) 

Bu1bourethl:al gland, ectasia 1 (50%) 

Lumen, concretion 1 (50%) 2 (40%) 

Lumen, dilatation ,1 (50%) 

Mucosa, hyperplasia 1 (50%) 1 (20%) 

Mucosa, metaplasia, squamous 2 (40%) 

Urinary bladder (49) (50) (50) (47) 

Concretion 1(2%) 10 (20%) 

Congestion 1 (2%) 

Dilatation , 1 (2%) 2 (4%) 7 (14%) 

Hemorrhage 1 (2%) 1 (2%) 4 (8%) 

Inflammation, subacute 3 (6%) 

Inflammation, suppurative 1 (2%) 

Mucosa, hyperplasia 14 (28%) 41 (82%) 1(2%) 
:m 

.............. 147 
146 Salicylazosulfapyridine, NTP TR 457 
VehicleControl 14mg/kg 161 mg/kg 337.5 mg/kg 

Early deaths 

15-Monfhinterim evaluahn 

Animals initially in study 

Disposition Summary 

Natural deaths 

Moribund 

Accidental deaths 

Terminal sacrifice 

Died last week of study 

Survivors 

10 

60 

2 

12 

1 

34 

1 

10 

60 

2 

13 

1 

33 

1 

10 

60 

5 

14 

31 

10 

60 

4 

11 

9 

26 

Animals examined microscopically 60 60 60 60 

Polyp stromal 

utenrs 

Adenoma 

Clitoral gland 

Genital System 

Skin 

btegusnentaey System 

Keratoacanthoma 

Multiple organsb 

Systemic Lesions 

Leukemia mononuclear 

wrinaey System 

Special Senses System 

Respiratory System 

Nervous System 

Musculoskeletal System 

Hematopoietic System 

General Body System 

Cardiovascular System 

Alimentaey System 

Systems Exmined With No Neoplasms Observed 
148 Salicylazosulfapyridine, NTP TR 457 

TABLEB11 

Summary of the Incidence of Neoplasms in Female Rats in the 2-Year Gavage Studyof Salicylazosulfapyridine(continued) 

Control Vehicle 168 84 mg/kg mg/kg 337.5 mg/kg 

2-Year Sihdy 

Alimentary System 

Intestine large, colon 

Schwannoma malignant 

Intestine small, duodenum 

. Leiomyoma 

Liver 

Carcinoma, metastatic, uterus 

Histiocytic sarcoma, metastatic, skin 

Hepatocellular adenoma 

Carcinoma, metastatic, uterus 

Mesentery 

1 (2%) 

1 (2%) 

(4) 

Fibroma 

Pancreas 

Pharynx 

Squamous cell carcinoma 

Squamous cell papilloma 

Stomach, forestomach 

Squamous cell papilloma 

Stomach, glandular 

Tongue 

Squamous cell papilloma 

Cardiovascular System 

Schwannoma malignant 

Heart (50) 

3 (6%) 

Endocrine System 

Adrenal cortex 

Adenoma 

Fibrous histiocytoma, metastatic, skin 

Adrenal medulla 

Ganglioneuroma 

Pheochromocytoma benign 

Bilateral, pheochromocytoma benign 

Carcinoma 

Adenoma 

Islets, pancreatic (50) 

1(2%) 

(50) 

1 (2%) 

Pars distalis, adenoma 

Pituitary gland (49) 

20 (41%) 

(50) 

13 (26%) 

Pars distalis, carcinoma 2 (4%) 

C-cell, carcinoma 

C-cell, adenoma 

Thyroid gland (50) 

3(6%) 

1 (2%) 

5 (10%) 

(50) (50)

2 (4%) 

Follicular cell, adenoma 2(4%) 

Follicular cell, carcinoma 1 (2%) 

General Body System 

Tissue NOS 
%Year Study (continued) 

Genital System 

Clitoral gland 

Adenoma 

Carcinoma 

Ovary 

Granulosa cell tumor benign 

Uterus 

Adenoma 

Carcinoma 

Polyp stromal 

Polyp stromal, multiple 

Sarcoma stromal 

Schwannoma malignant 

Hematopoietic System 

Bone marrow 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Thymus 

Integumentary System 

Mammary gland 

Adenoma 

Carcinoma 

Fibroadenoma 

Fibroadenoma, multiple 

Skin 

Histiocytic sarcoma 

Keratoacanthoma 

Sarcoma 

Squamous cell papilloma 

Subcutaneous tissue, fibrous histiocytoma 

Subcutaneous tissue, lipoma 

Musculoskeletal System 

Skeletal muscle 

Carcinoma, metastatic, uterus 

Rhabdomyosarcoma 

Nervous System 

Brain 

Astrocytoma malignant 

Carcinoma, metastatic, pituitary gland 

Glioma malignant, mixed 

Meningioma malignant 

Spinal cord 

Vehicle Control 

(50) 

2 (4%) 

18 (36%) 

4(8%) 

(50) 

1 (2%) 

3 (6%) 

(50) 

84 mglkg 

1 (2%) 

(1) 

1 (10%) 

(50) 

3 (6%) 

1 (2%) 

1 (2%) 

168 mg/kg 

7 (14%) 

1 (2%) 

1 (2%) 

(50) 

1 (2%) 

337.5 mg/kg 

(50) 

1(2%) 

3 (6%) 

1 (2%) 

1(2%) 

(1) 

1 (100%) 
150 Salicylazosulfapyridine, NTP TR 457 

TABLEB'l 

Summary of the Incidenceof Neoplasms in FemaleRats in the 2-Year Gavage Studyof Salicylazosulfapyridine(continued) 

&Year Study (continued) 

Respiratory System 

Lung 

Fibrous histiocytoma, metastatic, skin 

(50) 

Histiocytic sarcoma, metastatic, skin 

Sarcoma, metastatic, skin 

Squamous cell carcinoma 

Nose (50) 

Special Senses System 

Carcinoma 

Zymbal's gland (1)

1 (100%) 

Urinary System 

Kidney 

Renal tubule, carcinoma 

(50) (50) 

Transitional epithelium, papilloma 

Papilloma 

Urinary bladder (49) (50) 

Systemic Lesions 

Multiple orgiim 

Histiocytic sarcoma 

Leukemia mononuclear 

Lymphoma malignant 

Lymphoma malignant mixed 

Total animal:; with primary neoplasms' 

Neoplasm Summary 

15-Month interim evaluation 2 2 2 2 

2-Year study 44 41 47 31 

15-Month interim evaluation 

Total primary neoplasms 

2 2 2 2 

2-Year study 89 70 86 48 

l5-Month interim evaluation 

Total animals with benign neoplasms 

1 2 1 2 

2-Year study 41 30 44 25 

15-Month interim evaluation 

Total benign neoplasms 

1 2 1 2 

2-Year study 68 48 69 35 

15-Month interim evaluation 

Total animals with malignant neoplasms 

1 1 

2-Year study 21 20 16 12 

15-Month interim evaluation 

Total malignant neoplasms 

1 1 

2-Year study 21 21 17 13 
Vehicle Control 84 mglkg 148 mglkg 337.5 mglkg 

Neoplasm Summany (continued) 

Total animals with uncertain neoplasmsTotal metastatic neoplasms 

Total animals with metastatic neoplasms 

2-Year study 

2-Year study 

Total uncertainneoplasms 

benign or malignant 

2-Year study 

2-Year study 

3 

2 

a Number of animals examined microscopically at the site and the number of animals with neoplasm 

Primary neoplasms: all neopIasms except metastatic neoplasms 

Number of animals with any tissue examined microscopically 
Salicylazosulfapyridine,NTP TR 457 

2355566666666777777777777 

Number of'Days on Study 6309923466899003333333333 

0272825933412397888888888 ~ 

3443344444433444333344444 

CarcassID Number 9128810101099211889900022 

3289137631182679371604804 

Alimentary System 

Esophagus ......................... Intestine large, colon ......................... Schwannoma malignant X 

Intestine large, rectum ......................... Intestine large, cecum ++++++++++++M++++++++++++ 

Intestine small, duodenum .......................... Intestine small, jejunum ......................... Intestine small, ileum ......................... Liver ........................... Mesentery + 

Pancreas ......................... Pharynx 

Squamous cell papilloma 

Salivary glands ......................... Stomach, forestomach ......................... Stomach, glandular ......................... Tongue ~~ ~~ ~~ ~~~ ~~~ ~~ ~~ 

Cardiovascular System 

Blood vessel ......................... Heart ......................... Schwannoma malignant 

Endocrine System 

Adrenal cortf:x ......................... Adenoma X 

Adrenal medulla ......................... Ganglioneuroma X 

Bilateral, pheochromocytoma benign X 

Islets, pancreatic ......................... Adenoma 

Parathyroid gland ++++++++++M++++++++++++++ 

Pituitary gland ......................... Pars distalis, adenoma x x x x x x x x x x x x x .x 

Thyroid gland ......................... C-cell, adenoma X X 

General Body System 

None 

Genital System 

Clitoral gland ' ......................... Adenoma 

Carcinoma X 

Ovary ........................... Uterus ......................... Polyp stromal X x x 

+: Tissue examined microscopically M: Missingtissue X: Lesion present 

, A: Autolysis precludes examination I: Insufficient tissue Blank: Not examined 
Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Intestine large, colon 

Schwannoma malignant 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Mesentery 

Pancreas 

Pharynx 

Squamous cell papilloma 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Tongue 

Cardiovascular System 

Blood vessel 

Heart 

Schwannoma malignant 

Endocrine System 

Adrenal cortex 

Adenoma 

Adrenal medulla 

Ganglioneuroma 

Bilateral, pheochromocytoma benign 

Islets, pancreatic 

Adenoma 

Parathyroid gland 

Pituitary gland 

Pars djstalis, adenoma 

Thyroid gland 

C-cell, adenoma 

General Body System 

None 

Genital System 

Clitoral gland 

Adenoma 

Carcinoma 

Ovary 

uterus 

Polyp stromal 

7771117171111111711171717 

3333333333333333333333333 

8999999999999999999999999 

4333333333344444444444444 Total 

3888889999900001111222222 Tissues/ 

0245680451925690458123579 Tumors 

......................... 50 

......................... 50 

1 

......................... 50 

......................... 49 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

+ + + 4 < 

......................... 50 

+ 1 

X 1 

......................... 50 

......................... 50 

......................... 50 

+ 1 

......................... 50 

......................... 50 

X X X 3 

.......................... 50 

1 

......................... 50 

1 

1 

......................... 50 

X 1 

+M+++++++++++++++++++++++ 48 

......................... 50 

X X X X x x x xx xx 26 

......................... 50 

X 3 

......................... 50 

X X 2 

1 

......................... 50 

......................... 50 

X xx 6 
154 Salicylazosulfapyridine, NTP TR 457 

TABLEB2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Gavage Studyof Salicylazosulfapyridine: 

Vehicle Control (continued) 

Number of Days on Study 

Carcass IDNumber 

Hematopoietic System 

Bone marrow 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Thymus 

Integumentary System 

Mammary gland 

Carcinoma 

Fibroadenoma 

Fibroadenoma, multiple 

Skin 

Keratoacanthoma 

Squamous cell papilloma 

Musculoskeletal System 

Bone 

Skeletal muscle 

Nervous System 

Brain 

Respiratory System 

Lung

Nose 

Trachea 

Special Senses System 

Eve 

Urinary System 

Kidney 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Leukemia mononuclear 

2355566666666777777777777 

6309923466899003333333333 

027,2825,933412397888888888 

3443344444433444333344444 

91288101010.99211889900022 

3289137631182679371604804 

.......................... 

++ ++ ++ + + + ++ ......................... 

......................... 

......................... 

......................... 

......................... X 

x x x x xx x 

X X ......................... X 

X X 

......................... + + 

......................... 

......................... 

.......................... 

......................... 

+ + 

......................... 

++++++++++++++++++M++++++ 

......................... 

.x x xx x x x X
Carcass ID Number 

Hematopoietic System 

Bone marrow 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Thymus 

Integumentary System 

Mammary gland 

Carcinoma 

Fibroadenomaxxxx xx 

Fibroadenoma, multiple 

Skin 

Keratoacanthoma 

Squamous cell papilloma I 

Musculoskeletal System 

Bone 

Skeletal muscle 

Nervous §ystem 

Brain 

Respiratory System 

Lung 

Nose 

Trachea 

Special Senses System 

Eye 

Urinary System 

Kidney 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Leukemia mononuclear 

4333333,333344444444444444 

3888889999900001111222222 

0245680457925690458123579 

......................... + ......................... 

......................... 

......................... 

+++++++++++++++++M+++++++ 

......................... X 

xx xxx 

x x ......................... 

X 

......................... 

......................... 

......................... 

......................... 

......................... 

+ 

......................... 

......................... 

......................... 

X x x x X X 

Total 

Tissues/ 

Tumors 

50 

12 

50 

50 

50 

49 

50 

2 

18 

4 

50 

1 

3 

50 

2 

50 

50 

50 

50 

3 

50 

49 

50 

14 
156 Salicylazosulfapyridine,NTP TR 457 

TABLEB2: 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Gavage Study of Salicylazosulfapyridine: 84 mg/kg 

Number of Days on Study 

Carcass IDNumber 

Alimentary System 

Esophagus 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Leiomyoma 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Carcinoma, metastatic, uterus 

Mesentery 

Carcinoma, metastatic, uterus 

Fibroma 

Pancreas 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Tongue 

Squamous cell papilloma 

Cardiovascular System 

Blood vessel 

Heart 

Endocrine System 

Adrenal cortex 

Fibrous histiocytoma, metastatic, skin 

Adrenal medulla 

Islets, pancreatic 

Carcinoma 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Pars distalis, carcinoma 

Thyroid gland 

C-cell, adenoma 

C-cell, carcinoma 

General Body System 

Tissue NOS 

Genital System 

Clitoral gland 

Adenoma 

Carcinoma 

Ovary 

Granulosa cell tumor benign 

uterus 

Carcinoma 

Polyp stromal 

2233445556666777777717777 

1899370483489012333333333 

8056221432843374778888888 

44444,44'4.'44444444444444444 

488'5567757476678484455667 

9615983906689247382737160 ' 

~~~ ~ ~~~ 

......................... 

......................... 

......................... 

M++++++++++++++++++++++++ 

......................... 

......................... 

++M++++++++++++++++++++++ 

......................... X 

+ + ++ + 

X 

X .......................... 

......................... 

++++++++M++++++++++++++++ 

.......................... 

......................... 

......................... 

......................... X 

......................... 

......................... 

+++M+++++++++++++++++++++ 

++++++++++++++++++++++M++ 

X xxxxx xxxx 

X 

......................... X X X 

+ 

......................... X 

......................... 

......................... X 

X X X 
Number of Days on Study 

Carcass IUD Number 

Alimentary System 

Esophagus 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Leiomyoma 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Carcinoma, metastatic, uterus 

Mesentery 

Carcinoma, metastatic, uterus 

Fibroma 

Pancreas 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Tongue 

Squamous cell papilloma 

Cardiovascular System 

Blood vessel 

Heart 

Endocrine System 

Adrenal cortex 

Fibrous histiocytoma, metastatic, skin 

Adrenal medulla 

Islets, pancreatic 

Carcinoma 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Pars distalis, carcinoma 

Thyroid gland 

C-cell, adenoma 

C-cell, carcinoma 

General Body System 

Tissue NOS 

Genital System 

Clitoral gland 

Adenoma 

Carcinoma 

Ovary 

Granulosa cell tumor benign 

Uterus 

Carcinoma 

Polyp stromal 

7777777777777777777777777 

3333444444444444444444444 

8888000000000000000000000 

4444444444444444444444444 Total 

7888444455555666667778889 Tissues/ 

2045145812468034571572390 Tumors 

......................... 50 

......................... 50 

......................... 50 

......................... 49 

......................... 50 

X 1 

......................... 50 

......................... 49 

......................... 50 

1 

+ + 7 

1 

1 

......................... 50 

......................... 50 

......................... 49 

......................... 50 

+ 1 

X 1 

......................... 50 

......................... 50 

......................... 50 

1 

......................... 50 

......................... 50 

X 1 

......................... 49 

......................... 49 

x xx xx X X xx X 20 

X 2 

......................... 50 

X X 5 

X 1 

1 

......................... 50 

X 2 

xx 2 

......................... 50 

X 1 

........................... 50 

1 

X x x X 7 
158 Salicylazosulfapyridine,NTP TR 457 

TABLEB2 

Individual Animal Tumor Pathology of Female Rats in the 2-YearGavage Study of Salicylazosulfapyridine: 84 mgkg 

(continued) ,. .. 

2233445556666711771111711 

Number of Days on Study 1899310483489012333333333 

80'562214'32843374778888888 ~~~ 

4444444444444444444444444 

Carcass ID Number 488556115141661848445566~ 

9615983906689247382131160 

Genital System (continued) 

Vagina M + 

Hematopoietic System 

Bone marrow ......................... Lymph node + + ++ 

Lymph node, mandibular ......................... Lymph node, mesenteric ........................... . . Spleen ......................... Thymus ++++M++++++++++++++++++++ 

Integumentary System 

Mammary gland ......................... Adenoma 

. Fibroadenoma X x xx 

Fibroadenoma, multiple X 

Skin ......................... Subcutaneous tissue, fibrous histiocytoma X 

Musculoskeletal System 

Bone ......................... Skeletal muscle + 

Carcinoma, metastatic, uterus X 

.Nervous System 

Brain ......................... Carcinoma, metastatic, pituitary gland X X 

Glioma malignant, mixed X 

Meningioma malignant X 

Respiratory System 

Lug ......................... Fibrous histiocytoma, metastatic, skin X 

Nose ......................... Squamous cell carcinoma X 

Trachea ......................... 

Special Senses System 

Eye ++ + 

Zymbal's gland + 

Carcinoma X 

Urinary System 

Kidney ......................... Urinary bladder ......................... ~ ~ ~~~~~~~ ~~ 

Systemic Lcs'' Ions

Multiple organs .......................... Leukemia mononuclear X .x x x x 

Lymphoma malignant x 
i 

. ""---- ~ ~~ 

BaaBlcyn~QSPsnnnffa~~~~~e~ mp TR 05'7 

TABLIE BZ 

xmta~xmn~mbann~[hnmap~pauhapnw 

(continued) 

NUllbeP off Days Op1 StUdy 

cal-cas ED Nuberr 

enital System (continued) 

Vagina 

WematQpoietic System

Bone marrow 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Thymus 

I[ntegmenhry System 

Mammary gland 

Adenoma 

Fibroadenoma 

Fibroadenoma, multiple 

Skin 

Subcutaneous tissue, fibrous histiocytoma 

Musculoskeletal System

Bone 

Skeletal muscle 

Carcinoma, metastatic, uterus 

%WrQUS System

Brain 

Carcinoma, metastatic, pituitary gland 

Glioma malignant, mixed 

Meningioma malignant 

Respiratory System

Lung 

Fibrous histiocytoma, metastatic, skin 

Nose 

Squamous cell carcinoma 

Trachea 

Special Senses System 

Eye

Zymbal's gland 

Carcinoma 

Urinary System

Kidney 

Urinary bladder 

Systemic ILasiom 

Multiple organs 

Leukemia mononuclear 

Lymphoma malignant 

-- -~ ~~~ r -.v-!?z7L - ~~ _~" ~~ 

159 

~emde~m 2-y~ cl;r~age suudy Off Bannhcgrn~~Ptsnnnffa~y~~~~m~: 84mg/kg

7777777777777777777777777 

3333444444444444444444444 

8888000000000000000000000 

4444444444444444444444444 Total 

7888444455555666667778889 Tissues/ 

2045145812468034571572390 Tumors 

1 

......................... 50 

+ + 6 

++++++++++++++++M++++++++ 48 

......................... 50 

......................... 50 

......................... 49 

......................... 50 

X 1 

X X X X 8 

1 

......................... 50 

1 

......................... 50 

1 

1 

......................... 50 

X 3 

1 

1 

......................... 50 

1 

......................... 50 

1 

......................... 50 

+ + 5 

1 

1 

......................... 50 

......................... 50 

......................... 50 . 

x ,-. x x X 9 

1 
160 Salicylazosulfapyridine, NTP TR 457 

TABLEB2 

Individual Animal Tumor Pathology of Female Rats in the 2-Year Gavage Study of Salicylazosulfapyridine: 168 mg/kg 

Number of :Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Intestine large:, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatocellular adenoma 

Mesentery 

Pancreas 

Pharynx 

~quamouscell carcinoma 

Salivary glands 

Stomach, fort:stomach 

Stomach, glandular 

Tongue 

Cardiovascular System 

Blood vessel 

Heart 

Endocrine System 

Adrenal cortex 

Adenoma 

Adrenal medulla 

Pheochromocytoma benign 

Islets, pancreatic 

Adenoma 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Thyroid gland 

C-cell, adenoma 

Follicular cell, adenoma 

General Body System 

None 

Genital System 

Clitoral gland 

Adenoma 

Carcinoma 

Ovary 

Uterus 

Polyp stromal 

Schwannoma malignant 

Vagina 

5666666666666777777777777 

4122268999999002333333333 

4914577112556885333888888 

5555555555555555555555555 

2120312440133130134001122 

2868251077001456273393738 

......................... 

.......................... 

......................... 

......................... 

.......................... 

......................... 

......................... 

......................... 

+ + ......................... + 

X ......................... 

......................... 

......................... + 

............................ 

......................... 

......................... 

......................... 

......................... 

......................... 

.......................... x x xx x ' x x xx xx x ......................... X 

X 

......................... 

.......................... 

.......................... xx 

M 

' 

. 
Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatocellular adenoma 

Mesentery 

Pancreas 

Pharynx 

Squamous cell carcinoma 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Tongue 

Cardiovascular System 

Blood vessel 

Heart 

Endocrine System 

Adrenal cortex 

Adenoma 

Adrenal medulla 

Pheochromocytoma benign 

Islets, pancreatic 

Adenoma 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Thyroid gland 

C-cell, adenoma 

Follicular cell, adenoma 

General Body System 

None 

Genital System 

Clitoral gland 

Adenoma 

Carcinoma 

Ovary 

uterus 

Polyp stromal 

Schwannoma malignant 

Vagina 

7777777777777777777777777 

3333333333333333333333334 

8888999999999999999999990 

5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 Total 

3344000011122222333444454 Tissues/ 

3816124516904579469248905 Tumors 

......................... 50 

......................... 50 

+++++++++I+++++++++++++++ 49 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

X 1 

+ + 4 

......................... 50 

1 

1 

......................... 50 

......................... 50 

......................... 50 

1 

......................... 50 

......................... 50 

......................... 50 

X 1 

......................... 50 

X X X 3 

......................... 50 

X 1 

......................... 50 

......................... 50 

x xxx xx x x xx x x 24 

......................... 50 

X 2 

X 2 

......................... 50 

X X 2 

X 1 

......................... 50 

......................... 50 

X X X X X 7 

X 1 

+ 1 
162 Salicylazosulhpyridine, NTP TR 457 

TABLEB2 

Individual Animal Tumor Pathology OF Female Rats in the 2-Year Gavage StudyOF Salicylazosulfapyridine: 168mg/kg 

(continued) 

5666666666666777777777777 

Number of Days on Study 4122268999999002333333333 

4914577112556885333888888 

5555555555555555555555555 

Carcass ID Number 2120312440133130134001122 

2868251077001456273393738 

Hematopoietic System 

Bone marrow .......................... Lymph node ++ + ++ + 

Lymph node., mandibular .......................... Lymph node., mesenteric ++++++++++++M++++++++++++ 

Spleen ......................... Thymus ......................... 

Integumentary System 

Mammary gland ......................... Adenoma X 

Carcinoma X X 

Fibroadenoma x x x x X X X 

Fibroadenoma, multiple X X 

Skin ......................... Keratoacimnthoma X 

Subcutaneous tissue, lipoma X 

Musculoskeletal System 

Bone ......................... 

Nervous System 

Brain ......................... Astrocytoma malignant X ~~ 

Respiratory System 

Lung ......................... Nose ......................... Trachea ......................... ~~~ ~~ _____~~ ~~ 

Special Selnses System 

EY e 

Urinary System 

Kidney ......................... Renal tubule, carcinoma 

Ureter + 

Urethra + 

Urinary bladder ......................... Papilloma X 

Systemic 1Rsions 

Multiple organs +++ + + + + + + + + +++ + ++++++ + +++ 

Leukemia mononuclear xxx X xx 

Lymphoma malignantmixed X 
Nmbep of Days on Study 

Carcrass IUD Number 

Hematopoietic System 

Bone marrow 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Thymus 

Integumentary System 

Mammary gland 

Adenoma 

Carcinoma 

Fibroadenoma 

Fibroadenoma, multiple 

Skin 

Keratoacanthoma 

Subcutaneous tissue, lipoma 

Musculoskeletal System 

Bone I ~ ~ 

Nervous System 

Brain 

Astrocytoma malignant 

Respiratory System 

Lung

Nose 

Trachea 

Special Senses System 

Eye 

U~inslrySystem 

Kidney 

Renal tubule, carcinoma 

Ureter 

Urethra 

Urinary bladder 

Papilloma 

i Systemic Lesions 

Multiple organs 

Leukemia mononuclear 

Lymphoma malignant mixed 

7777777777777777777777777 

3333333333333333333333334 

8888999999999999999999990 

. 5555555555555555555555555 

3344000011122222333444454 

3816124516904579469248905 

......................... + + + ......................... 

......................... 

......................... 

......................... 

......................... 

X 

X 

xx X x xx xxxx

......................... 

......................... ~______ ~~ ~ ~ ~ 

......................... 

......................... 

......................... 

......................... 

+ + 

......................... X 

+ 

......................... X 

......................... X X 

Total 

Tissues/ 

Tumors 

50 

9 

50 

49 

50 

50 

50 

2 

3 

18 

2 

50 

1 

1 

50 

50 

1 

50 

50 

50 

2 

50 

1 

2 

1 

50 

2 

50 

8 

1 
164 Salicylazosulfapyridine, NTP TR 457 

Number of Days on Study 

CarcassID1 Number 

Alimentary System 

Esophagus 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Histiocytic sarcoma, metastatic, skin 

Mesentery 

Pancreas 

Salivary glands 

Stomach, forestomach 

Squamous cell papilloma 

Stomach, glandular 

Cardiovascular System 

Blood vessel 

Heart 

Schwannoma malignant 

Endocrine System 

Adrenal cortex 

Adenoma 

Adrenal medulla 

Islets, pancreatic 

Adenoma 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Thyroid gland 

C-cell, adenoma 

Fo!licular cell, carcinoma 

General Body System 

None 

Genital System 

Clitoral gland 

Adenoma 

Ovary 

Uterus 

Adenoma 

Polyp stromal 

Polyp stromal, multiple 

Sarcoma stromal 

1233333444455555666677777 

2145799019900006009900033 

24985385.65588888373634938 

6565665555555665555555555 

1916128666989006768678776 

9009306136517054674520972 

+++++++++++++++++M+++++++ 

.......................... 

......................... 

......................... 

.......................... 

.......................... 

++++++++++++++++.++++M++++ 

......................... X 

+ ......................... 

......................... 

......................... 

........................... 

.......................... 

.......................... .I

A 

......................... 

......................... 

......................... 

........................ 

......................... x xx X ......................... X 

......................... 

......................... 

......................... 

X 

X 
i 

TABLEB2 

IIundividnnaU Animal TU~CW PatU~~1~g-y off Female Rats in the %YearGavage Study of SaaicylazosraPhpyridine: 337.5 mgkg

(continued) 

~ Number of DaysStudy on 

I

I Carcass ID Number

I

I 

'I Alimentary System

Esophagus 1 Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum ! Intestine small, duodenum 

' Intestine small, jejunum 

Intestine small, ileum 

Liver 

Histiocytic sarcoma, metastatic, skin 

Mesentery 

Pancreas 

Salivary glands 

Stomach, forestomach 

Squamous cell papilloma I Stomach, glandular 

Cardiovascular System 

Blood vessel 

Heart 

Schwannoma malignant ~ ~~~ ~ 

Endocrine System 

Adrenal cortex 

Adenoma 

Adrenal medulla 

Islets, pancreatic 

Adenoma 

Parathyroid gland 

Pituitary>gland 

Pars distalis, adenoma 

Thyroid gland 

C-cell, adenoma 

Follicular cell, carcinoma 

General Body System 

None 

Genital System 

Clitoral gland 

Adenoma 

Ovary 

Uterus , 

Adenoma 

Polyp stromal 

Polyp stromal, multiple 

Sarcoma stromal 

7777777777777777777777777 

3333333333333333333333333 

8888888889999999999999999 

5555555565555555555556666 Total 

7788899906777788899990000 Tissues/ 

0827926928134535813481346 Tumors 

......................... 49 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

++++++++++++++++++M++++++ 48 

.......................... 50 

1 

+ + + 4 

......................... 50 

......................... 50 

......................... 50 

X 1 

......................... 50 

......................... 50 

......................... 50 

X X 3 

......................... 50 

X 1 

......................... 50 

......................... 50 

X 1 

......................... 48 

......................... 50 

x x xxxxx X X 13 

......................... 50 

X x x X 5 

X 1 

......................... 50 

X X 2 

......................... 50 

......................... 50 

X 1 

X X 3 

X 1 

1 
166 Salicylazosulfapyridine, NTP TR457 

TABLEB2. 

Individual Animal Tumor Pathologyof Female Rats in the 2-Year Gavage Studyof Salicylazosulfapyridine: 337.5 mg/kg 

(continued) 

Number of Days on Study 

Carcass ID1 Number 

Hematopoietic System 

Bone marrow 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Thymus 

Integumentary System 

Mammary gland 

Carcinoma 

Fibroadenoma 

Skin 

Histiocytic sarcoma 

Sarcoma 

Musculoskeletal System 

Bone 

Skeletal muscle 

Rhabdomyosarcoma 

Nervous System 

Brain 

Peripheral nerve 

Spinal cord 

Respiratory System 

Lung 

Histiocytic sarcoma, metastatic, skin 

Sarcoma, metastatic, skin 

Nose 

Trachea 

Special Senses System 

EY e 

Urinary System 

Kidney 

Transitional epithelium, papilloma 

Ureter 

Urinarv bladder 

Systemic lksions 

Multiple organs 

Histiocytic sarcoma 

Leukemia mononuclear 

Lymphoma malignant 

1233333444455555666677777 

2145799019900006009900033 

2498538565588888373634938 

6565665555555665555555555 

1916128666989006768678776 

9009306136517,054674520972 

......................... ++ + + + + ......................... 

.......................... 

......................... 

......................... 

++++++++++++++M++++++++++ 

X 

X x x.x x ......................... X 

X 

.......................... + 

X 

......................... + + 

+ + 

......................... X 

X 

......................... 

......................... 

.......................... 

+ + +. 

++++.+.+++++++++++++++++++t 

......................... x 

X X x 
Number of Days on Study 

carcapss E D Number 

Hematopoietic System 

Bone marrow 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Thymus 

Ihtegumentary System 

Mammary gland 

Carcinoma 

Fibroadenoma 

Skin 

Histiocytic sarcoma 

Sarcoma 

Musculoskeletal System 

Bone 

Skeletal muscle 

Rhabdomyosarcoma 

Nervous System 

Brain 

Peripheral nerve 

Spinal cord 

Respiratory System 

LungHistiocytic sarcoma, metastatic, skin 

Sarcoma, metastatic, skin 

Nose 

Trachea 

Special Senses System 

Eye 

Urinary System 

Kidney 

Transitional epithelium, papilloma 

Ureter 

Urinary bladder 

Systemic ][asions 

Multiple organs

Histiocytic sarcoma 

Leukemia mononuclear 

Lymphoma malignant 

1111111111171117111117117 

3333333333333333333333333 

8888888889999.999999999999 

5555555565555555555556666 Total 

1188899906111188899990000 Tissues/ 

0827926928134535813481346 Tumors ~~ ~~ ~ ~~ 

......................... 50 

+ 1 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

......................... 48 

1 

5 

......................... 50 

1 

1 

......................... 50 

1 

1 

......................... 50 

1 

2 

......................... 50 

,1 

1 

......................... 50 

......................... 50 

++ 2 

......................... 50 

X X 2 

+ 4 

......................... 50 

......................... 50 

1 

3 

X 1 
168 Salicylazosulfapyr,idine,NTP TR 457 

TABLEB:l 

Statisticall Analysis of Primary Neoplasms in Female Rats inthe 2-Year Gavage Study of Salicylazosulfapyridine 

Vehicle Control , 84 mg/kg 168 mg/kg 337.5 mglkg

Life table testd 

First incidence (days) 

Terminal rate' 

Adjusted rat,:b 

Overall rate" 

AdrenalMedulla: Benign Pheochromocytoma 

P=0.581N 

69 1 

0/35 (0%) 

2.6% 

1/50 (2%) 

0/34 (0%) 

0.0% 

0150 (0%) 

P=0.505N 

-

e 

P=O.283 

737 (T) 

3/31 (10%) 

9.7% 

3/50 (6%) . ' 

i 

0126 (0%) 

0.0% 

0/50 (0%)' 

P=0.539N 

-

Fisher exact test 

Cochran-Annita e testd 

Logistic regression testd 

% P=0.499N 

P=0.566N 

P=0.500N 

P=0.502N P=O.320 

P=O.309 P=0.500N 

P=0.525N 

Clitoral Gland: Adenoma or Carcinoma 

Fisher exact test 

Cochran-Annitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

P=0.344N 

P=0.458N 

P=0.493N 

663 

2/35 (6%) 

8.0% 

3/50 (6%) 

. 

. 

, 

. 

P=O.472 

P=O.481 

737 (T)

4/34 (12%) 

11.8% 

4/50 (8%) 

P=O.500 

P=0.644N 

P=O.621 

737 (T) 

3/31 (10%) 

9.7% 

3/50 (6%) 

P=0.661N 

P=0.610N 

P=0.631N 

737 (T) 

2/26 (8%) 

7.7% 

2/50 (4%) 

P=0.500N 

Fisher exact test 

Cochran-Annitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Heart: Malignant Schwannoma 

P=O.443 

P=O.347 

P=O.336 

737 (T) 

3/35 (9%) 

8.6% 

3/50 (6%) 

0134 (0%) 

0.0% 

0/50 (0%) 

P=0.121N 

P=0.126N 

P=0.126N 

- 0/31 (0%) 

0.0% 

0150 (0%) 

P=0.12lN 

P=0.143N 

P=0.143N 

-

P=O.539 

P=O.523 

733 

2/26 (8%) 

11.1% 

3/50 (6%) 

P=0.661N 

Fisher exact test 

Cochran-Armitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Mammary Gland: Fibroadenoma 

P=0.001N 

P=0.005N 

P=0.020N 

507 

17/35 (49%) 

54.3% 

22/50 (44%) 

P=0.007N 

P=0.008N 

724 

8/34 (24%) 

25.7% 

9/50 (18%) 

P=0.004N 

P=0.387N 

P=0.526N 

619 

11/31 (35%) 

48.4% 

20/50 (40%) 

P=0.420N 

P<0.001N 

P=0.004N 

603 

0/26 (0%) 

15.6% 

5/50 (10%) 

P<0.001N ' 

Fisher exact test 

Cochran-Arrnitage test 

Logistic regression test 

Life table test 

First incidence (days) 

Terminal rate 

Adjusted rate 

Overall rate 

Mammary Gland: Fibroadenoma or Adenoma 

P=0.001N 

P=0.005N 

P=0.020N 

507 

17/35 (49%) 

54.3% 

22/50 (44%) 

.P=0.013N 

P=0.014N 

724 . 

9/34 (26%) 

28.6% 

10150 (20%) 

P=0.009N 

P=0.469N 

P=O.550 

619 

11/31 (35%) 

49.6% 

21/50 (42%) 

P=0.500N 

P<0.001N 

P=0.004N 

603 

0/26 (0%) 

15.6% 

5/50 (10%) 

P<0.001N 
169 

Vehicle Control 

MammaryGland:Carcinoma 

Overall rate 2/50 (4%) 

Adjusted rate 5.6% 

Terminal rate 1/35 (3%) 

First incidence (days) 709 

Life table test P=O.574 

Logistic regression test P=0.545N 

Cochran-Armitage test P=0.556N 

Fisher exact test 

MammaryGland:AdenomaorCarcinoma 

Overall rate 2/50 (4%) 

Adjusted rate 5.6% 

Terminal rate 1/35 (3%) 

First incidence (days) 709 

Life table test P=O.546 

Logistic regression test P=0.543N 

Cochran-Armitage test P=0.522N 

Fisher exact test 

MammaryGland:Fibroadenoma,Adenoma,orCarcinoma 

Overall rate 24/50 (48%) 

Adjusted rate 58.0% 

Terminal rate 18/35(51%) 

First incidence (days) 507 

Life table test P=O.O24N 

Logistic regression test P=0.007N 

Cochran-Armitage test P=0.001N 

Fisher exact test 

Pituitary Gland (Pars Distalis): Adenoma 

Overall rate 26/50 (52%) 

Adjusted rate 57.6% 

Terminal rate 16/35(46%) 

First incidence (days) 598 

Life table test P=0.136N 

Logistic regression test P=0.051N 

Cochran-Armitage test P=O.WN 

Fisher exact test 

Pituitary Gland (Pars Distalis): Adenoma or Carcinoma 

Overall rate 26/50 (52%) 

Adjusted rate 57.6% 

Terminal rate 16/35(46%) 

First incidence (days) 598 

Life table test P=0.115N 

Logistic regression test P=0.039N 

Cochran-Armitage test P=0.006N 

Fisher exact test 

M mgkg 

01.50 (0%) 

0.0% 

0/34 (0%) -

P=0.243N 

P=0.249N 

P=0.247N 

1/50 (2%) 

2.9% 

1/34 (3%) 

737 (T) 

P=0.507N 

P=0.517N 

P=0.500N 

10150 (20%) 

28.6% 

9/34 (26%) 

724 

P=0.005N 

P=O.WN 

P=0.003N 

20/49 (41X) 

48.0% 

12/33 (36%) 

432 

P=0.265N 

P=0.332N 

P=0.180N 

22/49 (45%) 

53.0% 

14/33 (42%) 

432 

P=0.391N 

P=0.503N 

P=0.307N 

168mgkg 

3/50 (6%) 

7.9% 

1/31 (3%) 

544 

P=O.481 

P=O.474 

P=O.500 

5/50 (10%) 

13.0% 

2/31 (6%) 

544 

P=O.211 

P=O.201 

P=O.218 

23/50 (46%) 

53.0% 

12/31 (39%) 

544 

P=O.541 

P=0.482N 

P=0.500N 

24/50 (48%) 

57.6% 

l4/3 1 (45 %) 

619 

P=0.523N 

P=0.372N 

P=0.421N 

24/50 (48%) 

57.6% 

l4/3 1 (45 %) 

619 

P=0.523N 

P=0.372N 

P=0.421N 

337.5 mg/kg 

1/50 (2%) 

2.9% 

0126 (0%) 

568 

P=0.596N 

P=0.515N 

P=0.500N 

1/50 (2%) 

2.9% 

0126 (0%) 

568 

P=0.596N 

P=0.515N 

P=0.500N 

6/50 (12 %) 

18.0% 

0126 (0%) 

568 

P=0.005N 

P<0.001N 

P<0.001N 

13/50 (26%) 

44.4% 

10/26 (38%) 

693 

P=0.105N 

P=0.116N 

P=0.007N 

13/50 (26%) 

44.4% 

10/26 (38%) 

693 

P=O.lOSN 

P=O. 116N 

P=0.007N 
170 Salicylazosulfapyridine, NTP TR 457 

TABLEB:3 

Statistical Analysis of Primary Neoplasms in Female Rats in the 2-YearGavage Studyof Salicylazosulfapyridine(continued) 

VehicleControl .. 84 mgkg 168 mglkg 337.5 mglkg 

Adjusted rate 

Overall rate 

Skin: Squ.amous CellPapilloma 

8.6% 

3/50 (6%) 

0.0% 

0150 (0%) 

0.0% 

0150 (0%) 

0.0% 

Ob0 (0%) 

Life table test 

First incidence (days) 

Terminal rate 

P=0.063N 

737 (T) 

3/35 (9%) 0/34 (0%) 

P=O.l26N 

- 0131 (0%) 

P=0.143N 

- 0/26 (0%) 

P=0.178N 

-

Logistic regression test P-0.063N P=O.l26N P=0.143N P=O. 178N 

Cochran-Armitage test P=0.048N 

Fisher exact test P=0.121N P=0.121N P=O.l21N 

Skin: Squamous Cell Papilloma or Keratoacanthoma 

Adjusted rate 

Overall rate 

8.6% 

3l50 (6%) 

0.0% 

0150 (0%) 

2.9% 

1/50 (2%) 

0.0% 

Ob0 (0%) 

Life table test 

First incidence (days) 

Terminal rate 

P=0.117N 

737 (T) 

3/35 (9%) 0/34 (0%) 

P-O.126N 

- P=0.342N 

733 

0131 (0%) 0126 (0%) 

P=0.178N 

-

Logistic regression test P=O. 108N P=O.126N P=0.311N P=O. 178N 

Cochran-Armitage test P=O.O86N 

Fisher exact test, P=0.121N P=0.309N P=0.121N 

Thyroid Gland (C-cell): Adenoma 

Adjusted rate 

Overall rate 

7.8% 

3/50 (6%) 

12.9% 

5/50 (10%) 

5.6% 

2/50 (4%) 

17.4% 

5/50 (10%) 

First incidence (days) 

Terminal rate 

684 

1/35 (3%) 

544 

2/34 (6%) 

695 

1/31 (3%) 

495 

4/26 (15%) 

Life table test P=O.240 P=O.345 P=0.506N P=O.230 

Logistic regression test P=O.334 P-0.352 P=0.491N P=O.278 

Cochran-Armitage test P=O.375 

Fisher exact test P=O.357 P=O.SOON P=O.357 

Thyroid Gland (C-cell): Adenoma or Carcinoma 

Adjusted rate 

Overall rate 

7.8% 

3/50 (6%) 

15.6% 

6/50 (12%) 

5.6% 

2/50 (4%) 

17.4% 

5/50 (10%) 

First incidence (days) 

Terminal rate 

684 

1/35 (3%) 

544 

3134 (9%) 

695 

1/31 (3%) 

495 

4/26 (15%) 

Life table test P=O.280 P=O.236 P=0.506N P=O.230 

Logistic regression test P=O.379 P=O.234 P=0.491N P=O.278 

Cochran-Almitage test P=O.430 

Fisher exact test P=O.243 P=O.SOON P=O.357 

Adjusted rate 

Overall rate: 

Uterus:StromalPolyp 

16.2% 

6/50 (124%) 

19.3% 

7/50 (14%) 

22.6% 

7/50 (14%) 

14.7% 

4/50 (8%) 

Terminal rate 5/35 (14%) 5/34 (15%) 713 1 (23 %) 3/26 (1246) 

Life table test 

First incidence (days) 

P=0.484N 

635 

P=O.479 

693 

P=O.414 

737 (T) 

P=0.562N 

709 

Logistic regression test P=O.436N P=O.459 P=O.528 P=0.531N 

Fisher exact test 

Cochran-Armitage test P=0.278N 

P=O.5OO P=O.SOO P=O.37ON 
Wterus:StromalPolyporStromalSarcoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

All Organs: Mononuclear Cell Leukemia 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

AllOrgans:BenignNeoplasms 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

AllOrgans:MalignantNeoplasms 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

VehicleControl 

6/50 (12%) 

16.2% 

5/35 (14%) 

635 

P=O.499 

P=0.537N 

P=O.WN 

14/50 (28%) 

32.0% 

7/35 (20%) 

260 

P=0.018N 

P=0.001N 

P=0.003N 

41/50 (82%) 

87.2% 

29/35 (83%) 

507 

P=0.327N 

P=0.069N 

P=0.003N 

21/50 (42%) 

46.1% 

11/35 (31%) 

260 

P=0.158N 

P=0.012N 

P=0.020N 

14mgkg 

7/50 (14%) 

19.3% 

5/34 (15 R) 

693 

P=O.479 

P=O.459 

P=O.500 

9/50 (18%) 

23.7% 

6/34 (18%) 

396 

P=0.215N 

P=O. 156N 

P=0.171N 

30/50 (60%) 

69.6% 

21/34 (62%) 

432 

P=0.069N 

P=0.024N 

P=0.013N 

21/50 (42%) 

48.2% 

12/34 (35%) 

218 

P=O.521 

P=0.535N 

P=0.580N 

168 mg/kg 

7/50 (14%) 

22.6% 

7/31 (23%) 

737 (T) 

P=O.414 

P=O.528 

P=O.500 

8/50 (16%) 

19.3% 

2/31 (6%) 

544 

P=O.l49N 

P=O. 188N 

P=O. 114N 

44/50 (88%) 

93.5% 

28/31 (90%) 

544 

P=O.213 

P=O.435 

P=O.288 

16/50 (32%) 

38.2% 

7/31 (23%) 

544 

P=0.280N 

P=0.271N 

P=0.204N 

337.5 mg/kg 

5/50 (10%) 

16.6% 

3/26 (12%) 

375 

P=O.565 

P=O.589N 

P=O.SWN 

3/50 (6%) 

8.4% 

0/26 (0%) 

495 

P=0.026N 

P=0.002N 

P=0.003N 

25/50 (50%) 

73.4% 

17/26 (65%) 

495 

P=0.146N 

P=0.022N 

P<0.001N 

12/50 (24%) 

32.9% 

4/26 (15 %) 

358 

P=0.237N 

P=0.038N 

P=0.044N 
172 Salicylazosulfapyridine, NTP TR 457 

TABLEB:) 

Statistica:l Analysisof Primary Neoplasms in Female Rats in the 2-Year Gavage Studyof Saiicylazosulfapyridine(continued) 

. : 

Vehicle Control 84 mglkg 168 mglkg 337.5 mg/kg 

All Organs: Benign or Malignant Neoplasms 

Overall rate 44/50 (85%) 41/50 (82%) 47/50 (94%) 31/50 (62%) 

Adjusted rate 89.8% 83.7% 94.0% 79.2% 

Terminal rate 30/35 (86%) 26/34 (76%) 28/31 (90%) 18/26 (69%) 

First incidence (days) 218 260 544 358

Life table test P=O.434N P=0.469N P=O.228 P=0.373N 

Logistic regression test P=0.006N . P=0.293N P=O.263 P=0.023N 

Cochran-Armitage test P<0.001N 

~ 

Fisher exact test P=0.288N P=O.243 P=0.002N 

(T)Terminal sacrifice 

a Number of neoplasm-bearing animalslnumber of animals examined. Denominator is number of animals examined microscopically for adrenal gland, clitoral 

gland, heart, pituitary gland, thyroid gland,and uterus; for, other tissues, denominator is number of animals necropsied. . -

Kaplan-Meier estimated neoplasm incidence at the endof the study after adjustment for intercurrent mortality 

Observed incidence at terminalkill 

Beneath the vehicle control incidence are the P values associated with the trend test. Beneath the dosed group incidenceare the P values corresponding to 

pairwise comparisons beween the vehicle controls and that dosed group. The life table test regards neoplasms in animals dying prior to terminal killas 

being (directly or indirectly) the cause of death. The,logistic regression test regards these lesions as nonfatal. The Cochran-Armitage and Fisher exact tests 

compare directly the overall incidence rates. For all tests, a negative trend or a lower incidence in a dose group is indicated by N. .;. . . . e Not applicable; noneoplasmsinanimal group 
Overall Historical Incidence 

Range 

Standard deviation 

Total 

. . 

Incidence in Controls 

0%-2% 

0.8% 

31903 (0.3%) 

a Data as of 17 June 1994 

Incidence in Controls 

Overall Historical Incidence 

Total 

a Data as of 17 June 1994 

01920 (0%) 
-174 ' Salicylaiosulfapyridine,NTP TR.457 

TABLEB5 . .. ~

Summary of the Incidenceof Nonneoplastic Lesionsin Female Rab in the 2-Year Gavage Study 

of Salicylsrzosulfapyridinea 

Vehicle Control 84 mglkg 

Disposition Summary 

Animals initially in study 60 60 

IS-Month initerim evaluation 10 10 

Early deaths 

Accidental deaths 1 1 

Moribund 12 13 

Natural deaths 2 2 

Survivors 

Died last week of study 1 1 

Terminal sacrifice 34 33 

Animals examined microscopically 60 60 

15-Month Interim Evaluation 

Alimentary System 

Intestine large, colon 

Parasite metazoan 

Intestine large, rectum 

Hyperplasia, lymphoid 

Parasite metazoan 

Liver , Basophilic focus 

Clear cell focus 

Eosinophilic focus 

Granulom 1 (10%) 

Hepatodiaphragmatic nodule 3 (30%) 

Inflammation, chronic 1 (10%)

Mixed cell focus 

Mesentery 

Fat, necrosis 

Pancreas 

Atrophy 

Acinar cell, basophilic focus 

Stomach, forestomach 

Mineralization 

Cardiovascular System 

Heart 

Cardiomyopathy , 

Endocrine System 

Adrenal cortex (10) (10)

Accessory adrenal cortical nodule 1 (10%) 

Degeneration, fatty 1 (10%) 

Hyperplasia, focal 1 (10%) 

a Number of animals examined microscopically at the site and the number of animals with lesion 

168 337.5 mglkg

60 

10 

14 

5 

31 

60 

(10)

10 (100%) 

1 (10%) 

3 (30%) 

2 (20%) 

2 (20%) 

(1)

1 (100%) 

(10)

3 (30%) 

(10)

1 (10%) 

(10)

1 (10%) 

1 (10%) 

mgkg 

,. , 

60 

10 

9 . 

11 

4 

26 

60 

(9)

1 (11%) 

(9)

1 (11%) 

(10) 

5 (50%) 

2 (20%) 

1 (10%) 

(10) 

. I 

(10) 
Vehicle Control M mgkg 168 mglkg 337.9 mgkg 

Pars intermedia, cyst 

Pars distalis, hyperplasia, focal 

Pars distalis, cyst 

Pars distalis, angiectasis 

Ccell, hyperplasia 

Ultimobranchial cyst 

Thyroid gland 

1 (10%) 

1 (10%) 

6 (60%) 

1 (10%) 

(10) 

2 (20%) 

5 (50%) 

2 (20%) 

(10) 

2 (20%) 

1 (10%) 

1 (10%) 

5 (50%) 

1 (10%) 

(10) 

1 (10%) 

ahmitd System 

Clitoral gland 

Inflammation, chronic 

Inflammation, suppurative 

Ovary 

cyst 

uterus 

Hydrometra 

WC!m&@?tiC 

Bone marrow 

SyStem 

Pigmentation, hemosiderin 

Hematopoietic cell proliferation 

Pigmentation 

Hemorrhage 

Pigmentation 

Hemorrhage 

Mediastinal, pigmentation 

Mediastinal, hemorrhage 

Myelofibrosis 

Hypercellularity 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Hemorrhage 

Thymus 

Mwcoloskeletral System 

Bone 

Calvarium, osteopetrosis 
176 Salicylazosulfapyridine, NTP TR'457 

, , 'i 

TABLEBS ,. .. 

Summary of the Incidence of Nonneoplastic Lesions in FemaleRats in the2-YearGavage Study 

of Salicylazosulfapyridine(continued) 

VehicleControl ' ' . 84 mg/kg , . 188 mgkg 337.5 mglkg .. . 

IS-Month Interim Evaluation (continued) 

Respiratory System 

Lung

Infiltration cellular, histiocyte 

(10) 

1 (10%) 

(10) 

2 (20%) 

Inflammation, subacute 4 (40%) 

Metaplasia, osseous 

Alveolar epithelium, hyperplasia 3 (30%) 

Exudate 

Nose (10) (10) 

Foreign body 

Urinary System 

Kidney (10) (10) (10) 

Concretion 6 (60%) 

cyst 

Hydronephrosis 1 (10%) 

Inflammation, chronic 

Mineralization 9 (90%) 10 (100%) 9 (90%) 

Nephropathy 4 (40%) 6 (60%) 10 (100%) 

Transitional epithelium, hyperplasia 8 (80%) 

Urinary bladder (10) (10) (10) 

Mucosa, hyperplasia 2 (20%) 

Systems Examined With No Lesions Observed 

General Body System 

Integumentary System 

Special Senses System 

2-Year Study 

Aliientarly System 

Esophagus 

Diverticulum 

Perforation 

Intestine large, colon 

Parasite metazoan 

Intestine large, rectum 

Parasite metazoan 

Intestine large, cecum 

Hemorrhage 

Inflammation, subacute 

Parasite metazoan 1 (2%) 2 (4%) 
(OS) 

! 
Salicylazosulfapyridine, NTP TR 457 

TABLEB!; 

Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Gavage Study 

of ~Salicylrazosulfapyridine(continued) 

%Year Study (continued) 

Endocrine System 

Adrenal cortex 

Accessory adrenal cortical nodule 

Angiectasis 

cyst 

Degeneration, fatty 

Hematopoietic cell proliferation 

Hemorrhage 

Hyperplasia, diffuse 

Hyperplasia, focal 

Hypertrophy, focal 

Necrosis 

Adrenal medulla 

Hyperplasia 

Necrosis 

Pituitary gland 

Pars distalis, angiectasis 

Pars dista'lis, cyst 

Pars distalis, hyperplasia 

Pars distalis, hyperplasia, focal 

Pars intermedia, angiectasis 

Pars intermedia, cyst 

Thyroid gland 

Ultimobratnchial cyst 

C-cell, hyperplasia 

Follicle, cyst 

Follicular cell, hyperplasia 

General Body System 

None, 

Genital System 

Clitoral gland 

Ectasia 

Hyperplasia 

Inflammation, chronic 

Inflammation, suppurative 

Ovary 

cyst 

uterus 

cyst

Hydrometra 

Hyperplasia, cystic 

Endometrium, hyperplasia, focal 

Vagina 

Hyperplasia 

VehicleControl 84 mgkg 168 mglkg 

(50) 

8 (16%) 

(50) 

10 (20%) 

5 (10%) 1 (2%) 

1 (2%) 

10 (20%) 6 (12%) 

1 (2%) 

1(2%) 

6 (12%) 9 (18%) 

4 (8%) 5 (10%) 

1 (2%) 

2 (4%) 

(50) (50) 

2 (4%) 

(49) 

5 (10%) 

(50) 

7 (14%) 

26 (53%) 18 (36%) 

13 (27%) 12 (24%) 

3 (6%) 1 (2%) 

5 (10%) 2 (4%) 

1(2%) 

(50) 

3 (6%) 

(50) 

10 (20%) 10 (20%) 

4 (8%) 

(50) 

15 (30%) 

(50) 

17 (34%) 

2 (4%) 1 (2%) 

15 (30%) 8 (16%) 

2 (4%) 4 (8%) 

4 (8%) 

(50) (50)

1 (2%) 

(50) (50) 

3 (6%) 5 (10%) 

2 (4%) 2 (4%) 

1 (2%) 

(1) (1) 

1 (100%) 

337.5 mg/kg 

(50) 

16 (32%) 

2 (4%) ' -

7 (14%) 

(50) 

18 (36%) 

2 (4%) 

6 (12%) 

7 (14%) 

(50) 

4 (8%) 

(50) 
3-Year sway (continued) 

Hematopoietic System 

Bone marrow 

Hypercellularity 

Myelofibrosis 

Necrosis 

Lymph node 

Iliac, ectasia 

Iliac, hyperplasia, lymphoid 

Mediastinal, congestion 

Mediastinal, ectasia 

Mediastinal, hemorrhage 

Mediastinal, pigmentation 

Pancreatic, hemorrhage 

Pancreatic, hyperplasia, lymphoid 

Pancreatic, pigmentation 

Renal, hemorrhage 

Renal, hyperplasia, lymphoid 

Renal, pigmentation 

Lymph node, mandibular 

Congestion 

Ectasia 

Hemorrhage 

Hyperplasia, lymphoid

Hyperplasia, plasma cell 

Pigmentation 

Lymph node, mesenteric 

Ectasia 

Hemorrhage 

Hyperplasia, lymphoid 

Pigmentation 

Spleen 

Developmental malformation 

Fibrosis 

Hematopoietic cell proliferation 

Hemorrhage 

Necrosis 

Pigmentation, hemosiderin 

Lymphoid follicle. atrophy

Lymphoid follicle, hyperplasia 

Thymus 

Congestion 

cyst

Hemorrhage 

Cortex, atrophy 

Epithelial cell, hyperplasia 

Control Vehicle

(50) 

3 (6%) 

1 (2%) 

(12) 

6 (50%) 

7 (58%) 

(50) 

7 (14%) 

3 (6%) 

4 (8%) 

6 (12%) 

(50) 

2(4%) 

10 (20%) 

(50) 

3 (6%) 

25 (50%) 

1 (2%) 

1 (2%) 

38 (76%) 

1 (2%) 

1 (2%) 

(49) 

1 (2%) 

$4 mgkg 

(50)

3 (6%) 

1 (2%) 

(6) 

4 (67%) 

4 (67%) 

(48) 

7 (15%) 

3 (6%) 

5 (10%) 

1 (2%) 

14 (29%) 

(50) 

1(2%) 

4 (8%) 

1 (2%) 

9 (18%) 

(50) 

1 (2%) 

27 (54%) 

33 (66%) 

(49) 

1 (2%) 

1 (2%) 

168 mgkg 

1 (11%) 

3(33%) 

7 (78%) 

1 (11%) 

1 (11%) 

(50) 

3 (6%) 

2 (4%) 

4 (8%) 

6 (12%) 

(49) 

2 (4%) 

11 (22%) 

(50) 

2 (4%) 

1 (2%) 

36 (72%) 

1 (2%) 

35 (70%) 

337.5 mgkg 

(50)

2 (4%) 

(7) 

1 (14%) 

1 (14%) 

1 (14%) 

3 (43%) 

3 (43%) 

1 (14%) 

1 (14%) 

1 (14%) 

1 (14%) 

1 (14%) 

(50) 

1 (2%) 

1 (2%) 

2(4%) 

7 (14%) 

(50) 

6 (12%) 

2 (4%) 

(50) 

29 (58%) 

1 (2%) 

37 (74%) 

1 (2%) 

(50) 

1 (2%) 

6 (12%) 

1 (2%) 

1 (2%) 
180 Salicylazosulfapyridine,NTP TR 457 

TABLEB5 

Summary of the Incidence of Nonneoplastic Lesions in FemaleRats in the 2-YearGavage Study 

of Salicylazosulfapyridine(continued) 

Vehicle Control 84 mg/k 168 mgkg 337.5 mglkg 

&Year Study (continued) 

Integumentary System 

Mammary gland 

Ectasia 

Hyperplasia, lobular 

Skin 

Acanthosis 

Cyst epithelial inclusion 

Hyperkeratosis 

Inflammation, chronic 

Ulcer 

Subcutaneous tissue, inflammation, subacute 1 (2%) 

Musculoskeletal System 

Bone (50) (50) (50) (50)

Calvarium, osteopetrosis 4 (8%) 1 (2%) 1 (2%) 

Femur, osteopetrosis 5 (10%) 3 (6%) 2 (4%) 

Skeletal muscle (2) (1) (1)

Hemorrhage 1 (50%) 

Inflammation, chronic 1 (50%) 

Nervous System 

Brain (50) (50) (50) (50) 

Atrophy 1 (2%) 

Compression 10 (20%) 11 (22%) 10 (20%) 4 (8%) 

Hemorrhage 2 (4%) 2 (4%) 1 (2%) 

Hydrocephalus 3 (6%) 3 (6%) 4 (8%) 

Mineralization 1 (2%) 1 (2%) 

Necrosis 1 (2%) 1 (2%) 1 (2%) 

Respiratory System 

Lung (50) (50) (50) 

Congestion 3 (6%) 2 (4%) 7 (14%) 

Edem.a 1 (2%) 2 (4%) 4 (8%) 

Foreign b'ody 2 (4%) 2 (4.%) 8 (16%) 

Hemorrhage 2 (4%) 2 (4%) 

Infiltration cellular, histiocyte 14 (28%) 13 (26%) 9 (18%) 13 (26%) 

Inflammation, subacute 1 (2%) 5 (10%) 1 (2%) 2 (4%) 

Thrombosis 1 (2%) 

Alveolar epithelium, hyperplasia 2 (4%) 7 (14%) 4 (8%) 2 (4%) 

Nose (50) (50) (50) (50) 

Exudate 9 (18%) 9 (18%) 10 (20%) 5 (10%) 

Foreign body . 3 (6%) 6 (12%) 4 (8%) 2 (4%) 

Fungus 1 (2%) 2 (4%) 

Mucosa, hyperplasia 4 (8%) 2 (4%) 1 (2%) 1 (2%) 

Mucosa, metaplasia, squamous 1 (2%) 2 (4%) 1 (2%) 
I 

2-l'~~ Shdy (continued) 

Special Senses System 

Eye

Cataract 

Inflammation, chronic 

Inflammation, suppurative 

Phthisis bulbi 

Retina, atrophy 

urinary Bystem 

Kidney 

Concretion 

cyst 

Hemorrhage 

Hydronephrosis 

Infarct 

Inflammation, suppurative 

Mineralization 

Nephropathy

Papilla, necrosis 

Renal tubule, cytoplasmic alteration 

Renal tubule, dilatation 

Renal tubule, hyperplasia 

Renal tubule, necrosis 

Renal tubule, pigmentation 

Transitional epithelium, hyperplasia 

Ureter 

Concretion 

Mucosa, hyperplasia

Urethra 

Mucosa, hyperplasia 

Urinary bladder 

Concretion 

Mucosa, hyperplasia 

VehicleControl 

(3) 

1 (33%) 

1 (33%) 

1 (33%) 

2 (67%) 

(50) 

1 (2%) 

44 (88%) 

34 (68%) 

2 (4%) 

1 (2%) 

8 (16%) 

3 (6%) I 

(49) 

2 (4%) 

M mgkg 

(5) 

3 (60%) 

2 (40%) .~ 2 (40%) 

(50) 

9 (18%) 

1 (2%) 

47 (94%) 

36 (72%) 

2 (4%) 

1 (2%) 

6 (12%) 

7 (14%) 

(50) 

168 mg/kg 

(2) 

1 (50%) 

1 (50%) 

1 (50%) 

(50) 

34 (68%) 

3 (6%) 

2 (4%) 

1 (2%) 

43 (86%) 

41 (82%) 

1 (2%) 

1 (2%) 

2 (4%) 

23 (46%) 

(2) 

1 (50%) 

2 (100%) 

(1) 

1 (100%) 

(50) 

1 (2%) 

4 (8%) 

337.5 mglkg 

(50) 

37 (74%) 

1 (2%) 

1 (2%) 

10 (20%) 

3 (6%) 

2 (4%) 

41 (82%) 

44 (88%) 

1 (2%) 

1 (2%) 

6(12%) 

43 (86%) 

(4)

2 '(50%) 

4 (100%) 

(50) 

12 (24%) 
182 Salicylazosulfapyridine, NTP TR 457 

184 Salicylazosulfapyridine, NTP'TR 457 

TABLEC1 

Summary of the Incidence of Neoplasrns,inMale Mice in the 2-YearGavage Study of Salicylazosulfapyridinea 

Vehicle Control 675 mgkg 1,350 mglkg 2,700 mg/kg 

Disposition Summary 

Animals initially in study 60 60 60 60 

15-Month interim evaluation 10 10 10 10 

Early deaths 

Accidental deaths 2 

Survivors 

. Moribund 

Terminal sacrifice 

Natural deaths ' 3 

5 

40 

5 

4 

41 41 

6 

3 4 

46 

Animals examined microscopically 60 60 60 60 

15-Month Interim Evaluation 

Alimentary System 

Liver 

Hepatocellular carcinoma 

Hepatocellular adenoma 

Stomach, forestomach 

Squamous cell papilloma 

Respiratory System 

Lung (10) (10)

Alveolar/bronchiolar adenoma 1 (10%) 

Alveolaribronchiolar carcinoma 

Systems ExaminedWith No NeoplasmsObserved 

Cardiovascular System 

Endocrine System 

General Body System 

Genital System 

Hematopoietic System 

Integumentary System 

Musculoskeletal System 

Nervous System 

Special Senses System 

Urinary System 

2-Year Study 

Alimentary System 

Intestine large, colon 

Leiomyosarcoma 

Intestine large, cecum 

Adenocarcinoma ' 

Intestine small, jejunum 

Adenocarcinoma 

Intestine small, ileum 
%y@fW%Udy (continued) 

Alimentary System (continued) 

Liver 

Hemangioma 

Hemangiosarcoma 

Hepatoblastoma 

Hepatocellular carcinoma 

Hepatocellular carcinoma, multiple 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Histiocytic sarcoma 

Osteosarcoma, metastatic, bone 

Mesentery 

Pancreas 

Carcinoma, metastatic, lung 

Acinus, adenoma 

Stomach, forestomach 

Squamous cell papilloma 

Stomach, glandular 

Adenoma 

Cardiovascular System 

Blood vessel 

Aorta, alveolarhronchiolar carcinoma, 

metastatic, lung 

Endocrine System 

Adrenal cortex 

Carcinoma, metastatic, lung 

Capsule, adenoma 

Adrenal medulla 

Carcinoma, metastatic, lung 

Islets, pancreatic 

Adenoma 

Pituitary gland 

Pars distalis, adenoma 

Pars intermedia, adenoma 

Pars intermedia, carcinoma 

Thyroid gland 

Follicular cell, adenoma 

Follicular cell, carcinoma 

General Body System 

None 

Genital System 

Epididymis 

Histiocytic sarcoma 

Preputial gland 

Adenoma 

Prostate 

Vehicle Control . . 675mghg 1,350 mglkg 2,700 mglkg

1 (2%) 

11 (22%) 

4 (8%) 

11 (22%) 

21(42%) 

(50) 
186 Salicylazosulfapyridine, lVTPTR 457 

2-YetZt (continued) 

Hematopoietic System 

Bonemarrow , 

Histiocytic sarcoma 

Lymph node 

Mediastinal, alveolarlbronchiolar 

carcinoma, metastatic, lung 

Mediastinal, histiocytic sarcoma 

Pancreatic, histiocytic sarcoma 

Lymph node, mandibular 

Lymph node, mesenteric 

Hemangiosarcoma 

Histiocytic sarcoma 

Histiocytic sarcoma 

Hemangiosarcoma 

Spleen (50) 

1 (2%) 

Carcinoma, metastatic, lung 

Thymus (45) 

1 (2%) 

(43) 

Skin 

Integumentary System 

Subcutaneous tissue, fibrous histiocytoma 

(50) (50) 

1(2%) 

(50) (50) 

Subcutaneoustissue,hemangiosarcoma . 1 (2%) 

Subcutaneous tissue, osteosarcoma, 

metastatic, bone 1 (2%) 

Subcutaneous tissue, plasma cell tumor, 

benign 1 (2%) 

Musculoskeletal System 

Bone 

Vertebra, osteosarcoma 

Nervous System 

Brain 

Respiratory System 

Lung

Alveolarr'bronchiolar adenoma 

(50) 

9 (18%) 

Alveolar/bronchiolar adenoma, multiple 2 (4%) 

, 

Alveolar/bronchiolar carcinoma, multiple 

Alveolar/bronchiolar carcinoma . 3(6%) 

Carcinoma, metastatic, tissue NOS 

Osteosarcoma, metastatic, bone 

Hepatocellularcarcinoma,metastatic,liver 4 (8%) 2 (4%) 

1 (2%) 

Alveolar epithelium, alveolar/bronchiolar 

adenoma 1 (2%) 

Mediastinum, alveolar/bronchiolar carcinoma 1 (2%) 
Vehicle Control 675 mghg 

%4dy (continued) 

§pecial Senses System 

Harderian gland (3) (5)

Adenoma 2(67%) 4 (80%) 

Carcinoma 1 (33%) 

Urinary 'System 

Kidney 

Urinary bladder 

Hemangioma 

Systemic Lesions 

Multiple organsb 60) (50)

Histiocytic sarcoma 

Lymphoma malignant 5 (10%) 3 (6%) 

Lymphoma malignant lymphocytic 

Neoplasm Summary 

Total animals with primary neoplasms' 

15-Month interim evaluation 4 2 

2-Year study 40 45 

Total primary neoplasms 

15-Month interim evaluation 4 2 

2-Year study 62 I7 

Total animals with benign neoplasms 

15-Month interim evaluation 4 2 

2-Year study 26 36 

Total benign neoplasms 

15-Month interim evaluation 4 2 

2-Year study 35 48 

Total animals with malignant neoplasms 

15-Month interim evaluation 

2-Year study 25 26 

Total malignant neoplasms 

15-Month interim evaluation 

&Year study 27 29 

Total animals with metastatic neoplasms 

%Year study 5 2 

Total metastatic neoplasms 

%Year study 8 2 

a Number of animals examined microscopically at the site and the number of animals with neoplasm 

Number of animals with any tissue examined microscopically 

Primary neoplasms: all neoplasms except metastatic neoplasms 

1,350 mglkg 

(1)

1 (100%) 

(50)

2 (4%) 

1 (2%) 

3 

41 

4 

77 

2 

32 

2 

46 

2 

28 

2 

31 

2 

5 

2,700 mglkg 

(2)

2 (100%) 

(50) 

1 (2%) 

1 (2%) 

2 

45 

2 

75 

2 

43 

2 

56 

14 

19 

1 

1 
188 'Salicylizosulfapyridine,.NTPTR 457 

TABLEC2, 

Individual1 Animal Tumor Pathologyof Male Mice in the 2-Year Gavage Study of Salicylazosulfapyridine: . ' ' ' . . i., 

Vehicle Clontrol 

Number of Days on Study 

Carcass . . ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver' . 

Hemangioma 

Hemangiosarcoma 

Hepatocellular carcinoma 

Hepatocellular carcinoma, multiple 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Mesentery 

Pancreas 

Carcinoma, metastatic, lung 

Acinus, adenoma 

Salivary glands 

Stomach, forestomach 

Squamous cell papilloma 

Stomach, glandular 

Cardiovascular System 

Blood vessel 

Heart 

Endocrine System 

Adrenal cortex 

Carcinoma, metastatic, lung 

Capsule, adenoma 

Adrenal medulla 

Carcinoma, metastatic, lung 

Islets, pancreatic 

Adenoma 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Pars intermedia, carcinoma 

Thyroid gland 

Follicular cell, adenoma 

General Body System 

None 

+: Tissue examined microscopically 

A: Autolysis precludes examination 

~~~~ ______ ~~ 

1344666666777777777777777 

5522115668222222222233:33-.3' 

0100018269888888888800000 

0000000000000000000000000 

1300134012001122334400001 1 

2325704838495926250513670 

......................... 

+++A+++++++++++++++'++++++ 

A++A+++++++++++++++++++++ 

A++A+++++++++++++++++++++ 

: 

A++A+++++A+++++++++++++++ . 

A++A+++++A+++++++++++++++. 

A++A+++++A+++++++++++++++ 

A++A+++++A+++++++++++++++.. 

......................... 

X X 

x x xx x X ' X .I

A 

x x X X x 

. . . . 

+ + + ......................... X . .. 

X . i .

.. . 

......................... 

......................... X 

......................... 

.......................... 

......................... 

......................... 

X 

.......................... X 

......................... 

+++++++++++M+++++++++++++. 

M+++++++++M++++++++++++++ 

X 

......................... 

M: Missingtissue X: Lesion present 

I: Insufficient tissue Blank: Not examined 
TABLE c2 

IIEIMI"U~ AW~ hIUhdOgj' ODB 

Vehhcne CounUrd (continued) 

Number o f Days on Study 

CmcassIDNumber 

I 

Gallbladder 

Esophagus 

Alhentaa~ysystem 

Intestine small, duodenum 

Intestine large, cecum 

Intestine large, rectum 

Intestine large, colon 

Intestine small, ileum 

Intestine small, jejunum 

Liver 

Hepatocellular adenoma, multiple 

Hepatocellular adenoma 

Hepatocellular carcinoma, multiple 

Hepatocellular carcinoma 

Hemangiosarcoma 

Hemangioma 

Pancreas 

Mesentery 

Carcinoma, metastatic, lung 

Acinus, adenoma 

Stomach, forestomach

Salivary glands 

Stomach, glandular 

Squamous cell papilloma 

I ~ ~ ~ ~~ 

CardiovmculaaPsystem 

Blood vessel 

Heart 

IEwdcPcPine system 

Adrenal cortex 

Adrenal medulla 

Capsule, adenoma 

Carcinoma, metastatic, lung 

Carcinoma, metastatic, lung 

Adenoma 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Pars intermedia. carcinoma 

Follicular cell, adenoma 

Thyroid gland 

Geneml Body System

None 

MLQ ~rn ~-VWG~VX~su~dg.OITBanicgm~~nnnffa~~~~~iune: 

lllllilllll777l777l777l77 

3333333333333333333333333 

0000000000000000000011111 

0000000000000000000000000 Total 

1111222222233333344444445 Tissues/ 

j 1468013451914678912367890 Tumors 

......................... 50 

......................... 49 

.......................... 48 

......................... 48 

........................... 47 

......................... 47 

......................... 47 

......................... 47 

......................... 50 

x 1 

2 

x x x x x 12 

. . 1 j 

x x x x X X X 12 

x 1 

+ 4 

......................... 50 i 

1 j 

1 1 ......................... 50 1 

......................... 50 j xx 3 I ......................... 50 j 

~~ ~- ~~ ~~ ~~ ~ ~ 

......................... 50 

......................... 50 

......................... 50 

1 

1 

......................... 50 

1 

......................... 50 

x 1 

+++++++++++++++++++++M+++ 48 

+M++++++++++MM++++++++M++ 44 

x 1 

1 

......................... 50 

X 1 
190 Salicylazosulfapyridine, NTP TR 457 

TABLEC2 

Individual1 Animal Tumor Pathology of Male Mice in the 2-Year Gavage Study of Salicylazosulfapyridine: 

Vehicle Control (continued) -. 

1344666666111111111111111 

Number of Days on Study 5522115668222222222233333 

0100018269888888888800000 

"0000000000000000000000000 

Carcass ID Number 1300134012001122334400001 

2325104838495926250513670 ~~ ~ 

Genital System 

Coagulating gland + 

Epididymis ......................... Preputial gland ......................... Prostate ......................... Seminal vesicle ......................... Testes ......................... 

Hematopoietic System 

Bone marrow ......................... Lymph node + 

Lymph node, mandibular .......................... Lymph node, mesenteric ......................... Spleen ......................... Hemangiosarcoma X 

Thymus ++M+M+M+++M++++++++M+++++ 

Carcinoma, metastatic, lung X 

Integumentary System 

Mammary gland .......................... Skin ......................... Subcutaneous tissue, hemangiosarcoma 

Musculoskeletal System 

Bone .......................... 

Nervous System 

Brain ......................... Spinal cord + 

Respiratory System 

Lung ......................... Alveolarlbronchiolar adenoma X X 

Alveolarlbronchiolar adenoma, multiple 

Alveolaribronchiolar carcinoma X. X X 

Hepatocellular carcinoma, metastatic, liver X 

Nose ......................... Trachea ......................... 

Special Senses System 

Ear 

Harderian gland M 

Adenoma 

Carcinoma 

Urinary System 

Kidney ......................... Urinary bladder .......................... ~~ ___~ 

Systemic l~sions 

Multiple organs .......................... Lymphoma malignant X X 
.. 

Number OP Days on Study 

Genital System 

Coagulating gland 

Epididymis 

Preputial gland 

Prostate 

Seminal vesicle 

Testes 

HematQpQi@tiCsystem 

Bone marrow 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Hemangiosarcoma 

Thymus 

Carcinoma, metastatic, lung 

htegmentary system 

Mammary gland 

Skin 

Subcutaneous tissue, hemangiosarcoma 

Musculoskeletal System 

Bone 

Respiratory system 

Lung 

Alveolar/bronchiolar 

Alveoladbronchiolar 

Alveolar/bronchiolar 

inoma, Hepatocellular 

Nose 

Trachea 

Sp@cidsenses System

Far 

Harderian gland 

Adenoma 

Carcinoma 

urinary system 

Kidney 

Urinary bladder 

systemic hSiOnS 

Multiple organs 

Lymphoma malignant 

7777777777777777777777777 

3333333333333333333333333 

0000000000000000000011111 

0000000000000000000000000 Total 

1111.222222233333344444445 Tissues/ 

I468013457914678912367890 Tumors 

+ + + + 5 

......................... 50 

++M++++++++++++++++++++++ 49 

......................... 50 

......................... 50 

......................... 50 

......................... 50 

+ + 3 

......................... 50 

......................... 50 

......................... 50 

1 

......................... 45 

1 

......................... 50 

......................... 50 

X 1 

......................... 50 

......................... 50 

1 

......................... 50 

xxx adenoma X
 X 9

X X 2 

carcinoma 3 

xx X 4 

......................... 50 

......................... 50 

+ 1 

+ ++ 3 

X X 2 

X 1 

......................... 50 

......................... 50 

......................... 50 

xx X 5 
.192 Salicyl&osulfapyridine; NTP TR 457 

TABLEC2 i . Individual Animal Tumor Pathology of Male Micein the 2-Yk Gavage Study of Salicylazosulfapyridine: . . 

675 mglkg 

Number of IDays on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large:, colon 

Leiomyosarcoma 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatoblastoma 

Hepatocellular carcinoma 

Hepatocellular carcinoma, multiple 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Mesentery 

Pancreas 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Cardiovascular System 

Blood vessel 

Heart 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Pars intermedia, adenoma 

Thyroid gland 

General Body System 

None 

Genital System 

Coagulating gland 

Epididymis 

Preputial gland 

Prostate 

Seminal vesicle 

Testes 

0255666677777777777777777 

7868156602222222222333333 

5878125608888888888222222 

0010000000000000011000000 

780896867677788990066666.6 

8359042165149481506236789 

......................... 

A+A++++++.++++++++++++++++ 

A+A++++A+++++++++++++++++ 

X 

A++++++A+++++++++++++++++ 

A+A+++AA+++++++++++++++++ -. 

A+++++AA+++++++++++++++++. 

A+A+++AA+++++++++++++++++ 

A+A+++AA+++++++++++++++++ 

......................... 

x x . X X -

X x x 

X X xx 

x xxxx xxx xxxx 

+ + + + ......................... 

......................... 

.......................... .. 

......................... 

.......................... 

......................... 

++++++++++++++M++++++++++++ . .

++++++++++++++M++++++++++ 

......................... 

+M++++++++++M++++++++++++ 

M+++++MM+++++++++++++++++ . . X ......................... .. 

.......................... 

+++M+++++++++++++++++++++ '' 

......................... 

......................... 

......................... 
77777777777777777'77777777 

Number of Days on Study 3333333333333333333333333 

2222222222222222222222222 ~~ ~ ~ ~ ~~ ~ ~~~~~~ ~ ~ 

. ,. 

0000000~0000000000111 11 11 1 Total 

, 7777788888999999900000001 Tissues/ 

0235701567234678912347890 Tumors 

. & . 

Gallbladder 

Esophagus 

Alimentary System ......................... 

......................... 50 

48 

Intestine large, rectum 

Leiomyosarcoma 

Intestine large, colon 

Intestine small, duodenum 

Intestine large, cecum 

......................... 

......................... 

.......................... 

......................... 46 

48 

1 

47 

47 

Intestine small, ileum 

Intestine small, jejunum 

......................... 

......................... 46 

46 

Liver ......................... 50 

Hepatocellular adenoma, multiple 

Hepatocellular adenoma 

Hepatocellular carcinoma, multiple 

Hepatocellular carcinoma 

Hepatoblastoma 

Pancreas 

Mesentery 

xx x x xx x

X 

X 

x xx x xx x 

xxx x X xx X X 

......................... 

21 

11 

4 

11 

1 

50. 

4 

Stomach, forestomach 

Salivary glands 

......................... 

......................... 50 

50 

Stomach, glandular ......................... 50 

Blood vessel 

Cardiovascular System ......................... 50 

Heart ......................... 50 

Adrenal cortex 

Endocrine System .......................... 49 

Adrenal medulla ......................... 49 

Pituitary gland 

Parathyroid gland 

Islets, pancreatic 

Pars intermedia, adenoma 

++++++++++M++++++++++++++ 

+.++++++++++++++++++++M+++ 

.......................... 

46 

47 

50 

1 

Thyroid gland ......................... 50 

Genital System 

Coagulating gland + 1 

Epididymis ......................... 50 

Preputial gland ......................... 49 

Prostate ......................... 50 

Seminal vesicle ......................... 50 

Testes ......................... 50 
194 Salicylkosulfapyridine, NTP TR 457 

TABLEC2, 

Individual1 Animal Tumor Pathology.of Male Mice in the 2-Year Gavage Study of Salicylazosulfapyridine: 

675 mg/kg (continued) 

0255666611111111111111111 

Number of Days on Study I868156602222222222333333 

581812560888888888,8222222 

0010000000000000011000000 

Carcass ID Number 1808968616117889900666666 

8359042165149481506236789 

Hematopoietic System 

Bone marrow ......................... Lymph node + .+ + 

Lymph node, mandibular ......................... Lymph node., mesenteric ......................... Spleen ......................... Thymus ++M++++M+M++++++++M++++++. 

Integumentary System 

Mammary gland ......................... Skin ......................... Subcutaneous tissue, fibrous histiocytoma X ~~ ~~~ ~ 

Musculoskeletal System 

Bone ......................... ~~~~ ________ ~~ 

Nervous System 

Brain ......................... 

Respiratory System 

Lung ......................... Alveolar/bronchiolar adenoma X xx X 

Alveolarhronchiolar carcinoma X X x x X 

Hepatocellular carcinoma, metastatic, liver X 

Alveolar epithelium, 

alveoladbronchiolar adenoma X 

Nose ......................... Trachea .......................... 

Special Senses System 

EY e Harderian g:land + + + 

Adenoma X X 

Urinary System 

Kidney ......................... Urinary bladder .......................... 

Systemic Lesions 

Multiple organs ......................... Lymphoma malignant X X 
n95 

Number of Days QII Study 

I 

-I 

-- I Hematopoietic System

i Bone marrow 

Lymph node 

Lymph node, mandibular I- Lymph node, mesenteric 

--I 1 Spleen 

Thymus I

I ~~ ~~ ~ ~ 

i Integumentary System 1 Mammary gland 

Skin 

Subcutaneous tissue, fibrous histiocytoma 

I Musculoskeletal System

I Bone

I Nervous System -~ I Brain =I 

-1 1 Respiratory System 

I Lung 

-i 

Alveolar/bronchiolar adenoma 

Alveolarlbronchiolar carcinoma 

Hepatocellular carcinoma, metastatic, liver 

Alveolar epithelium, 

alveolar/bronchiolar adenoma 

i Nose 

Trachea I

' Special Senses System 1 Eye -: 1 Harderian gland 

I Adenoma 

1 urinary ~yitem 

Kidney ' 

.. 

I Urinary bladder 

j Systemic bions 

Multiple organs I Lymphoma malignant 

7777777777777777777777777 

3333333333333333333333333 

2222222222222222222222222 

~-- ~p ~ ~ ~ ~~ ~~p -~ ~ ~~~~~~~ ~ ~ -

000000000000000001 11 11 11 1 Total 

7777788888999999900000001 Tissues/ 

0235701567234678912347890 Tumors 

......................... 50 

3 

+++++++++++M+++++++++++++ 49 

......................... 50 

......................... 50 

++M+++++++M++++++++++++M+ 43 ____ ~~ ~ ____ ~ ~-p ~ -

......................... 50 

......................... 50 

1 

......................... 50 

......................... 50 

......................... 50 

xx X X X X 10 

xx X 8 

X 2 

1 

......................... 50 

......................... 50 

+ 1 

+ + 5 

X X 4 

......................... 50 

......................... 50 

......................... 50 

X 3 
196 Salicylazosulfapyridine,.NTPTR 457 

TABLEC;! 

Individual AnimalTumor Pathology of Male Mice in the 2-YearGavage Study of Salicylazosulfapyridine: 

1,350 mgr'kg 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine larj,re,rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Adenocarcinoma 

Intestine small, ileum 

Liver 

Hepatocellular carcinoma 

Hepatocellular carcinoma, multiple 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Histiocytic sarcoma 

' Osteosarcoma,metastatic,bone 

Mesentery 

Pancreas 

Salivary glands 

Stomach, forestomach 

Squamous cell papilloma 

Stomach, glandular 

Cardiovascular System 

Blood vessel 

Aorta, alveolar/bronchiolar carcinoma, 

metastatic, lung 

Heart 

Endocrine System 

Adrenal cortex 

Capsule, adenoma 

Adrenal medulla 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Thyroid gland 

General Body System 

None 

Genital System 

Epididymis 

Histiocytic sarcoma 

Preputial gl,and 

Prostate 

Seminal vesicle 

Testes 

. . 

45566666777.77777777777777 

0001478802222222222333333 

2451881128888888888111111 

1111111.111111111111111111 

6245323522233444566222233 

8797942583936048316125601 . , 

.......................... 

++++++++++++M++++++++++++ 

, 

+AA+++++A++++++++++++++++. 

+AA+++++A++++++++++++++++ 

+AA+++A+A+++++++++++++.+++ 

+AA+++++A++++++++++++++++< 

+AA+++++A++++++++++++++++ 

+AA+++A+A++++.+++++.+++++++ 

......................... x x x xxxxx-xxx x 

X 

x x xx xxxx 

X X ,x X 

. 'X . . 

.......................... 

......................... 

+++++.+++A++++++'++++++++++ 

++A+++++A++++++++++++++++ 

......................... 

X ......................... 

.......................... X .......................... 

......................... 

......................... 

++++++++M+++++,+++++++++++ 

......................... 

.......................... 

+++++++++M+++++++++++++++ 

........................... 

........................... 

......................... 
1111111111111111111111111 Total 

3333444444555555566666661 Tissues/ 

4518123567012468902345190 Tumors 

Ahenbry System 

Esophagus ......................... 50 

Gallbladder ......................... 49 

Intestine large, colon ......................... 41 

Intestine large, rectum ......................... 41 

Intestine large, cecum ......................... 46 

Intestine small, duodenum ......................... 41 

Intestine small, jejunum ......................... 41 

Adenocarcinoma X 1 

Intestine small, ileum ......................... 46 

Liver ......................... 50 

Hepatocellular carcinoma xx x x X X 18 

Hepatocellular carcinoma, multiple X x x X 5 

Hepatocellular adenoma x x x X X 13 

Hepatocellular adenoma, multiple x x x xx xxx x x X 15

Histiocytic sarcoma X 1. 

Osteosarcoma, metastatic, bone 1 

Mesentery + 1 

Pancreas ......................... 50 

Salivary glands ......................... 50 

Stomach, forestomach ......................... 49 

Squamous cell papilloma x 1 

Stomach, glandular ......................... 48 

Crardiov~cularSystem 

Blood vessel ......................... 50 

Aorta, alveolar/bronchiolar carcinoma, 

metastatic, lung 1 

Heart ......................... 50 

EPIdwrhe system 

Adrenal cortex +++++++++++++++M+++++++++ 49 

Capsule, adenoma x 2 

Adrenal medulla +++++++++++++++M+++++++++ 49 

Islets, pancreatic ......................... 50 

Parathyroid gland +++++++-++++M+++++++++++++ 49 

Pituitaly gland M++M+++++++++++++++++++++ 41 

Thyroid gland ......................... 50 

Gen@ralBody system \ 

None 

GePIitPl system 

Epididymis ......................... 50 

Histiocytic sarcoma x 1 

Preputial gland ......................... 49 

Prostate ......................... 50 

Seminal vesicle ......................... 50 

Testes ......................... 50 
198 Saliciylazosulfapyridine,NTP TR 457 

TABLEC2 

Individual Animal Tumor Pathology of Male Mice in the2-YearGavage Studyof Salicylazosulfapyridine: 

1,350 mg;/kg (continued) 

Number of Days on Study 

Carcass IID Number 

Hematopoietic System 

Bone.marrow 

Histiocytic sarcoma 

Lymph node 

Mediastinal, alveolar/bronchiolar carcinoma, 

metastatic, lung 

Mediastinal, histiocytic sarcoma 

Pancreatic, histiocytic sarcoma 

Lymph node, mandibular 

Lymph node, mesenteric 

Histiocytic sarcoma 

Spleen 

Histiocytic sarcoma 

Thymus 

Integumentary System 

Mammary gland

Skin 

Subcutaneous tissue, osteosarcoma, 

metastatic, bone 

Musculoskeletal System 

Bone 

Vertebra, osteosarcoma 

Nervous System 

Brain 

Respiratory System 

Lung

Alveolar/bronchiolar adenoma 

Alveolarhonchiolar adenoma, multiple 

Alveolarhronchiolar carcinoma 

Osteosarcoma, metastatic, bone 

Mediastinum, alveolarhronchiolar carcinoma 

Nose 

Trachea 

Special Senses System 

Harderian gland 

Adenoma 

u?inary System 

Kidney 

Urethra 

Urinary bladder 

Systemic :Lesions 

Multiple organs 

Histiocytic sarcoma 

Lymphoma malignant 

. .. 

4556'666677777777777777777 

000147880222222222233333333 

2451881128888888888111111 ~~ 

1111111~111111111111111111 

6245323322233444566222.233 

8797942583936048316125601 

......................... 

+ + 

X 

.......................... 

......................... 

++++++++A++++++++++++++++ 

M+++++++M+++++++++++++M++ 

.......................... 

......................... 

X 

........................... X 

......................... 

......................... X xx X x xx 

xx 

X X 

X 

X .......................... 

.......................... 

+ +,.+ + 

+ 

+ + + +'+ + + + + + + + + + + + + +.+'+ + 

.......................... 

.......................... 
I

i 

:

i TABLE c2 

I : I[l”dUd hhdTUD!lMlQlrIhthOkDgJ‘ Off “kMice hthe %~CSIU’ (hVS@t &JJ~JJ Off Bankyn~QPsUnff~~~~d~~~: : ! 1,350 mgkg (continued) j ~. 7777777777777717777771777 ~~ i Number of Days on Study 3333333333333333333333333 ~~ 

z : 

i 1111111111112222222222222 ~~ 

= . 

.- 111111111~1111111111111111 Total II 

carcsnss m Number 3333444444555555566666667 Tissues/ ~= 

i =! 4578123567012468902345790 Tumors 

Hematopoietic System 

. .i Bone. marrow ......................... 50 - ii Histiocytic sarcoma -X 1 

Lymph node + 3 

; Mediastinal, carcinoma, alveolar/bronchiolar I metastatic, lung 1 i -i 

- 1 Mediastinal, histiocytic sarcoma X 1 

i Pancreatic,sarcoma histiocytic X 1

I Lymphmandibular node, +++++M+++++++++++++++++++ 49 i Lymphmesenteric node, ~~ ! ......................... 50

i Histiocytic sarcoma X 1 

! Spleen ......................... 49 ~~ 

i

\ Histiocytic sarcoma X 1 

=~ 

i i Thymus .......................... 46 I -

Integumentmy System 

Mammary gland ......................... 50 

Skin ......................... 50 

Subcutaneous tissue, osteosarcoma, 

metastatic, bone 1 

~. ~ ~~~ ~____ ~ ~~ ~~_______ ~~~ 

-: I 

: Musculoskeletal System ; 

21 

..~ i Bone ......................... 50 ;: Vertebra, osteosarcoma 1 

i 

~;j ~- i 

i 

~~~ 

~~ i :~ I -1 j 

=. i ~~~ I

=i 

~~ I 

~:~1 

=I 

-I 

~~~=! 

~~ 

.~~ i ~~ : 

i ~. 

.. I 

Newoussystem

Brain 

RespiratorySystem 

LungAlveolar/bronchiolar adenoma 

Alveolarhronchiolar adenoma, multiple Alveolar/bronchiolar carcinoma 

Osteosarcoma, metastatic, bone 

Mediastinum, alveolarhronchiolar carcinoma 

Nose 

Trachea 

Special Senses System

Harderian gland 

Adenoma 

urinary System 

Kidney 

Urethra 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Histiocytic sarcoma 

Lymphoma malignant 

......................... 

........................... x x xx 

X 

......................... 

......................... 

+ 

X 

......................... 

......................... 

......................... x X 

X 

50 

50 

11 

3 

2 

1 

1 

50 

50 

1 ’ 

1 

50 

1 

50 

50 

2 

1 
200 Salicylazosulfapyridine,NTP TR 457 

TABLEC2 

Individual Animal Tumor Pathology 'ofMale Miwin the 2-Year Gavage Study of Salicylazosulfapyridine: ' 

2,700mg/kg 

004677~771~7777777777777777 

Number OF Days on Study 0094222222222233333333333 

6777888888888811111111111 

- ' , 1122111122222211111111111 

Carcass II)Number 8921889900122388888899999 

5435493827604012367801256 

Alimentary System 

Esophagus .......................... Gallbladder ++++++++++++++++++M++++++ 

Intestine large, colon ......................... Inkstine large, rectum .......................... Intestine large, cecum .......................... Adenocarcinoma 

Intestine small, duodenum ......................... Intestine small, jejunum ......................... Intestine small, ileum ' ......................... Liver ......................... Hepatocellular carcinoma X X X 

Hepatocellular adenoma x x X X xx xx 

Hepatocellular adenoma, multiple x xx xx xx x xx x x x X 

Mesentery + 

Pancreas . ......................... Salivary glands ......................... Stomach, forestomach .......................... Stomach, glandular .......................... Adenoma 

Cardiovascular System 

Blood vessel .......................... Heart ........................... .. 

Endocrine System 

Adrenal cortex ......................... Adrenal medulla 

~ 

.......................... Islets, pancreatic ......................... Adenoma X 

Parathyroid gland +++.++++++++'+'+M+++++++++++' 

Pituitary gland ++M+-+++++M++++++++M++++++ 

Thyroid gland .......................... Follicular cell, adenoma 

Follicular cell, carcinoma ~ ~~~~~~~~ 

General Body System 

None 

Genital System 

Coagulating gland 

Epididymis ......................... Preputial gland ......................... Adenoma 

Prostate ......................... Seminal vesicle ......................... Testes ......................... 
Number of Days on Study 

Carcass I[I[D Number 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Adenocarcinoma 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatocellular carcinoma 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Mesentery 

Pancreas 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Adenoma 

Cardiovascular System 

Blood vessel 

Heart 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Islets, pancreatic 

Adenoma 

Parathyroid gland 

Pituitary gland 

Thyroid gland 

Follicular cell. adenoma 

Follicular cell, carcinoma 

General Body System 

None 

Genital System 

Coagulatinggland 

Epididymis 

Preputial gland 

Adenoma 

Prostate 

Seminal vesicle 

Testes 

7777777777777777777777777 

3333333333333333333333333 

1111111111111111111122222 

1122222222222222222222222 

9900000000111112222211122 

7901345689012345678978912 

......................... 

+++++++++++++++++++++M+++ 

......................... 

......................... 

......................... X 

......................... 

........................... 

.......................... 

......................... X xx X X 

xx X X x xx 

X x x xx x x x X xxxx 

......................... 

......................... 

......................... 

......................... X 

.......................... 

......................... 

......................... 

......................... 

......................... 

.......................... 

.......................... 

......................... X 

X 

+ .......................... 

......................... x ......................... 

......................... 

......................... 

Total 

Tissues/ 

Tumors 

50 

48 

50 

50 

50 

1 

50 

50 

50 

50 

8 

15 

27 

1 

50 

50

3 I, 

1’ 

50 

50 

50 

50 

50 

1 

49 .. 

47 

50 

1. 

1 

1 

50 

50 

1 

50 

50 

50 
202 Salicylazosulfapyridine,NTP TR 457 

TABLEC:l 

Individual Animal Tumor Pathology of Male Mice in the Gavage Study of Salicylazosulfapyridme: 2-Year 

2,700 mg/kg . . (continued) 

Number of Days on Study 

Carcass ID Number 

Hematopoiietic System 

Bone marrow 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Hemangiosarcoma 

Spleen 

Hemangiosarcoma

Thymus 

Integumentary System 

Mammary gland

Skin 

Subcutaneous tissue, plasma celltumorbenign 

Musculoskeletal System 

Bone 

Nervous System 

Brain 

Respiratory System 

LungAlveolar/bronchiolar adenoma 

Alveolar/bronchiolar adenoma, multiple 

Alveolar/bronchiolar carcinoma 

Alveolar/bronchiolar carcinoma, multiple 

Carcinoma, metastatic, tissue NOS 

Nose ' 

Trachea 

Special Senses System 

Harderian gland 

Adenoma 

Urinary System 

Kidney 

Urinary bladder 

Hemangioma 

Systemic Lesions 

Multiple organs 

Lymphoma malignant 

Lymphoma malignant lymphocytic 

0046777777777777777777777 

009422222222223333333.3333 

6777888888888811111111111 

1122111122222211111111111 

892188990012238888889999.9 ...' 

5435493827604012367801256 

......................... 

+M+++++++++++++++++++++++ 

......................... 

......................... 

+.++M++++M+++++M++++++++++ 

......................... 

......................... 

- X ~ ~ ~ ~~~~~~ ~ ~ ~~ ~~ 

......................... 

., 

.......................... 

......................... X X 

X 

X X 1 . 

x .......................... 

......................... 

+ + 

X x ' . 

......................... 

.......................... X 

......................... 
!

1

i

!

j

i 

i

: 

j ~ ~ ~~~~ ~~~~~ 

j We~W~p~ietk System

j Bone marrow 

i Lymph node 

i Lymph node, mandibular 

I Lymph node, mesenteric I

i

i Hemangiosarcoma 

j Spleen 

Hemangiosarcoma 

Thymus i

i -

htewentq Sys.tem i Mammary gland I Skin i Subcutaneous tissue, plasma cell tumor benign I 

i MlascoloskeletaPSystem 1 Bone I wewrolas system I Brain I

I

i Respiratory System 

Lung i Alveolarlbronchiolar adenoma 

Alveolarlbronchiolar adenoma, multiple 

Alveolarlbronchiolar carcinoma i Alveolarlbronchiolar carcinoma, multiple I Carcinoma, metastatic, tissueNOS I Nose 

Trachea I

I Special Semw Sptem I Harderian gland 

Adenoma I 

I -

I ukhq System

Kidney 1 

Urinary bladder I Hemangioma ~ ~~~ ~

i 

Systemic ILesiom 1 Multiple organs !

i Lymphoma malignant 

Lymphoma malignant lymphocytic 

1122222222222222222222222 

9900000000111112222211122 

7901345689012345678978912 ~ ~ ~~ 

......................... + + ++ ......................... 

......................... X 

......................... X 

+++,+++++++++++++++++++++M 

......................... 

......................... 

......................... 

......................... 

......................... X X 

X 

X X 

X 

......................... 

.......................... 

......................... 

......................... 

~~ ~ 

......................... X 

X 

Total 

Tissues/ 

Tumors 

50 

4 

49 

50 

1. 

50 

1 

46 

50 

50 

1 

50 

50 

50 

4 

2 

4 

1 

1 

50 

50 

2 

2 

50 

50 

1 

50 

1 

1 
c204 Salicylazosulfapyridine,NTP TR 457 

TABLEC3 

Statistical Anhlysisof PrimaryNeoplasms in MaleMiceinthe2-Year'GavageStudy"ofSalicylazosulfapyridine ' ' 

: Vehicle Control 

HarderianGland:Adenoma 

Overall ratea 2/50 (4%) 

Adjusted rateb 5.0% 

Terminal rate' 2/40 (5%) 

First incidenc:e (days) 728 (T)

Life table testd P=0.371N 

Logistic regression testd P=0.371N 

Cochran-Annita e testd P=0.431N

Fisher exact lest %

Harderian(Gland:AdenomaorCarcinoma 

Overall rate 3/50 (6%) 

Adjusted rate '7.5% 

Terminal rate 3/40 (8%) 

First incidence (days) 728 (T) 

Life table test P=O.240N 

Logistic regression test P=O.240N 

Cochran-Armitage test P=0.292N 

Fisher exact test 

Liver:HepatocellularAdenoma 

Overall rate 13/50 (26%) 

Adjusted rate 32.5% 

Terminal rate 13/40 (33%) 

First incidence (days) 728 (T) 

Life table test P<0.001 

Logistic regression test P <0.001 

Cochran-Armitage test P<O.001, 

Fisher exact test 

Liver:HepatocellularCarcinoma 

Overall rate 13/50 (26R) 

Adjusted rate 29.2% 

Terminal rate 9/40 (23%) 

First incidence (days) 420 

Life table test P=0.094N 

Logistic regression test P=0.154N 

Cochran-Armitage test P=O.l66N 

Fisher exact test 

Liver: Hepatocellular Adenoma or Hepatocellular Carcinoma 

Overall 24/50 (48%) 

Terminal 

Adjusted . . 20140(50%) 

54.3% 

First 420 

Life P=O.ooQ 

regression Logistic test- P<0.001 

test Cochran-Armitage P<0.001 

Fisher exact test 

675 mg/kg 

4/50 (8%) 

9.8% 

4/41 (10%) 

728 (T)

P=O.348 

P=O.348 

P~O.339 

'4150 (8%) 

9.8% 

4/41 (10%) 

728 (T)

PsO.514 

P~O.514 

P=O.500 

32/50 (64%) 

76.2% 

31/41 (76%) 

666 

P<O.001 

P<O.001 

P <0.001 

15/50 (30%) 

33.8% 

12/41 (29%) 

567 

P=O.45 

P10.415 

P=O.412 

38/50 (76%) 

84.4% 

34/41 (83%) 

567 

P=O.O08 

P=O.ooQ 

P=O.o04 

1,350 mgkg 

.. . 

1/50 (2%) 

2.4% 

1/41 (2%) 

728 (T) 

P=0.491N 

P=0.491N 

P=O.500N 

1/50 (2%) 

2.4% 

1/41 (2%) 

728 (T)

P=0.296N 

P=O.Z%N 

Pe0.309N 

,. . 

28/50 (56%) 

66.6% 

27/41 (66%) 

678 

P=O.o02 

P=O.o02 

P=O.o02 

.. ,. 

23/50 (46%) 

51.0% 

19/41(46%) 

61 1 

P=O.O50 

P=O.O35 

P=O.O30 

38/50 (76%) 

82.6% 

33/41 (80%) 

61 1 

P=O.OlO 

P-0.005 

P=O.o04 

2,700 mg/kg 

2/50 (4%) 

4.3% 

2/46 (4%) 

728 (T) . 

P=0.644N 

P=0.644N 

P=0.691N 

2/50 (4%) 

4.3% 

2/46 (4%) 

728 (T) -

P=0.436N 

P=O.436N 8 , 

P=O.500N 

42/50 (84%) 

87.5% 

40146 (87%) 

497 , ' 

P<O.001 

P<O.001 

P<O.001 ' 

. .. 

8/50 (16%) 

17.4% 

8/46 (17%) 

728 (T)

P=O.106N 

P=0.159N 

P=0.163N 

4/50 (8896) 

91.7% 

42/46 (91%) 

497 

P=O.O01 

P<0.001 

P<0.001 

i 
Liver: Hepatocellular Carcinomaor Hepatoblsastoma 

Overall rate 13/50 (26%) 15/50 (30%) 23/50 (46%) 8/50 (16%) 

Adjusted rate 29.2% 33.8% 51.0% 17.4% 

Terminal rate 9/40 (23 %) 12/41 (29%) 19/41(46%) 8/46 (17%) 

First incidence (days) 567 420 61 1 728 (T) 

P=O.O50 

P-0.035 

P=O.O30 

38/50 (76%) 

82.6% 

33/41 (80%) 

61 1 

P=O.OlO 

P=O.o05 

P=O.W 

14/50 (28%) 

33.2% 

13/41 (32%) 

648 

P=O.346 

P=O.361 

P=O.322 

2/50 (4%) 

4.9% 

2/41 (5%) 

728 (T) 

P=O.489N 

P=0.483N 

P=0.500N 

15/50 (30%) 

35.6% 

14/41 (34%) 

648 

P=O.533 

P=O.551 

P=O.500 

P=0.106N 

P=0.159N 

P=0.163N 

44/50 (88%) 

91.7% 

42/46 (91 %) 

497 

P=O.ool 

P<O.ool 

P<O.oOl 

6/50 (12X)

13.0% 

6/46 (13%) 

728 (T)

P=0.081N 

P=0.08lN 

P=O. 143N 

5/50 (10%) 

10.9% 

5/46 (1 1 46) 

728 (T) 

P=O.434 

P=O.392 

P=O.357 

11/50 (22%) 

23.9% 

11/46 (24%) 

728 (T) 

P=0.192N 

P=0.218N 

P=0.322N 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

Lung: Alveolarhronchiolar Adenoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

Lung: Alveolarhonchiolar Carcinoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

Life table test P=O.WN P=O.445 

Logistic regression test P=0.154N P=O.415 

Cochran-Armitage test P=0.166N 

Fisher exact test . P-0.412 

Liver: Hepatocellular Adenoma, Hepatwellular Carcinoma.or He~at~blmt~ma 

Lung: Alveolar/bronchiolar Adenoma or Carcin~~t~a 

Overall rate 14/50 (28%) 

Adjusted rate 34.1 % 

Terminal rate 13/40 (33%) 

First incidence (days) 689 

Life table test P=O.WN 

Logistic regression test P=O.l25N 

Cochran-Armitage test P=0.185N 

Fisher exact test 

24/50 (48%j 

54.3% 

20140 (50%) 

420 

P=O.W 

P<O.ool 

P<O.ool 

11/50 (22%) 

27.5% 

11/40 (28%) 

728 (T) 

P=0.068N 

P=0.078N 

P=0.132N 

3/50 (6%) 

7.3% 

2/40 (596) 

689 

P-0.512N 

P=O.557 

P=O.533 

38/50 (76%) 

84.4% 

34/41 (83%) 

567 

P=O.oo8 

P=O.W 

P=O.o04 

11/50 (22%) 

26.8% 

11/41 (27%) 

728 (T)

P=0.572N 

P=0.572N 

P=0.595N 

8/50 (16%) 

18.4% 

6/41 (15%) 

588 

P=O.115 

P=O. 104 

P=O.loO 

18/50 (36%) 

41.7% 

16/41 (39%) 

588 

P=O.289 

P=O.284 

P=O.260 
206 Salicyl&iosulfapyridine, NTP TR 457 

TABLEC3 

Statistical Analysis'of Primary Neoplasms in Male Mice in the 2-Year Gavage Study of Salicylazosulfapyridine (continued) 

Vehicle Control 675 mgkg 1,350 mg/kg 2,700 mgntg

StomachO?orestomach): SquamousCell Papilloma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test ,

Fisher exact test 

All Organs: Hemangiosarcoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Annitage test 

Fisher exact test 

All Organs: Hemangioma or Hemangiosarcoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Amlitige test <-

Fisher exact test 

All Organs: Malignant Lymphoma (Lymphocyticor Undifferentiated Cell Type) 

1/50 (2%) 

2.4% 

1/41 (2%) 

728 (T)

P=0.296N 

P=O.296N 

P=O.309N 

0/50 (0%) 

0.0% 

0/41 (0%) -

P=0.117N 

P=0.117N 

P=0.121N 

0150 (0%) 

0.0% 

0141 (0%j -

P=0.060N 

P=O.WN' 

P=O.O59N 

1/50 (2%) 

2.4% 

1/41(2%) 

728 (T)

P=0.098N 

P=0.095N 

P=O.l02N 

32/50 (64%)

74.3% 

30/41 (73%) 

648 

P=O.168 

P=O.194 

P=O.156 

0150 (0%) ,' 

. 

0.0% 

0/46(0%) " 

-

P=0.098N 

P=0.098N 

P=0.121N 

1/50 (2 I%) 

2.2% 

1/46 (2%). 

728 (T) ' 

P=0.257N 

P=0.257N 

P=0.309N 

2/50 (4%)' 

'4.3% ' ' 

2/46 (4%) 

728 (T) ' 

P=0.275N 

P=0.275N 

P=O.339N 

2/50 (4%) 

4.3% ' 

2/46 (4 %) 

728 (T) 

P=0.169N 

P=0.198N 

P=0.218N 

43/50 (86%) 

89.6% 

41/46 (89%) 

497 

P=O.003 

P<O.001 

P <0.001 

3/50 (6%) 0150 (0%) 

7.5% 0.0% 

3/40 (8%) 014 1 (096) -e 728 (T)

P=O.O74N P=0.117N 

P=0.074N P=0.117N 

P=O.O86N 

P=O.l21N 

3/50 (6%) 0/50 (0%) 

7.5% 0.0% 

3/40 (8%) 0/41 (0%) 

728 (T) -

P=O.219N P=O. 117N 

P=0.219N P-0.117N 

P=0.247N 

P=0.121N 

4/50 (8 %) 0150 (0A ) 

.lO.O% 0.0% 

4/40 (10%) 0/41 (0%)

728 (T) - P=0.294N P=0.060N 

P=O.294N P=0.060N 

P=0.337N 

P=0.059N 

Overall 

Adjusted 

Terminal rate 

First 

Life test 

ression Logistic 

Cochran-Amlitage 

Fisher 

All Organs: Benign Neoplasms 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Amlitage test 

Fisher exact test 

5/50 (10%) 

12.1% 

4/40 (10%) 

666 

106N P=O. 

118N P=O. 

P=O.l35N 

26/50 (52%) 

65.0% 

26/40 (65 %)

728 (T) 

P=O.008 

P=O.o22 P=O.001 

P<O.001 

3/50 (6%) 

7.3% 

3/41 (7%) 

728 (T) 

P=0.344N 

P=0.343N 

P=0.357N 

36/50 (72%) 

85.7% 

35/41 (85 %) 

666 

P=O.O21 

P=O.O32 

208 Salicylazosulfapyridine, NTP TR 457 

TABLE C4a1 .. . 

Historical Ihcidenceof Hepatocellular Neoplasms in Male B6C3Fl Mice Receiving CornbyOilGavagea 

') , . . AdenomaAdenoma Carcinoma

Incidence in Controls 

or Carcinoma 

Overall Historical Incidence 

Range 

Standard dwiation 

Total 

13.4% 

2541763 (33.3%) 

14%-58% 8%-24% 

4.5% 

1271763 (16.6%) 

14.6% 

3401763 (44.6%) 

25 % -72X 

a Data as of 17 June 1994 

Historical Incidenceof Splenic Hemangiosarcoma in Male B6C3Fl Mice Receiving Corn Oil by Gavagea 

TABLE C4b 

./ 

, ' : 

Incidence in Controls 

Overall Historical Incidence . . 

a 

Range 

Standard deviation 

Total 

Data as of 17 June 1994 

0%-8% 

2.7% 

221759 (2.9%) 

, 

I 

s 
Control Vehice 675 mglkg 1,350 mglkg 2,7063 mglkg

Disposition Summary 

Animals initially in study 60 60 60 60 

15-Month interim evalualion 10 10 10 10 

Early deaths 

Accidental deaths 2 

Moribund 5 4 3 4 

Natural deaths 3 5 6 

Survivors 

Terminal sacrifice 40 41 41 46 

Animals examined microscopically 60 60 ’ 6 0 60 

IS-iWOpkEh BpkEe~h.EV&WdiOpk 

Alimentary System 

Liver 

Basophilic focus 

Clear cell focus 

Cytologic alterations 

Eosinophilic focus 

Hematopoietic cell proliferation 

Vacuolization cytoplasmic 

Pancreas 

Inflammation, subacute 

Salivary glands 

Vacuolization cytoplasmic 

Submandibular gland, depletion secretory 

Stomach, forestomach 

Hyperplasia 

Stomach, glandular 

Atypia cellular 

cyst 

Erosion 

Mineralization 

~~~~~~~~~~~~~~~ 

Endocrine System 

Adrenal cortex 

Hyperplasia 

Hypertrophy

Capsule, hyperplasia 

Parathyroid gland 

cyst 

Thyroid gland 

Follicle, cyst 

Follicle, degeneration 

a Number of animals examined microscopically at the site and the number of animals with lesion 
210 Salicylazosulfapyridine,NTP TR 457 

TABLEC5 

Summary of the Incidence of Nonneoplastic Lesions in Male Micein the 2-YearGavage Study 

of Salicylazosulfapyridine (continued) . 

I .. 

ControlVehicle 675 mg/kg 1,350 mg/kg 

15-Month Interim Evaluation (continued) 

Genital System 

Preputial gland (10) (10) (10)

Duct, cyst 4 (40%) 8 (80%) 10 (100%) 

Testes (10) (10) (10)

Atrophy 1 (10%) 

Thrombosis 1 (10%) 

Hematopoietic System 

Lymph node, mesenteric (10) (10) (10)

Angiectasis 1 (10%) 

Spleen (10) (10) (10)

Hematopoietic cell proliferation 2 (20%) 8 (80%) 10 (100%) 

Pigmentation, hemosiderin 4 (40%) 10 (100%) 

Thymus (9) (10) (10) .. 

cyst 1 (10%) 3 (30%) 

Integumentary System 

Skin (10)

Subcutaneous tissue, edema 

Subcutaneous tissue, hemorrhage 

Respiratory System 

Lung (10) (10) (10) 

Alveolus, cellular. infiltrationhistiocyte 1 (10%)

Nose (10) (10) (10) 

Glands, cyst 4 (40%) 

Nasolacrimal duct, exudate 1 (10%) 

Olfactory epithelium, degeneration 1 (10%) 2 (20%) 8 (80%) 

Olfactory epithelium, hyperplasia 1 (10%) 

Urinary System 

Kidney (10) (10) (10) 

Mineralization 8 (80%) 3 (30%) 6 (60%) 

Pelvis, inflammation, subacute 1 (10%) 

Renal tubule. casts 1 (10%) 

Renal tubule, regeneration 7 (70%) 6 (60%) 8 (80%) 

Urinary bladder (10) (10) (10) 

Inflammation, chronic 2 (20%) 

Transitional epithelium, hyperplasia 1 (10%) 

Systems Examined With No Lesions Observed 

Cardiovascular System 

General Body System 

Musculoskeletal System 

Nervous System 

Special Senses System 

. 

2,700 m'gkg 

(10) 

7 (70%) 

(10) 

.. 

' (10) 

1 (10%) 

(10) 

10 (100%) 

10 (100%) 

(10) 

1 (10%) 

(10) 

1 (10%) 

(10) 

2 (20%) 

(10) 

2 (20%) 

1 (10%) 

4 (40%) 

(10) 
-I

I

- IIIi

- i1

I ~. i 

; 

! ~~ ! I1j

1 =: :: 

3-YeaP st” 

Mientaq System 

Esophagus 

Hemorrhage 

Gallbladder

Fibrosis ., 

Inflammation, suppurative 

Epithelium, hyperplasia 

Intestine large, cecum 

metazoan Prasite

Intestine small, duodenum 

Dilatation 

Inflammation, subacute 

Necrosis 

Ulcer 

(50) 

1 (2%) 

(49) 

(47) 

1 (2%) 

(47) 

(50) 

(48) 

1 (2%) 

(46) 

(47) 

(50) 

(49) 

1 (2%) 

1 (2%) 

(46) 

(47) 

1 (2%) 

1 (2%) 

1 (2%)

i Intestinejejunum small, (47) (46) j 

(50) 

(48) 

(50) 

(50) 

1 (2%) 

(47) (50) _ 

1 Peyer’s patch, hyperplasia, lymphoid 2 (4%) Intestine small, ileum (47) (46) (46) (50):I- Inflammation, chronic 1 (2%) 

-I Liver (50) (50) (50) (50) Angiectasis 1 (2%) 1 (2%) Basophilic focus 1 (2%) -= 2 (4%) 

Eosinophilic focus 5 (10%) 

~~ :I 1 (2%) I

i 3 (6%) Clear cell focus 14 (28%) 4 (8%) 8 (16%) 

Clear cell focus. multiple 1 (2%) 3 (6%)

Developmental malformation 1 (2%) 

17 (34%) 17 (34%) 13 (26%)

Eosinophilic focus, multiple 2 (4%) 3 (6%) 9 (18%) 

Fatty change, focal 1 (2%) Hemorrhage 1 (2%) 1 (2%)

-:I Hepatodiaphragmatic nodule 1 (2%) Inflammation, chronic 1 (2%) 

2 (4%) 

i Inflammation, subacute =I 3 (6%) 1 (2%), 1 (2%)

Mineralization 2 (4%) 3 (6%) .I~ ~~ 

I Mixed cell focus 2 (4%) 4 (8%) 1 (2%)

Necrosis -::I 3 (6%) 6 (12%) 5 (10%) 3 (6%) Pigmentation, bile 1 (2%) 

-~:i Pigmentation, hemosiderin 1 (2%) Vacuolization cytoplasmic 3 (6%) 5 (10%) 1 (2%) Bile duct. cyst 2 (4%)

Bile duct, hyperplasia 1 (2%)

Mesentery (4) (4) (1) (1) Hemorrhage 1 (25%) 

Inflammation, subacute 1(25%)

r; I Fat, necrosis 3 (75%) 2 (50%) 1 (100%) 1 (100%) 

r-I 

=r I Pancreas (50) (50) (50) (50) Inflammation, subacute 1 (2%) .. - Necrosis 1 (2%) ~~. 

. -- Polyarteritis 1 (2%) ~~ Acinus, atrophy 2 (4%) .~ 

~.. 

Acinus, depletion secretory 1 (2%)

Duct, cyst 1 (2%) 

.~~ .. 

--= 
212 Salicylazosulfapyridine,NTP TR 457 

TABLEC5 : .’. Summary of the Incidenceof Nonneoplastic Lesionsin Male Micein the 2-Year Gavage Study 

of Salicyllazosulfapyridine(continued) 

Control Vehicle 675 mg/kg 1,350 mgkg 2,700 mgkg

2-Year Study (continued) 

Alimentary System(continued) 

Stomach, forestomach (49)

cyst 

Edema 

Hyperplasia 10 (20%) 

Inflammation, subacute 2 (4%) 

Mineralization 

Ulcer 

Stomach, glandular 

cyst 

Hyperplasia 

Inflammation, subacute 

Mineralization 9 (19%)

Necrosis 1 (2%) 

Pigmentation 

. Polyarteritis 1 (2%)

Ulcer 

CardiovascularSystem 

Heart 

Cardiomyopathy 

Embolus 

Fibrosis 1 (2%) 

Mineralization 1 (2%) 

Polyarteritis 1 (2%) 

Endocrine System 

Adrenal cortex 

Accessory adrenal cortical nodule 

Embolus 

Hemorrhage 1 (2%) 

Hyperplasia 8 (16%) 8 (16%) 6 (12%) 

Hypertrophy 11 (22%) 13 (27%) 12 (24%) 

Necrosis 

Vacuolization cytoplasmic 1 (2%) 

Capsule, hyperplasia 1 (2%) 4 (8%) 

Adrenal medulla (50) (49) (50) 

Embolus 1 (2%) 

Hemorrhage 

Hyperplasia 

Islets, pancreatic 

Embolus 

Hyperplasia 3 (6%) 

Parathyroid gland (49) 

cyst 1 (2%) 

Pituitary gland (47) 

Pars distalis, cyst 

Pars distalis, hyperplasia 

214 Salicylazosulfapyridine,NTP TR 457 

TABLEC!j 

Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 2-Year Gavage Study 

of Salicylazosulfapyridine(continued) 

. . . .

ControlVehice 675 mg/kg 1,350 mg/kg 2,700 mg/kg

&Year s&dy (continued) 

Hematopoietic System (continued) 

Lymph node, mandibular (50) (49) (49) (49) Hyperplasia, lymphoid 1 (2%) 1 (2%)

Lymph node, mesenteric (50)

Angiectasis 

(50) (50) 

17 (34%)

(50) 

17 (34%) 18 (36%) 12 (24%) 

Hyperplasia, lymphoid 4 (8%) 3 (6%) 3 (6%) 

Infiltration cellular, histiocyte 1 (2%) 

Necrosis 1 (2%) 

Spleen (50) (50) (49) 

3 (6%) 

Atrophy 5 (10%) 1 (2%) 2 (4%) 

(50) 

Congestion 

Hematopoietic cell proliferation 

Hyperplasia, lymphoid 

Pigmentation, hemosiderin 

Capsule, inflammation, subacute 

Thymus 

Atrophy 

cyst 

Polyarteritis 

Thymocyte, necrosis 

Integumentary System 

Skin 

Subcutaneous tissue,; inflammation, chronic 

Subcutaneous tissue, inflammation, subacute 

Subcutaneous tissue, metaplasia, osseous 

Subcutaneous tissue, mineralization 

MusculoskeletalSystem 

Bone 

Cranium, hyperostosis 

Nervous System 

Brain 

Compression 

Embolus 

Hydrocephalus 

Respiratory System 

Lung 

Congestion 

Hyperplasia, macrophage 

Alveolar epithelium, hyperplasia 

Alveolus, foreign body 

Alveolus, hemorrhage 

Alyeolus, infiltration cellular, histiocyte 

Mediastinum, hemorrhage 

Mediastinum, inflammation, chronic 

1 (2%) 

13 (26%) 

7 (14%) 

47 (94%) 

(46) 

1 (2%) 

1 (2%) 

1 (2%) 

(50) 

1 (2%) 

1 (2%) 

11 (22%) 

5 (10%) 

2 (4%) 

(45) 

3 (7%) 

2 (4%) 

1 (2%) 

(50) 

1(2%) 

1 (2%) 

1 (2%) 

16 (32%) 

12 (24%) 

25 (50%) 

1 (2%) 

(43) 

2 (s%j 

3 (7%) 

1 (2%) 

(50) 

1 (2%) 

20 (41%) 

10 (20%) 

32 (65%) 

(46) 

1 (2%) 

4 (9%) 

(50) 

(50) 

1 (2%) 
2-yelW &4@ (continued) 

Respiratory System(continued) 

Nose 

Lumen, inflammation, subacute 

Glands, fibrosis 

Glands, cyst 

(50)

1 (2%) 

(50) 

1 (2%) 

1 (2%) 

(50) 

Respiratory epithelium, hyperplasia 1 (2%) 

Special Senses System 

EY e 

Cornea, inflammation, subacute 

Inflammation, chronic 

Harderian gland (3) 

Urinary System 

Kidney 

Bacterium 

(50) 

1 (2%) 

(50) 

Fibrosis 1 (2%) 

Glomerulosclerosis 2 (4%) 

Infarct 2 (4%) 1 (2%) 

Infiltration cellular, lymphocyte 

Inflammation, chronic, suppurative 1 (2%) 

Inflammation, subacute 

Mineralization 

Metaplasia, osseous 

40 (80%) 33 (66%) 

2 (4%) 

Polyarteritis 

Cortex, cyst 7 (14%) 1 (2%) 

Cortex, medulla, inflammation, suppurative 

Pelvis, necrosis 

Renal tubule, casts 12 (24%) 3 (6%) 

Transitional epithelium, hyperplasia 

Renal tubule, regeneration 

Renal tubule, degeneration 

40 (80%) 

1 (2%) 

24 (48%) 

1 (2%) 

Hemorrhage 

Urethra (1) 

1 (100%) 

Inflammation, subacute 1 (100%) 

Dilatation 

Urinary bladder 

1 (2%) 

(50) (50) 

1 (2%) 

Inflammation, chronic 1 (2%) 

Inflammation, subacute 

Transitional epithelium, hyperplasia 
216 Salicylazosulfapyridine, NTP TR 457 

Disposition Summary 

Animals initially in study 

15-Monfh interimevddn 

Early deaths 

Accidental deaths 

Moribund 

Natural deaths 

Survivors 

Died last week of study 

Terminal sacrifice 

Animals examined microscopically 

n5-mpath medm mdum-opa 

Aliientary System 

Liver 

Hepatocellular carcinoma 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Stomach, forestomach 

Squamous cell papilloma 

Genital System 

Ovary 

Cystadenoma 

Respiratory System 

Lung

Alveolar/bronchiolar adenoma 

60 

10 

2 

5 

2 

41 

60 

(10) 

1 (10%) 

1 (10%) 

(10) 

(10) 

1 (10%) 

Systems Exmined With No NeoplQsmsObserved 

Cardiovascular System 

Endocrine System 

General Body System 

Hematopoietic System 

Integumentary System 

Musculoskeletal System 

Nervous System 

Special Senses System 

Urinary System 

60 

10 

6 

3 

41 

60 

(10) 

(10) 

1 (10%) 

(10) 

60 

10 

1 

8 

4 

1 

36 

60 

(10) 

1 (10%) 

(10) 

60 

10 

2 

6 

4 

38 

60 

(10) 

1 (10%) 

1 (10%) 

1 (10%) 

(10) 
220 Salicylazosulfapyridine,NTP TR 457 

TABLEDl1 

Summary of the Incidenceof Neoplasms in Female Mice in the 2-YearGavage Study of Salicylazosulfapyridine (continued)' 

VehicleControl . . . 675 mgkg . . 1,350 mgkg 2,700 mgkg 

2-Year Study 

Alimentary System 

Intestine large, rectum 

Histiocytic sarcoma 

Leiomyosarcoma 

Intestine large. cecum 

Leiomyosarcoma 

Intestine small, jejunum 

Hemangiosarcoma 

Intestine small, ileum 

Liver 

Hemangioma 

Hemangiosarcoma 

Hepatocellular carcinoma 

Hepatocellular carcinoma, multiple 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Histiocytic sarcoma 

Osteosarcoma, metastatic, bone 

Mesentery 

Histiocytic sarcoma 

Leiomyosarcoma, metastatic, uterus 

Pancreas 

Hemangioma 

Salivary glands 

Stomach, forestomach 

Squamous cell papilloma 

Stomach, glandular 

Cardiovascular System

Heart 

Endocrine System 

Adrenal cortex 

Adenoma 

Leiomyosarcoma, metastatic, intestine large, 

rectum 

Adrenal medulla 

Pheochromccytoma benign 

Pituitary gland 

Pars distalis, adenoma 

Pars intermedia, adenoma 

Thyroid gland

.Histiocytic sarcoma 

Follicular cell, adenoma 

Follicular cell, carcinoma 

General Body System

None 

(49) 

8 (16%) 

1 (2%)

15 (31%) 

13 (27%) 

(49) 

(49) 

2 (4%) 
%YWP S&a"' (continued) 

Ovary 

Genital System 

Thecoma malignant 

Teratoma malignant 

Luteoma 

Leiomyosarcoma, metastatic, ute~s 

Granulosa cell tumor benign 

Granulosa cell tumor malignant 

Cystadenoma 

1 (2%) 

3 (6%) 

1 (2%) 

1 (2%) 

UteNS 

Polyp stromal 

Leiomyosarcoma 

Histiocytic sarcoma 

Bone marrow 

Hematopoietic System 

Histiocytic sarcoma 

Hemangiosarcoma 

Renal, teratoma malignant, metastatic, ovary 

Renal. histiocytic sarcoma 

Mediastinal, histiocytic sarcoma 

Inguinal, mast cell tumorNOS 

Inguinal, histiocytic sarcoma 

Iliac, histiocytic sarcoma 

Histiocytic sarcoma 

Lymph node, mesenteric 

Histiocytic sarcoma 

Lymph node, mandibular 

Histiocytic sarcoma 

Hemangiosarcoma 

Hemangioma 

Spleen 

Lymph node 

Thymus 

1 (9%) 

1 (9%) . 

Integumentary System 

Adenoacanthoma 

Mammary gland (50) 

Carcinoma 

Subcutaneous tissue, histiocytic sarcoma 

Subcutaneoustissue,hemangiosarcoma 

Subcutaneous tissue, hemangioma 

Subcutaneoustissue,fibrosarcoma 

Sguamous cell papilloma 

Skin 

1 (2%) 

1 (2%) 

1 (2%) 

(50) ' 
222 Salicylazosulfapyridine,NTP TR 457 

Control Vehice 675 mg/kg 1,350 mg/kg 2,700 mg/kg

&Year Study . "(continued) 

Musculoskeletal System

Bone (50) (50)

Cranium,, osteosarcoma 

Vertebra, osteosarcoma 1 (2%)

Skeletal muscle (1) Fibrosarcoma 

Leiomyosarcoma, metastatic, uterus 

Nervous System 

Brain (50)

Osteosarcoma, metastatic, bone 

Spinal cord (2)

Osteosarcoma, metastatic, bone 

Respiratory System

Lung (50) (50)

Adenoacanthoma, metastatic, mammary gland 

Alveolar/bronchiolar adenoma 2 (4%) 4 (8%) 

Alveolarhronchiolar carcinoma 1 (2%) 

Alveolarhronchiolar carcinoma, multiple 

Granulosa cell tumor malignant, metastatic, 

ovary 

Hepatocellular metastatic, carcinoma, liver 2 (4%)

sarcoma Histiocytic 1 (2%) 

Leiomyosarcoma, metastatic, uterus 

Special Senses System 

Harderian gland (2) (1) (2)

Adenoma 2 (100%) 1 (100%) 2 (100%) 

Urinary System 

Kidney 

Histiocyticsarcoma 

Urinary bladder 

Systemic Lesions 

Multiple organsb 

Histiocytic sarcoma 

Lymphoma malignant 
Neoplasm Sum~na~gr 

Total animals with primary neoplasms' 

15-hlonth interim evaluation 3 1 1 2 

2-Year study 34 37 42 36 

Total primary neoplasms 

15-Month interim evaluation 4 1 1 3 

2-Year study 57 72 84 67 

Total animals with benign neoplasms 

15-Month interim evaluation 3 1 1 2 

2-Year study 22 32 34 32 

Total benign neoplasms 

15-Month interim evaluation 3 1 1 2 

2-Year study 35 48 49 46 

Total animals with malignant neoplasms 

15-Month interim evaluation 1 1 

2-Year study 19 19 26 17 

Total malignant neoplasms 

15-Month interim evaluation 1 1 

2-Year study 22 24 34 21 

Total animals with metastatic neoplasms 

2-Yearstudy . 2 3 3 2 

Total metastatic neoplasms 

2-Year study 2 4 6 2 

Total animals with uncertain neoplasms-

- benign or malignant 

2-Year study 1 

Total uncertain neoplasms 

2-Year study 1 

a Number of animals examined microscopically at the site and the number of animals with neoplasm 

Number of animals with any tissue examined microscopically ' Primaryneoplasms:allneoplasmsexceptmetastaticneoplasms 
224 Salicylazosulfapyridine,NTP TR 457 

TABLED2 

Individual Animal Tumor.Pathology of Female Micein the 2-Year Gavage Study of Salicylwosulfapyridne: . 

Vehicle Control 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejumun 

.Intestinesmall, ileum 

Liver 

Hemangioma 

Hemangiosarcoma 

Hepatocellular carcinoma 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Histiocytic sarcoma 

Mesentery 

Pancreas 

Salivary glands 

Stomach, forestomach 

Squamous cell papilloma 

Stomach. glandular 

Cardiovascular System 

Blood vessel 

Heart 

Endocrine System

Adrenal cortex 

Adrenal medulla 

Pheochromocytoma benign 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Pars intermedia, adenoma 

Thyroid . -

gland

Histiocytic sarcoma 

Follicular cell, adenoma . . 

General Body System 

None 

+: Tissue examined microscopically 

A: Autolysisprecludesexamination 

.0034-455777777777777777777 

1722719023333333333333.333 

4449685835555555555566666 

3333333333333333'333333333 

05353141400122233344000~00 

4234600713940481253712567 

~ 

......................... 

++++++A++++++++++++++++++ 

++++++A+++.++++++++++++++.+ 

++++++A++++++.++++++++++++ 

++++++A++++++++++++++++++ , 

.......................... 

++++++A++++++++++++++++.++ 

++++++A++++++++++++++++++, 

......................... X 

x . .. . 

X xx X 

X. 

+ + ......................... 

++++M++++++++++.++++++++++ 

......................... X .......................... 

.......................... 

......................... 

......................... 

......................... X ......................... 

M+++++++++++M++M+++++++++ 

+++++++++++++++++++M+++++ 

'X x xx L X ......................... 

M: Missing tissue X: Lesionpresent 

I:Insufficient tissue Blank: Not examined 
Number of Days on Study 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hemangioma 

Hemangiosarcoma 

Hepatocellular carcinoma 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Histiocytic sarcoma 

Mesentery 

Pancreas 

Salivary glands 

Stomach, forestomach 

Squamous cell papilloma 

Stomach, glandular 

Cardiovascular System 

Blood vessel 

Heart 

Endocrine system 

Adrenal cortex 

Adrenal medulla 

Pheochromocytoma benign 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Pars distalis. adenoma 

Pars intennedia, adenoma 

Thyroid gland 

Histiocytic sarcoma 

Follicular cell, adenoma 

Generd Body System 

None 

1111111111111111111111111 

3333333333333333333333333 

6666666666666666666666666 

3333333333333333333333333 Total 

0111111122222223333344444 Tissues/ 

8123568912356190478924568 Tumors 

......................... 50 

......................... 49 

......................... 49 

......................... 49 

......................... 49 

......................... 50 

.......................... 49 

......................... 49 

.......................... 50 

1 

X 2 

x X 2 

x xx X x 9 

xx 3 

X 1 

+ + + + + I ......................... 50 

......................... 49 

......................... 50 

x x x x 5 

......................... 50 

......................... 50 ' 

......................... 50 

.......................... 50 

.......................... 50 

1 

......................... 50 

+'++++++++++++++++M+MMM+M+ 42 

......................... .49 : 

x xx I 

1 

......................... 50 

X 1 

X 1 
226 Salicylazosulfapyridine, NTP TR 457 

TABLED;! 

Individual AnimalTumor Pathology of Female Mice in the 2-Year GavageStudy of Salicylazosulfapyridine: 

Vehicle Control (continued) 

0034455777777777777777177 

Number of Days on Study 1722719023333333333333333 

4449685835555555555566666 

3333333333333333333333333 

Carcass ID Number 0535314140012223334400000 

4234600713940481253712567 

Genital System 

Clitoral gland ......................... Ovary +++.+++++++++++++++++++++M 

Cystadenoma 

Granulosa cell NmOr benign 

Uterus ......................... Histiocytic sarcoma 

Hematopoietic System \

Blood + 

Bone marmw ......................... Histiocytic sarcoma 

Lymph node + + + + 

Iliac, histiocytic sarcoma 

Mediastinal, histiocytic sarcoma 

Renal, histiocytic sarcoma 

Lymph node, mandibular ++++M++++++++++++++++++++ 

Histiocytic sarcoma 

Lymph node, mesenteric ++++++A++++++++++++++++++ 

Histiocytic sarcoma 

Spleen ......................... Histiocytic sarcoma 

Thymus ......................... 

Integumentary System

Mammarygland - ......................... Skin ......................... Subcutaneous tissue, fibrosarcoma X 

Subcutaneous tissue, hemangioma X 

Subcutaneous tissue, hemangiosarcoma 

Musculoskeletal System

Bone ......................... Skeletal muscle + 

Nervous System 

Brain ......................... Spinal cord + + ~~ ~~ .~ ~~ ~~ ~~~ ~~ ~ ~~ ~ 

Respiratory System 

Lung ......................... Alveolar/bronchiolar adenoma X X 

Alveolar/bronchiolar carcinoma 

Hepatocellular carcinoma, metastatic, liver 

Histiocytic sarcoma 

Nose ......................... Trachea ......................... 
TABLIE ID2 

Inndividund AnnimmaU TM~CDI~ Pathokqgy off Femmde Mice inn Uhe %Pear Gavage St~dy~ff SdicyUa;e~~Uffa~yg7kidlrme: 

vehkk ahlI~h'Q~(continued) 

> 

Number of Days on Study 

@arcsassID Number 

Genital System 

Clitoral gland 

Ovary 

Cystadenoma 

Granulosa cell tumor benign 

Uterus 

Histiocytic sarcoma 

Hematopoietic System 

Blood 

Bone marrow 

Histiocytic sarcoma 

Lymph node 

Iliac, histiocytic sarcoma 

Mediastinal, histiocytic sarcoma 

Renal, histiocytic sarcoma 

Lymph node, mandibular 

Histiocytic sarcoma 

Lymph node, mesenteric 

Histiocytic sarcoma 

Spleen 

Histiocytic sarcoma 

Thymus 

Integumentary System 

Mammary gland 

Skin 

Subcutaneous tissue, fibrosarcoma 

Subcutaneous tissue, hemangioma 

Subcutaneous tissue, hemangiosarcoma 

Musculoskeletal System 

Bone 

Skeletal muscle 

Nervous System 

Brain 

Spinal cord 

Respiratory System 

Lung

Alveolarlbronchiolar adenoma 

Alveolarlbronchiolar carcinoma 

Hepatocellular carcinoma, metastatic, liver 

Histiocytic sarcoma 

Nose 

Trachea 

1111111111171711111171711 

3333333333333333333333333 

6666666666666666666666666 

3333333333333333333333333 Total 

0111111122222223333344444 Tissues/ 

8123568912356790478924568 Tumors 

......................... 50 

......................... 49 

X 1 

X 1 

......................... 50 

X 1 

1 

......................... 50 

X 1 

++ + + 8 

X 1 

X 1 

X 1 

......................... 49 

X 1 

......................... 49 

X 1 

......................... 50 

X 1 

......................... 50 

......................... 50 

......................... 50 

1 

1 

X 1 

......................... 50 

1 

......................... 50 

L 

......................... 50 

2 

X 1 

X X 2 

X 1 

......................... 50 

......................... 50 

d 
228 Salicylazosulfapyridine,NTP TR 457 

TABLED2 

Individual AnimalTumor Pathology of Female Mice in the2-YearGavage Studyof Salicylazosulfapyridine: 

Vehicle Control(continued) 

Number of Days on Study 

Carcass II)Number 

Special Senses System 

Ear 

Harderian gland 

Adenoma 

Urinary System 

Kidney 

Histiocytic sarcoma 

Urinary bladder 

Systemic Lesions 

Multiple organs 

Histiocytic sarcoma 

Lymphoma malignant 

00344551'111171'11111111111 

112211902333'3333333333333 

4449685835555555'555566666 

3333333333333333333333333. 

0535314140012223334400000, 

4234600113940481253712561 

+ 

+ 

X 

......................... 

......................... 

......................... 

xxxxx X 
229 

TABLE ID2 

IIndividuaU Anhd TWORPath~U~gy~ f Femlnne f Mice in the %Year Gavage Study ~ff S~UnnicyUazosanUffalpyridliane: 

V@"k COUltUd (continued) 

Number of Days on Study 

6666666666666666666666666 

3333333333333333333333333 

1111111111111111111111111 

Carcass IUD Number 

3333333333333333333333333 

0111111122222223333344444 

8123568912356190418924568 

Total 

Tissues/ 

Tumors ~ ~~ ~~ ~~ ~~ ~ 

Harderian gland 

Ear 

Special Senses System 

Adenoma X 

.+ 

2 

2 

1 

Kidney 

Urinary System . , 

.......................... 50 

Histiocytic sarcoma ., . X 1 

Urinary bladder .......................... 50 

Systemic Lesions 

Multiple organs ......................... 50 ,. 

Histiocytic sarcoma X X 2 

Lymphoma malignant x xxxx x X 13 
,230 .Salicylazosulfapyridine,NTP TR 457 

TABLE112 

Individual Animal Tumor Pathology of Female Mice in.the 2-Year Gavage Studyof Salicylazosulfapyridine: 

675 mglkg 

2244556777777777777777777 

Number of Days on Study 2979352233333333333333333 

47139'.103,25555555555777788 

3343333333333333344333333 

Carcass ID Number 7708,99968.6677889900666666 

0117028314848384948125679 

Alimentary System 

Esophagus .......................... Gallbladder .......................... Intestine large, colon ......................... ' 

large, , 

Intestine large, cecum ++++++A++++++++++++++++++ 

Intestine small, duodenum ++++++A++++++++++++++++++ 

Intestine small, jejunum ++++++A++++++++++++++++++ 

Intestine small, ileum ++++++A+.+++++++++++++++++ 

Liver ......................... Hemangiosarcoma X 

Hepatocellular carcinoma X xx X X 

Hepatocellular carcinoma, multiple X X 

Hepatocellular adenoma x x X X x x 'X 

Hepatocellular adenoma, multiple x xx X X 

Osteosarcoma, metastatic, bone X 

Mesentery + + + + 

Pancreas +++++A++++.+++++++++++++++ 

Salivary glands .......................... Stomach, forestomach ++++++A++++++++++++++++++ 

Squamous cell papilloma X 

Stomach, glandular ++.++++A++++++.++++++++++++ 

Intestinerectum ......................... 

Cardiovascular System 

Blood vessel .......................... Heart ......................... ' 

Endocrine System 

Adrenal cortex ......................... Adenoma 

Adrenal medulla .......................... Pheochromocytoma benign X X 

Islets, pancreatic .......................... Parathyroid gland M+++-+++++++++++++++++++++ 

Pituitary gland +++"MI++++++++++++++++++ 

Pars distalis, adenoma X X X 

Thyroid gland ......................... 

General Body System 

None 

Genital System 

Clitoral gland ......................... Ovary +++++++++++++++++++M+++++ 

Cystadenoma xx 

Granulosa cell mmor -malignant 

Luteoma 

Thecoma malignant X 

uterus ......................... 

X 
 

Number of Days on Study 

Carcrsss m Number 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hemangiosarcoma 

Hepatocellular carcinoma 

Hepatocellular carcinoma, multiple 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple xx 

Osteosarcoma, metastatic, bone 

Mesentery 

Pancreas 

Salivary glands 

Stomach, forestomach ~~uamous cell papilloma 

Stomach, glandular 

Cardiovascular System 

Blood vessel 

Heart 

Endocrine System 

Adrenal cortex 

Adenoma 

Adrenal medulla 

Pheochromocytoma benign 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland

Pars distalis, adenoma 

Thyroid gland 

General Body System 

None 

Genital System 

Clitoral gland 

Ovary 

Cystadenoma 

Granulosa cell tumor malignant 

Luteoma 

Thecoma malignant 

uterus 

7771111711111117117771777 

3333333333333333333333333 

8888888888888888888888888 

33333333.33333333344444444 Total 

1771778888889999900000001 Tissues/. 

2356190245691356702356790 Tumors 

......................... 50 

M++++++++++++++++++++++++ 49 

......................... 50 

......................... 50 

......................... 49 

......................... 49 

......................... 49 

......................... 49 

......................... 50 

1 

x X X 8 

2 

x x xxxx x xx 16 

x xx X x 12 

1 

+ + + 4- + + 10 

......................... 49 

.......................... 50 

......................... 49 

1 

......................... 49 

......................... 50 

......................... 50 

......................... 50 

x 1 

......................... 50 

L 

......................... 50 

++++++++M++++++++++++++++ 48 

++++++++++++++++++++++M++ 45 

x x x X 7 

......................... 50 

......................... 50 

......................... 49 

X X X 5 

X 1 

1 

1 

......................... 50 
'232 '' Salicylazosulfapyridine,'NTP TR 457 

TABLED2 

Individual Animal Tumor Pathologyof Female Mice in the 2-Y.k Gavage StudyofSalicylazosulfapyridine: 

675 mglkg (continued) 

224455677777777777777-7777 

Number of Days on Study 2979352233.33333333333.3'333 

4713910325555555555777788 I.. , . ... _.,. I . . 

. . 3343333333'333'333344333333 

Carcass ID Number 7708999686677889900666666 

0117028314848384948125679 . . .. . .. I 

Bone marrow 

Hematopoietic System 

......................... 

Spleen 

Lymph node, mesenteric 

Lymph node, mandibular 

Lymph node 

Thymus 

Hemangiosaicoma 

......................... 

........................... 

......................... 

+ +. 

++++++++'++++++M++++++++++ 

+ + ++ 

X X 

Skin

Manimary gland 

integumentary System 

......................... 

........................... 

Subcutaneous tissue. fibrosarcoma 

ihsculoskeletal System 

Bone ........................... Vertebra, osteosarcoma X .. . 

NeervOus System 

Brain ......................... Spinal cord + 

Osteosarcoma, metastatic. bone X 

Respiratory System 

Lung ......................... Alveolarhronchiolar adenoma X X 

Alveolarhronchiolar carcinoma X 

Granulosa cell Nmor malignant, 

metastatic, ovary 

Hepatocellular carcinoma, metastatic, liver 

Nose ........................... Trachea .......................... 

Special Senses System 

None 

Urinary System

Kidney .......................... Urinary bladder .......................... .. . . . 

Systemic Lesions 

Multiple organs ......................... Lymphoma malignant X X X 
San~cyn~QSannff~~ydnrr~~~~e~ II'R 233 

TABLE ID2 .. 

IUM"U~U ma^ MQU P~UI~MJUQ~ ~f ~~ema~e ice im the 2-ywdnrr ~avavanp SUU~~ of BaUicyu~~Unfal~idime: 

675Mmg/kg (continued) 

111111'1111111111111111111 

Nmber of Days on Study 3333333333333333333333333 

8888888888888888888888888 

.. . 

33333.33333333333344444444 TotalCarcass IID Number . 1111178888889999900000001 Tissues/ 

2356190245691356102356190 Tumors 

Hematopoietic System 

Bone marrow ......................... 50 

Lymph node + 1 

Lymph node, mandibular ......................... 50 

" 

Lymph node, mesenteric ......................... 50 

Spleen ......................... 50 

Hemangiosarcoma 2' 

Thymus ......................... 49 

htegumemtarySystem 

Mammary gland ......................... 50 

Ski ......................... 50 . Subcutaneous tissue, fibrosarcoma X 1 

Musculoskeletal System 

Bone ......................... 50 

Vertebra, osteosarcoma .1 

Nervous System 

Brain ......................... 50 

Spinal cord 1 

Osteosarcoma, metastatic, bone 1, 

Respiratory System .. 

Lung ......................... 50 ' 

Alveolar/bronchiolar adenoma x 3 : 

Alveolar/bronchiolar carcinoma 1 ,' 

Granulosa cell tumor malignant, 

metastatic, ovary X 1. 

Hepatocellular carcinoma, metastatic, liver X 1 

Nose ......................... 50 

Trachea ......................... 50 

Special Senses Bystem

None 

Urinary System 

Kidney ......................... 50 

Urinary bladder ......................... 50 . 

tsystemic ILesions ' . 

Multiple organs ......................... 50 

Lymphoma malignant X x X 6 
234 Salicylazosulfapyridine, NTP TR 457 

TABLED2 

Individual Animal Tumor Pathology ofFemale Micein the 2-Year Gavage Study of Salicylazosulfapyridine: 

1,350 mg/kg 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

bPhW 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Histiocytic sarcoma 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatocellular carcinoma 

Hepatocellular carcinoma, multiple 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Histiocytic sarcoma 

Mesentery 

Histiocytic sarcoma 

Leiomyosarcoma, metastatic, WNS 

Pancreas 

Hemangioma 

Salivary glands 

Stomach, forestomach 

Squamous cell papilloma 

Stomach, glandular ~~ ~~ 

CardiovascularSystem 

Blood vessel 

Heart 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Thyroid gland

Follicular cell, adenoma 

General Body System

None 

Genital System 

Clitoral gland 

Ovary . 

.Cystadenoma 

Leiomyosarcoma, metastatic, uterus 

Luteoma 

Teratoma malignant 

,0134456666667777777777777 

0712681666893633333333333 

9160588013632555555555577 

4444444444444444444444444 

2247633552245233444556622 

9539941583684406047260512 

......................... 

+++++++A+++A+++++++++++++ 

++++++A++++++++++++++++++ 

,++++++A++++++++++++++++++ 

++++++A++++A+++++++++++++ 

++++++A++++A+++++++++++++ 

++++++A+;+++A+++++++++++++ 

++++++A++++A+++++++++++++ 

......................... X X xx x 

X 

X xx X 

X X x x 

X

+ + 

X 

X .......................... X ......................... 

......................... 

......................... ~~ - ~~ ~-~- ~ ~- ~ ~ ~~ ~ ~~ ~ ~ ~ ~ ~~ 

......................... 

......................... 

......................... 

......................... 

......................... 

......................... 

......................... X X X 

......................... X x 

......................... 

++++++++++++++++++++++++M 

X xx 

X 

X 
Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Histiocytic sarcoma 

Intestine large, cecum 

Intestine small, duodenum 

Intestine small, jejunum 

Intestine small, ileum 

Liver 

Hepatocellular carcinoma 

Hepatocellular carcinoma, multiple 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Histiocytic sarcoma 

Mesentery 

Histiocytic sarcoma 

Leiomyosarcoma, metastatic. WNS 

Pancreas 

Hemangioma

Salivary glands 

Stomach, forestomach 

Squamous cell papilloma

Stomach, glandular 

Cardiovascular System 

Blood vessel 

Heart 

Endocrine System 

Adrenal cortex 

Adrenal medulla 

Islets. pancreatic 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Thyroid gland 

Follicular cell, adenoma 

General Body System 

None 

._ Genital System ~_I 

~~~ 

c= Clitoral gland ~ 

~~~ Ovary - .~ Cystadenoma 

~

. Leiomyosarcoma, metastatic, uterus .=" - ~~~ ~ 

Luteoma ~ 

Teratoma malignant 

4444444444444444444444444 

2233333344444555556666666 

1823578912569013191234618 

......................... 

......................... 

......................... 

......................... X 

........................... 

.......................... 

......................... 

......................... 

......................... X x x x 

xxx X .X x X' x,x 

xx x x x X x .X 

X 

+ 

......................... 

. . . . . . . . . . . . . . . . . . . . . . . . . . 

......................... A 

......................... 

......................... 

......................... 

......................... 

......................... 

......................... 

.......................... 

............................ X x x X 

.......................... 

......................... 

.......................... X X 

X X X 

Total 

Tissues1 

Tumors 

50 

48 

49 

49 

1 

48 

48 

48 

48 

50 

9 

1 

13 

12 

2 

3 

1 

1 

50 

1 

50 

50 

1 

50 

50 

50 

'50' 

50 

50 

50 

'50 

I 

50 

2 

50 

49 

5 

1 

3 

1 
236 Salicylazosulfapyridine,NTP TR 457 

TABLED2 

Individurll AnimalTumor Pathology of Female Mice in the %Year Gavage Studyof Salicylazosulfapyridine: , . 

1,350 :mg/kg(continued) . ,, 

013445666666,7777777777777 

Number off Days on Study 0712681666893333333333333. . ' . 

9160588013632555555555577 - . . ,. 

444444444444,4444444444444 

Carcass IDNumber 2'247633552245'2334445566.22 

9539941583684406047260512 

Genital System -\ ', (continued) 

Uterus .......................... Histiocytic sarcoma X X 

Leiomyosarcoma X 

Hematopoietic System 

Bone marrow ......................... . . Hemangiosarcoma 

Lymph node + + ++ + + + 

Inguinal, histiocytic sarcoma X 

Inguinal, mast cell tumor NOS 

Renal, teratoma malignant, 

metastatic, ovary X 

Lymph node, mdibular ......................... Lymph node, mesenteric ......................... Spleen ............................ . Hemangioma X 

Hemangiosarcoma X 

Thymus +++++++++++++++++++++++M+ ~ ~ ~~ ~ ~~ ~ ~~~~~~~~ 

: .. . , Integumentary System .. 

Mammary ghand ......................... Adenoacanthoma 

Carcinoma 

Skin ......................... Subcutaneous tissue, fibrosarcoma X 

Subcutaneous tissue, hemangiosarcoma X 

.. Subcutaneous tissue, histiocytic sarcoma X 

Musculoskeletal System 

Bone ......................... Skeletal muscle + + 

Fibrosarcoma X ,' 

, Leiomyosarcoma, metastatic, uterus X 

Nervous System 

Brain .......................... 

Respiratory System 

Lung ......................... Adenoacanthoma, metastatic, mammary gland 

Alveolar/bronchiolar adenoma . x ' 

Alveolar/bronchiolar carcinoma, multiple 

Histiocytic sarcoma X 

Leiomyosarcoma, metastatic, uterus X 

Nose ......................... Trachea ......................... 

Special Senses System 

Harderian gland + 

Adenoma X 
I 

I 

os 

I 

E 

I 

I 

I 

os 

os 

os ......................... ura3sffg snouaN U!Uff 

OS 

2 

I 

I 

......................... 

euro3lesolq!~ 

swan ‘apasaam ‘mo~resoLmo!q 

ura$sLs [e~a[aqso[n~sn~ 

auoa 

al3snur iaataqs 

. 

I 

P 

6P 

1 

os 

OS 

os 

......................... x X X 

......................... 

......................... 

......................... 

I 

os 

I 

I1 

I 

I x 

+ + ++ 

x .......................... 

I

I 
II 238 Salicylazosulfapyridine,NTP TR 457 

TABLED2 

Individual Animal Tumor Pathologyof Female Micein the 2-Year Gavage Studyof Salicylazosulfapyridine: 

1,350 mg/kg (continued) 

Number of Days on Study 

Carcass ID Number 

Urinary System 

Kidney 

Urinary blatlder 

Systemic Lesions 

Multiple organs 

Histiocytic sarcoma 

Lymphoma malignant 

0134456666667777777777777 

0712681666893333333333333 

9160588013632555555555577 

4444444444444444444444444 

2247633552245233444556622 

9539941583684406047260512 

......................... 

......................... 

......................... X X X . . xx xx 
Number of Days on Study 

carcass m NumbeP 

UrinaRy System 

Kidney

Urinary bladder 

Systemic Lesions 

Multiple organs 

Histiocytic sarcoma 

Lymphoma malignant 

~ ~ ~~~~ ~ 

7777777777777777777777777 

3333333333333333333333333 

777777777777777777777777.7 

4444444444444444444444444 

. 2233333344444555556666666 

7823578912569013791234678 

......................... 

......................... 

......................... X 

X X X 

Total 

Tissues/ 

Tumors 

50 

50 

50 

4 

7 
240 'Salicylazosulfapyridine,NTP TR 457 

TABLED2 

IndividualAnimal Tumor Pathology of Female Mice in the'2-Year Gavage Study of Salicylazosulfapyridine:, .: . 

. . 

:' 

' . ' 

. . 

. . 

2,700 mg/kg 

Number of Days on Study 

Carcass ID Number 

Alimentary System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Leiomyosarcoma 

Intestine large, cecum 

Leiomyosarcoma 

Intestine small, duodenum 

Intestine small, jejunum 

Hemangiosarcoma 

Intestine small, ileum 

Liver 

Hepatocellular carcinoma 

Hepatocellular carcinoma, multiple 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Mesentery 

Pancreas 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Cardiovascular System 

Blood vessel 

Heart 

Endocrine System 

Adrenal cortex 

Leiornyos;moma, metastatic, 

intestine large, rectum 

Adrenal medulla 

Pheochromocytoma benign 

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Pars intennedia, adenoma 

Thyroid gland 

Follicular cell, adenoma 

Follicular cell. carcinoma 

General Body System 

None 

~~ ~~ ~~ ~~ ~~~ 

0000566666667777777777777 

0677800155693333333333333 

2702334059055555555555666 

4554545454554445555555444 

9019190829128890111222888 

8679244841974953016038123 

L 

......................... 

++A++++++++++++++++++++++ 

+AA+A+++A++++++++++++++++ 

+AA+A+++A+++++++++++++++'+ 

+AA+A+++A++$+++++++++++++ 

+AA+A+++A++++++++++++++++ 

+AA+A++.+'A++++++++++++++.+-+ 

X 

+AAMA+++A++++++++++++++++ 

++A++++++++++++++++++++++ 

X 

X 

X xxx x x xxx 

xx xx 

+ + + 

++A++++++++++++++++++++++ 

.......................... 

++++++++A++++++++++++++++ 

++++++++A++++++++++++++++ 

......................... 

......................... 

......................... 

......................... 

.......................... 

+++++++++M+++++++++++++++ 

++++M+++++++++++++M++++++ 

X 

......................... X 

li 
Number of Days on Study 

carcass m Number 

Aliientq System 

Esophagus 

Gallbladder 

Intestine large, colon 

Intestine large, rectum 

Leiomyosarcoma 

Intestine large, cecum 

Leiomyosarcoma 

Intestine small, duodenum 

Intestine small, jejunum 

Hemangiosarcoma 

Intestine small, ileum 

Liver 

Hepatocellular carcinoma 

Hepatocellular carcinoma, multiple 

Hepatocellular adenoma 

Hepatocellular adenoma, multiple 

Mesentery 

Pancreas 

Salivary glands 

Stomach, forestomach 

Stomach, glandular 

Cardiovascular System 

Blood vessel 

Heart 

Endocrine System 

Adrenal cortex 

Leiomyosarcoma, metastatic, 

intestine large, rectum 

Adrenal medulla 

Pheochromocytoma benign

Islets, pancreatic 

Parathyroid gland 

Pituitary gland 

Pars distalis, adenoma 

Pars intermedia, adenoma 

Thyroid gland 

Follicular cell, adenoma 

Follicular cell, carcinoma 

General Body System 

None 

7777777777777777777777777 

3333333333333333333333333 

6666666666666677777777777 

4444444455555555555555555 Total 

8889999900000001111222223 Tissues/ 

5670236701257893458125690 Tumors 

......................... 50 

......................... 49 

......................... . . 4 6 

......................... 46 

X 1 

......................... 46 

X 1 

.......................... 46 

......................... 46 

1 

45 -

......................... 49 .......................... 

x X xx xx X 8 

. 1 

X X X xx x 15 

X xx x xx 

+

xx X 

+ 

13 

5 

. 

......................... 49. 

......................... 50 

......................... 49 

......................... 49 

......................... 50, 

. ......................... 50 . 

......................... 50 

X 1 

......................... 50 

X 1 

......................... 50 

......................... 49 1 

M+++++++++++++++M++++++++ 46 

X 2 

X X 2. 

......................... 50 

1 

X 1 
Salicylazosulfapyridine,NTP TR 457 

TABLICD2 

Individual Animal Tumor Pathology of Female Mice inthe 2-Year Gavage Study of Salicylazosulfapyridine:. 

2,700mg/kg (continued) 

Number of Days on Study 

Carcass II)Number 

Genital System 

Clitoral gland

Ovary 

Cystadenoma 

Luteoma 

Uterus 

Polyp stromal 

Hematopoietic System 

Bone marrow 

Lymph node 

Lymph node, mandibular 

Lymph node, mesenteric 

Spleen 

Hemangiosarcoma 

Thymus 

Integumentary System 

Mammary gland 

Skin 

Squamous cell papilloma 

Musculoskeletal System 

Bone 

Cranium, osteosarcoma 

Skeletal muscle 

Nervous System 

Brain 

Osteosarcoma, metastatic, bone 

Spinal cord 

Respiratory System 

Lung 

Alveolar/bronchiolar adenoma 

Nose 

Trachea 

Special Senses System 

Harderian gland 

Adenoma 

Urinary System 

Kidney 

Urinary bladder 

Systemic Lesions 

Multiple organs

Lymphoma malignant 

0000566666667777777777777 

0677800155693333333333333 

2702334059055555555555666 ~~~ ~~__ __

455454545455-4445555555444 

9019190829128890111222888 

8679244841974953016038123 

.......................... 

++A++++++++++++++++++++++ 

X 

++A++++++++++++++++++++++ 

......................... ++ + 

++A++++++++++++++++++++++ 

++A+++++M++++++++++++++++ 

++A++++++++++++++++++++++ 

++++++++++++++M++++++++++ 

......................... 

......................... X 

.......................... X 

+ 

......................... X 

++ 

......................... X ......................... 

......................... 

++A++++++++++++++++++++++ 

......................... 

......................... X X 

.. 

. . 
SaPBicyu~QsPsanh~ygrrri8inne, mTR 454 243 

Tmm ID2 

Inndividud Anninnd "MMIIQICPaUh~bgy~ff FemaUe Mice inn the %YearGavage Study of S~UacyUazosuUff~lpy~~i~inne: 

2,7m (continued) 

1111111177771771711771111 

Number of Days on Study 3333333333333333333333333 

6666666666666671717171777 

4444444455555555555555555 Total 

Carcass ID Number 8889999900000001111222223 Tissues/ 

5610236101251893458125690 Tumors 

Genital System 

Clitoral gland ......................... 50 

Ovary ......................... 49 

Cystadenoma X 2 

Luteoma X 1 

Uterus ......................... 49 

Polyp stromal X 1 

Hematopoietic System 

Bone marrow ......................... 50 

Lymph node + 4 

Lymph node, mandibular ......................... 49 

Lymph node, mesenteric ......................... 48 

Spleen ......................... 49 

Hemangiosarcoma x 1 

Thymus ......................... 49 

Integumentary System 

Mammary gland ......................... 50 

Skin ......................... 50 

Squamous cell papilloma X 2 

Musculoskeletal System 

Bone ......................... 50 

Cranium, osteosarcoma 1 

Skeletal muscle 1 

Nervous System 

Brain ......................... 50 

Osteosarcoma, metastatic, bone 1 

Spinal cord 2 

Respiratory System 

Lung ......................... 50 

Alveolarhronchiolar adenoma X X X 4 

Nose ......................... 50 

Trachea ......................... 50 

Special Senses System 

Harderian gland + + 2 

Adenoma X X 2 

Urinary System 

Kidney ......................... 49 

Urinary bladder ......................... 50 

Systemic Lesions 

Multiple organs ......................... 50 

Lymphoma malignant X X x x 6 
244 Salicylazosulfapyridine,NTP TR 457 

TABLE:D3 

Statistical Analysisof Primary Neoplasms in Female Mice in the2-YearGavage Study of Salicylazosulfapyridine 

Liver:HepatocellularAdenoma 

Overdl ratea 

Adjusted rateb 

Tenninal rate' 

First incidence (days) 

Life table testd 

Logistic regression testd 

Cochran-Armita e testd 6 Fisher exact test 

Liver:HepatocellularCarcinoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

Liver: Hepatocellular Adenoma br Carcinoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

Lung: Alveolar/bronchiolar Adenoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Amlitage test 

Fisher exact test 

Lung: Alveolar/bronchiolar Adenoma or Carcinoma 

Overall rate 

Adjusted rate 

Terminalrate ' 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

VehicleControl 

12/50 (24%) 

29.3% 

12/41 (29%) 

735 (T)

P<O.001 

P=O.o02 

P=O.o04 

2/50 (4X) 

4.9% 

2/41 (5%) 

735 (T)

P=O.O51 

P=O.O59 

P~O.071 

14/50(28X) 

34.1 % 

14/41 (34%) 

735 (T)

P-0.002 

P-0.005 

P=O.O11 

2/50 (4%) 

4.9% 

2/41 (5%) 

735 (T) 

P~O.237 

P-0.254 

P=O.273 

3/50 (6%) 

7.3% 

3/41 (7%) 

735 (T) 

P=O.395 

P=O.418 

P=O.446 

675 mg/kg 

28/50 (56%) 

63.6% 

25/41 (61%) 

539 

P=O.001 

P=O.001 

P=O.001 

10150 (20%) 

23.7% 

9/41 (22%) 

55 1 

P=O.O16 

P=O.O17 

P=O.O14 

32/50 (64%) 

71.1% 

28/41 (68%) 

539 

P<O.001 

P<O.001 

P<O.001 

3/50 (6%) 

7.0% 

2/41 (5%) 

620 

P=O.502 

P=O.507 

P=O.500 

4/50 (8%) 

9.4% 

3/41 (7%) 

620 

P=O.502 

P=O.509 

P=O.500 

1,350 mg/kg 

25/50 (50%) 

65.7% 

24/37 (65%) 

618 

P=O.001 

P=O.002 

P-0.006 

10/50(20%) 

27.0% 

10137 (27%) 

735 (T) 

P=O.o09 

P=O.009 

P=O.O14 

28/50 (56%) 

73.6% 

27/37 (73%) 

618 

P<O.001 

P=O.001 

P=O.o04 

3/50 (6%) 

8.1% 

3/37 (8%) 

735 (T) 

P=O.453 

P=O.453 

P=O.500 

4/50 (8%) 

10.8% 

4/37 (1 1 %) 

735 (T) 

P=O.444 

P=O.444 

P=O.500 

2,700 mg/kg 

28/49 (57%) 

71.7% 

27/38 (71X) 

655 

P<O.001 

P<O.001 

P<O.001 

9/49 (18%) 

23.7% , 

9/38 (24%) 

735 (T) 

P=O.O19 

P=O.O19 

P=O.O23 

29/49 (59%) 

74.3% 

28/38 (74%) 

655 

P <0.001 

P<O.001 

P=O.002 

4/50 (8 %) 

10.5% 

4/38 (1 1 W) 

735 (T) 

P=O.3O2 

P=0.3M 

P=O.339 

4/50 (8 %) 

10.5% 

4/38 (1 1 X) 

735 (T) 

P=O.458 

P=O.458 

P=O.500 

, 

, 
Ovary:Luteoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

Ovary:Cystadenoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Lifetest table . 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

PituitaryGlaxid(Pars Distalis): Adenoma 

Overallrate , 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

Spleen:Hemangiosarcoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

Stomach (Forestomach): Squamous Cell Papilloma 

Overall rate, 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

VehicleControl 

0149 (0%) 

0.0% 

0/40(0%) 

-e 

P=O.331 

P=O.331 

P=O.351 

1/49 (2%) 

2:5% 

1/40(3%) 

735 (T) 

P=O.524 

P=O.548 

P=O.558 

7/49 (14%) 

17.5% 

7/40 (18%) 

735 (T) 

P=0.097N 

P=0.079N 

P=0.069N 

0/50 (0%) 

0.0% 

0/41 (0%) 

-

P=O.391 

P=O.411 

P=O.414 

5/50 (10%) 

12.2% 

5/41 (12%) 

735 (T) 

P=0.020N 

P=O.02ON 

P=O.O16N 

6% mgkg 

1/49 (2 %) 

2.5% 

1/40 (3%) 

735 (T)

P=O.500 

P=O.500 

P=O.500 

5/49 (10%) 

12.5% 

5/40 (13%) 

735 (T)

P=O.103 

P=O.103 

P=O.102 

7/45 (16%) 

17.5% 

7/40 (18%) 

735 (T) 

P=O.615 

P=O.615 

P=O.545 

2/50 (4%) 

4.7% 

1/41 (2%) 

620 

P=O.242 

P=O.235 

P=O.247 

1/50 (2%) 

2.4% 

1/41 (2%) 

735 (T) 

P=0.103N 

P=0.103N 

P=0.102N 

1,350 mgkg 

3/49 (6%) 

8.3% 

3/36 (8 %) 

735 (T) 

P=O.103 

P=O.103 

P=O.121 

5/49 (10%) 

13.1% 

4/36 (1 1 %) 

618 

P=O.O87 

P=O.100 

P=O.102 

7/50 (14%) 

17.8% 

5/37 (14%) 

663 

P=O.553 

P=O.603 

P=O.SSrlN 

4/50 (8 X) 

10.3% 

3/37 (8%) 

66 1 

P=O.O55 

P=O.o61 

P=O.O59 

1/50 (2%) 

2.7% 

1/37 (3%) 

735 (T) 

P=O.l28N 

P=0.128N 

P=0.102N 

2,7QQmgkg 

1/49 (2%) 

2.6% 

1/38 (3 %) 

735 (T)

P=O.490 

P=O.490 

P=O.500 

2/49 (4%) 

5.3% 

2/38 (5 %) 

735 (T) 

P=O.482 

P=O.482 

P=O.500 

2/46 (4%) 

5.0% 

1/35 (3%) 

603 

P=O. 112N 

P=O.lMN 

P=0.095N 

1/49 (2%) 

2.6% 

1/38 (3%) 

735 (T) 

P=O.485 

P=O.485 

P=O.495 

0150 (0%) 

0.0% 

0138 (0%) -

P=O.WN 

P=O.WN 

P=0.028N 
-- 

246 Salicylazosulfapyridine, NTP TR 457 

TABLEI)3 

Statistical Analysisof Primary Neoplasms in Female Mice in the2-YearGavage Studyof Salicylazosulfapyridine(continued) 

All Organs:Hemangiosarcoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Annitage test 

Fisher exact test 

All Organs: Hemangioma or Hemangiosarcoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Almitage test 

Fisher exact test 

AU Organs: Malignant Lymphoma 

Overall rate 

I VehicleControl 

3/50 (6%) 

7.3% 

3/41 (7%) 

735 (T)

P=0.522N 

P=0.495N 

P=0.479N 

4/50 (8 %) 

9.8% 

4/41 (10%) 

735 (T)

P-0.439N 

P==0.406N 

P-0.389N 

13/50 (26%) 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

AU Organs: Histiocytic Sarcoma 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

All Organs: Benign Neoplasms 

Overall rate 

Adjusted rate 

Terminal rate 

First incidence (days) 

Life table test 

Logistic regression test 

Cochran-Armitage test 

Fisher exact test 

675 mg/kg 

2/50 (4%) 

4.7% 

1/41 (2%) 

620 

P=0.498N 

P=0.493N 

P=O.5oON 

2/50 (4%) 

4.7% 

1/41 (2%) 

620 

P=0.337N 

P=O.329N 

P=0.339N 

6/50 (12%) 

1,350 mg/kg 

4/50 (8%) 

10.3% 

3/37 (8%) 

661 

PzO.450 

P=O.487 

P=OSoO 

6/50 (12%) 

15.1% 

4/37 (1 1 %) 

661 

P=O.316 

P=O.356 

P=O.370 

2,700 mgkg 

2/50 (4X) 

5.3% 

2/38 (5 %) 

735 (T) . 

P=0.535N 

P=0.535N 

P=O.5oON 

2/50 (4%) 

5.3% 

2/38 (5 %) 

735 (T)

P=0.372N 

P=0.372N 

P=0.339N 

7/50 (14%) 6/50 (12I )

Adjusted rate 30.2% 13.7% . 17.4% 14.7%

Terminal rate 11/41 (27%) . 4/41.(10%) 5/37 (14%) 4/38 (1 1 %) 

First incidence (days) 708 . 493 618 583 

Life table test P=0.068NP=O.l13N P=O.l60N P=0.095N 

P=O.O83N ' 

P=0.076N 

2/50 (4X) 

4.9% 

2/41 (5%) 

735 (T)

P=0.366N 

P=0.338N 

P-0.337N 

22/50 (44 96) 

53.7% 

22/41 (54%) 

735 (T)

P-0.009 

P-0.017 

P=O.O49 

P=0.057N 

P=0.062N 

0150 (0R) 

0.0% 

0/41 (0%) -

P=0.238N 

P=0.238N 

P=0.247N 

32/50 (64%) 

72.7% 

29/41 (71%) 

539 

P=O.O30 

P=O.O32 

P=O.O35 

P=0.112N 

4/50 (8%) 

9.9% 

2/37 (5 46) 

420 

P=O.303 

P=O.337 

P=O.339 

34/50 (68%) 

82.9% 

30/37 (8146) 

618 

P=O.o02 

P=O.O03 

P=O.O13 

P=0.069N 

0150 (0%) 

0.0% 

0/38 (0%) -

P=0.255N 

P=0.254N 

P=0.247N 

32/50 (64%) 

79.9% 

30/38 (79%) 

603 

P=O.o(n 

P=O.OlO 

P=O.O35 
PIP'O=d 

95E'O=d 

VIZ'O=d 

E85 

(%P@ OElZE 

X9'58 

(%ZL)OSl9E 

NBIP'O=d 

NLW'O=d 

NIZS'O=d 

E85 

(%LE) 8ElPI 

%I'IP 

(%*E) OSILI 

OSO'O=d 

SEO'O=d 

510'0=d 

I LI 

(%68) LElEE 

%€'I6 

(%V8) OSlZP 

PII'O=d 

01I'O=d 

Su)'O=d 

ILI 

(I6PP)LE181 

%VLS 

(%ZS) 05l9Z 

I 

OEE'O=d 

6LE'O=d 

ISE'O=d 

E6P 

(Y9L) IPIIE 

%L'8L 

(YPL) OSlLE 

NZOS'O=d 

NLSS'O=d 

N695'O=d 

E6V 

t%VQ WVI 

%E'IP 

(1%)05/61 

19E'O=d 

Z9Z'O=d 

LSI'O=d 

6ZV 

(Y9L) IVlIE 

YZ'LL 

(Y$9)OS/PE 

NZEVO=d 

NLWO=d 

SOS'O=d 

6ZP 

(Y6E) 19/91 

II'EP 

(%8E) OS161 
248 Salicyliosulfapyridine, NTP TR a57 

TABLIED4a 

Historical Incidence of Hepatocellular Neoplasmsin'Female B6C3Fl Mice Receiving Corn Oil.by Gavagea 

Incidence in Controls 

. . 

Adenoma Carcinoma or Adenoma Crcinoma 

Overall Historical Incidence .. 

Total 991759 (13.OX) 381759 (5.0%) 1311759 (17.3%) . Standarddeviation 8.4% 3.7% 9.8% 

, Range 2%-28% 0%-14% 4%-37% 

a Data as of 17 June 1994 

TABLED4b . 

Historical Incidence of Splenic Hemangiosarcoma in Female B6C3F, Mice Receiving Corn Oilby Gavagea 

Incidence in Conkols 

I . 

Overall Historical Incidence 

Total 61756 (0.8%) 

' Standard deviation 1.3% 

Range 0%-4% 

a Data as of 17 June 1994 
Disposition Summary 

Animals initially in study 

15-Month interim evalualion 

Early deaths 

Accidental deaths 

Moribund 

Natural deaths 

Survivors 

Died last week of study 

Terminal sacrifice 

Animals examined microscopically 

Alimentary System 

Liver 

Cytologic alterations 

Eosinophilic focus 

Inflammation, subacute 

Mixed cell focus 

Vacuolization cytoplasmic 

Mesentery 

Fat, necrosis 

Pancreas 

Acinus, atrophy 

Stomach, forestomach 

Hyperplasia 

Inflammation, subacute 

Stomach, glandular 

cyst 

Hyperplasia 

Mineralization 

Endocrine System 

Adrenal cortex 

Accessory adrenal cortical nodule 

Pituitary gland 

Pars distalis, hyperplasia 

Thyroid gland 

Follicle, degeneration 

Genital System 

Clitoral gland 

Duct, cyst 

Ovary 

cyst 

UkNS 

Dilatation 

Hyperplasia, cystic 

60 60 

10 10 

2 

5 6 

2 3 

41 41 

60 60 

(10) (10) 

1 (10%) 

(10) (10) 

1 (10%) 

(10) (10) 

2 (20%) 4 (40%) 

60 

10 

1 

8 

4 

1 

36 

60 

(10) 

(10) 

(10)

3 (30%) 

60 

10 

2 

6 

4 

38 

60 

1 (10%) 

3 (30%) 

(10) 

(10) 

1 (10%) 

(10) 

4 (40%) 

a Number of animals examined microscopically at the site and the number of animals with lesion 
250 Salicylazosulfapyridine,NTP TR 457 

TABLE:D5 

Summary of the Incidenceof Nonneoplastic Lesions in Female Mice in the 2-YearGavage Study 

of Salicylazosulfapyridine(continued) 

675ControlVehicle mgkg 1,350 mgkg 2,700 mag 

15-Month Interim Evaluation (continued) 

Hematopoietic System

Lymph node, mandibular (10)

Pigmentation 

Lymph node, mesenteric (10) Necrosis 2 (20%)

Spleen (10) (10) (10)

Hematopoietic cell proliferation 2 (20%) 7 (70%) 10 (100%)

Hyperplasia, lymphoid 2 (20%)

Pigmentation, hemosiderin 10 (100%) 10 (100%) 

Thymus (10) (10) (10) 

cyst 2 (20%)

Thymocyte, necrosis . 1 (10%) 

Respiratory System 

Lung (10) (10) (10) (10)

Intlammation, subacute 1 (10%) 

Alveolar epithelium, hyperplasia 1 (10%) 1 (10%) 

Alveolus, infiitration cellular, histiocyte 2 (20%) 1 (10%) 

Nose (10) (10) (10) (10)

Glands, cyst 2 (20%) 4 (40%)

Olfactory epithelium, degeneration 8 (80%) 10 (100%) 10 (100%) 6 (60%) 

Urinary System 

Kidney 

Fibrosis 

Glomerulosclerosis 

Metaplasia, osseous 

Mineralization 

Renal tubule, casts 

Renal tubule, regeneration 

Systems Examined With No Lesions Observed 

Cardiovascular System 

General Body System 

Integumentary System 

Musculoskeletal System 

Nervous System . . 

Special Senses System 

2-Year Study 

Alimentary System 

Esophagus

Inflammation, chronic 

Perforation 

Intestine large, cecum 

Edema 

Infiammation, subacute 
2- St"' (continued) 

Alimentary System (continued) 

Intestine small. duodenum 

Hyperplasia 

Necrosis 

Intestine small, jejunum 

Dilatation 

Peyer's patch, hyperplasia, lymphoid 

Intestine small, ileum 

Dilatation 

Inflammation, subacute 

Liver 

Angiectasis 

Basophilic focus 

Clear cell focus 

Congestion 

Depletion glycogen 

Eosinophilic focus 

Eosinophilic focus, multiple 

Fibrosis 

Hematopoietic cell proliferation 

Hemorrhage 

Hyperplasia, lymphoid 

Inflammation, chronic 

Inflammation, subacute 

Mineralization 

Mixed cell focus 

Necrosis 

Pigmentation, bile 

Thrombosis 

Vacuolization cytoplasmic 

Bile duct, cyst

Mesentery 

cyst 

Inflammation, subacute 

Polyarteritis 

Fat, inflammation, subacute 

Fat, necrosis 

Pancreas 

Edema 

Fibrosis 

Hemorrhage 

Inflammation, chronic 

Inflammation, subacute 

Polyarteritis 

Acinus, atrophy 

Acinus, depletion secretory 

Acinus, necrosis 

Duct, cyst

Salivary glands 

Acinus, atrophy 

1 (2%) 

1 (2%) 

1 (2%) 

15 (31%) 

4 (8%) 

1 (2%) 

3 (6%) 

2 (4%) 

1 (2%) 

1 (20%) 

4 (80%) 

(49) 

. 1 (2%) 

1 (2%) 
152 Salicylazosulfapyridine,'NTP TR 457 

TABLED!; 

Summary of the Incidence of Nonneoplastic Lesions'in Female Mice in the 2-Year Gavage Study 

of Salicylazosulfapyridine(continued) 

Vehicle Control 675 mg/kg 1,350 mg/kg 2,700 mg/kg 

2-Year Study (continued) 

Stomach, forestomach 

Alimentary System(continued) 

Ulcer 

Polyarteritis . 

Mineralization 

Inflammation, subacute 

Hyperplasia 

Edema 

,Diverticulum 

Edema 

cyst 

Stomach, glandular 

(50) 

1 (2%) 

2 (4%) 

1 (2%) 

1(2%) 

2 (4%) 

12 (24%) 

2 (4%) 

(50) 

(49) 

1(2%) 

1 (2%) 

6 (12%) 

(49) 

1 (2%) 

4 (8%) 

(49) 

1 (2%) 

4 (8%) 

1(2%) 

Erosion 

Mineralization 

Inflammation,subacute 

Hyperplasia 

3 (6%) 8 (16%) 

2 (4%) 

1(2%) 

4 (8%) 2 (4%) 

Heart 

Cardiovascular System 

Cardiomyopathy 

(50) 

1(2%) 

Congestion 

Fibrosis 

Degeneration 

1 (2%) 

1 (2%) 

1 (2%) 

Foreignbody . 1 (2%) 

Hemorrhage 1 (2%) 

Pericardium, inflammation, chronic 

.Mineralization 

1 (2%) 

1 (2%) 1 (2%) 

Endocrine System . . 

Adrenal cortex 

Accessory adrenal cortical nodule 

(50) 

cyst 1(2%) 1(2%) 

Hematopoietic cell proliferation 

Vacuolization cytoplasmic, focal

Hypertrophy 

Hyperplasia 

1(2%) 

2 (4%) 

1 (2%) 

1 (2%) 

3 (6%) 

1 (2%) 

4 (8%) 

. 

. 

' 

. 

Capsule, hyperplasia 1(2%) 

Spindle cell, hyperplasia 1 (2%) 

X-zone, vacuolization cytoplasmic 

Hyperplasia 

Adrenal medulla (50) 

2 (4%) 

Hyperplasia 

Islets, pancreatic 

Pars distalis, cyst 

Pituitary gland 

, (50) 

(49) 

1 (2%) 

Pars intermedia, hykrplasia 

Pars distalis, hyperplasia 12 (24%) 

Follicular cell, hyperplasia 

Follicle, degeneration 

Follicle, cyst 

Polyarteritis 

Thyroid gland (50) 

20 (40%) 

1 (2%) 

6 (12%) 

1 (2%) 

2 (4%) 

6 (12%) 

3 (6%) 

6 
(%Z) I 
254 Salicylazosulfapyridine,NTP TR457 

TABLE:DS 

Sumrnary of the Incidence of NonneoplasticLesions in Female Mice in the2-YearGavage Study 

of Salicylazosulfapyridine(continued) 

&Year Study (continued) 

Integumentary System 

Mammary gland 

Duct, cys! 

Skin 

Cyst epithelial inclusion 

Hyperkeratosis 

Inflammation, chronic 

Inflammation, subacute 

Subcutaneous tissue, abscess 

Subcutaneous tissue, edema 

Subcutaneous tissue, fibrosis 

Subcutaneous tissue, foreign body 

Subcutaneous tissue, hemorrhage 

Musculoskeletal System 

Bone 

Cranium, fibrous osteodystrophy 

Cranium, hyperostosis 

Femur, fibrous osteodystrophy 

Skeletal muscle 

Abscess 

Foreign body 

Nervous System 

Brain 

Compression 

Cyst epithelial inclusion 

Hemorrhage 

Vacuolization cytoplasmic 

Spinal cord 

Vacuolizxtion cytoplasmic 

RespiratoIy System 

Lung

Congestion 

Foreign body 

Hemorrhage 

Hyperplasia, macrophage 

Inflammation,subacute 

Pigmentation 

Alveolar epithelium, hyperplasia 

Alveolus, infiltration cellular, histiocyte 

Mediastinum, inflammation, chronic 

Nose 

Glands, mineralization 

Lumen, exudate 

Lumen, foreign Body 

Olfactory epithelium, degeneration 

Respiratory epithelium, mineralization 

Control Vehice

1 (2%) 

(50) 

1 (2%) 

3 (6%) 

(1) 

1 (100%) 

1 (100%) 

(50) 

1 (2%) 

1 (2%) 

(2)

1 (50%) 

(50) 

1 (2%) 

2 (4%) 

1 (2%) 

(50) 

1 (2%) 

675 mgkg 1,350 mg/kg 2,700 mgkg 

(50) 

1 (2%) 

1 (2%) 

(50) 

(50) (50) (50) 

2 (4%) 

1 (2%) 1 (2%) 

1 (2%) 

1 (2%) 

(1) (2) 

1 (2%) 

(50) 

256 Salicylazosulfapyridine,NTP TR 457 
 ON^^ MUTAGENICITY TEST PROTOCOL ...................................... 258 

CW~E IHL~STEROVARYCELLCYTOGENETICS PROTOCOLS ............................ 258 

MQUSE E%RIPIIERAL BLOOD MICRONUCLEUS TEST PROTOCOL ............................ 259 

MQWSE BONE MARROW CHROMOSOMAL hERRATIONS 1['EST PROTOCOLS .................... 260 

IWAT AP~DMourn Bow M~OW MICRONUCLEUS TEST PROTOCOL ........................ 260 

MQWSE BQNE MARROW MICRONUCLEUS TEST WITH KWETQCHORE ANALYSIS PROTOCOL ......... m 

~SWS ............................................................... 26n 

TABLEIEI 

TABLEIE2 

TABLEE3 

TABLEE4 

TABLEIE4 

TABLEIE6 

TABLEIE7 

TABLEE8 

TABLE IE9 

TABLEIEl0 

Mutagenicity off SaPicyPazosaaUhnr~dinne in Salmonella typhimlsvium ............... 264 

IInadnctionaoff Sister Chromatid Exchanges inn Chinese Hamster Ovary CeUb 

by SaLyBazosuUfapyr~dinae.......................................... 265 

Indwctiow off ChromosomaP A0errationnsinn Chinese Hamster Ovary CeUb 

by SaBicyU~osuBffapylridlinae .......................................... 266 

Frequency off Micronuclei in Mouse PeripheraP Bl~~odl lE@hroc@es 

Following Treatment with SaUani~yUa~osuBffa~pyr~~inae by Gavage ffor I3 Weeks ......... 26'7

Indnctiona off Chromosomal A0errationnsinn Bone Marrow CeUUs off MaPe Mice 

Treated Once with SalicyBazosnaUhpyrid~nneby Gavage: Standard lh-docd .......... 268 

Frequency off Micronuclei in PoUychromatic Bone Marrow CeUb dMaUe Mice 

Treated Twice with SalicyU~osnan~apyr~dinne by Gavage: InitiaU Assay ............. 269

Freqwenncy of IKinetochore Positive and Kinetochore NegaUive Micronnucnei 

in Pdgr~hromatk Bone Marrow CeUlsoffMaUe Mice Treated Three Times 

with SaUicyUazosullffapyridineby Gavage ................................. 270 

Frequency off MicronwcPei ina Pdychr~matic Bone Marrow CeUUs ~ f Mice f 

Treated with SaPicyPazosnnPffapyrid~nneby Gavage: Splbseqwennt Assay .............. 271 

IInadwctionoff ChromosomaP A0errationas inn Bone Marrow CePb off Mde Mice 

Treated with SaUacyPazosoBffaWridineby Gavage: N~~~standard lh-~t~~d ........... 2'72

Freqwency of Micronwclei in PolychromaticBonae Marr~wCeUh off MaUe Rats 

Treated Three Times with SalicyB~osaaU~apyridi~e by Gavage ................... 273
258 Salicylazosulfapyridine,. T P N
 TR 457

GENETIC TOXICOLOGY 

SALMONELLAMUTAGENICITYESTPROTOCOL 

Testing was performed as reported by Zeiger ef al. (1988). Salicylazosulfapyridine was sent to the 

laboratory as a coded aliquot from Radian Corporation (Austin, TX). It was incubated with the Salmonella 

typhimunum tester strains TA97, TA98, TA100, and TA1535 either in buffer or S9 mix (metabolic 

activation enzymes and cofactors from 'Aroclor 1254-induced male Sprague-Dawley rat or Syrian hamster, 

liver) for 20 minutes at 37" C. Top agar supplemented with I-histidine and d-biotin was added, and the 

contents of the tubes were mixed and poured onto the surfaces of minimal glucose agar plates. Histidineindependent mutant colonies arising on these plates were counted following incubation for 2 days at 370 C. 

Each trial consisted of triplicate plates of concurrent positive and negative controls and of five doses of 

salicylazosulfapyridine. The high dose was limited by toxicity. . ' 

In this assay, a positive response is defined as a reproducible, dose-related increase in histidine-independent 

(revertant) colonies in any one straidactivation combination. An equivocal response is defined as an 

increase in revertants which is not dose-related, is not reproducible, or is of insufficient magnitude to 

support a determination of mutagenicity. A negative response is obtained when no increase in revertant 

colonies is observed following chemical treatment. There is no minimum percentage or fold increase 

required for a chemical to be judged positive or weakly positive. 

CIHINESE mS"ER-OVARY CELL CYTOGENETICS PROTOCOLS 

Testing was performed as reported by Bishop ef aI. (1990). Salicylazosulfapyridine was sent to the 

laboratory as a coded aliquot by Radian Corporation. It was tested in cultured Chinese hamster ovary 

(CHO) cells for induction of sister chromatid exchanges (SCEs) and chromosomal aberrations (Abs) both in 

the presence and absence of Aroclor 1254-induced male Sprague-Dawley rat liver S9 and cofactor mix. 

Cultures were handled under gold lights to prevent photolysis of bromodeoxyuridine-substitutedDNA; 

Each test consisted of concurrent solvent and positive controls and of at least three doses of 

salicylazosulfapyridine;the high dose was limited by toxicity. A single flask per dose was used. 

Sister Chromatid Exchange Test: In the SCE test without S9, CHO cells were incubated for 26 hours with 

salicylazosulfapyridine in supplemented McCoy's 5A medium. Bromodeoxyuridine (BrdU) was added 

2 hours after culture initiation. After 26 hours, the medium containing salicylazosulfapyridine was removed 

and replaced with fresh medium plus BrdU and Colcemid, and incubation was continued for 2 hours. Cells 

were then harvested by mitotic shake-off, fixed, and stained with Hoechst 33258 and Giemsa. In the SCE 

test with S9, cells were incubated with salicylazosulfapyridine,serum-free medium, and S9 for 2 hours. The 

medium was then removed and replaced with medium containing serum and BrdU and no 

salicylazosulfapyridine, and incubation proceeded for. an additional 26 hours, with Colcemid present for the 

final 2 hours. Harvesting and staining were the same as for cells treated without S9. All slides were scored 

blind and those from a single test were read by the same person. Fifty second-division metaphase cells 

were scored for frequency of SCEskell from each dose level. 

Statistical analyses were conducted on the slopes of the dose-response curves and the individual dose points 

(Galloway ef al., 1987). An SCE frequency 20% above the concurrent solvent control value was chosen as 

a statistically conservative positive response. The probability of this level of difference occurring by chance 

at one dose point is less than 0.01; the probability for such a chance occurrence at two dose points is less 

than 0.001. An increase of 20% or greater at any single dose was considered weak evidence of activity; 

increases at two or more doses resulted in a determination that the trial was positive. A statistically 
S~~~~yl~QSlalhn~~~~@, TW45’7 259 

significant trend (P<O.O05) in the absence of any responses reaching 20% above background led to a call 

of equivocal. 

ChromosomalAbewtarkions TesS: In the Abs test without S9, cells were incubated in McCoy’s 5A medium 

with salicylazosulfapyridine for 10 hours; Colcemid was added and incubation continued for 2 hours. The 

cells were then harvested by mitotic shake-off, fixed, and stained with Giemsa. For the Abs test with S9, 

cells were treated with salicylazosulfapyridine and S9 for 2 hours, after which the treatment medium was 

removed and the cells incubated for 11 hours in fresh medium, with Colcemid present for the final 2 hours. 

Cells were harvested in the same manner as for the treatment without S9. 

Cells were selected for scoring on the basis of good morphology and completeness ofkaryotype 

(21 f 2 chromosomes). All.slides were scored blind and those from a single test were read by the same 

person. Two hundred first-division metaphase cells were scored at each dose level. Classes of aberrations 

included “simple” (breaks and terminal deletions), “complex” (rearrangements and translocations), and 

“other” (pulverized cells, despiralized chromosomes, and cells containing 10 or more aberrations). 

Chromosomal aberration data are presented as percentage of cells with aberrations. To arrive at a statistical 

call for a trial, analyses were conducted on both the dose-response curve and individual dose points. For a 

single trial, a statistically significant (PiO.05) difference for one dose point and a significant trend 

(PsO.015) were considered weak evidence for a positive response; significant differences for two or more 

doses indicated the trial was positive. In the absence of a statistically significant increase at any one dose 

point, a positive trend led to an equivocal call (Galloway et al., 1987). Ultimately, the trial calls were 

based on a consideration of the statistical analyses as well as the biological information available to the 

reviewers. 

MOUSE rnIwnPrnWIL BLOOD M(cRO~(cLEUSTIEST rnOTOCOL 

A detailed discussion of this assay is presented in MacGregor et al. (1990). Peripheral blood samples were 

obtained from male and female B6C3F, mice at the end of the 13-week toxicity study (Table FA). Smears 

were immediately prepared and fixed in absolute methanol, stained with a chromatin-specific fluorescent 

dye mixture of Hoechst 33258/pyronin Y (MacGregor et al., 1983), and coded. Slides were scanned at 

630 X or 1,000 X magnification using a semi-automated image analysis system to determine the frequency of 

micronuclei in at least 2,000 polychromatic erythrocytes (PCEs) and 10,000 normochromatic erythrocytes 

(NCEs) in each of 10 animals per dose group. The criteria of Schmid (1976) were used in defining 

micronuclei, with the additional requirement that micronuclei exhibit %e’ characteristic fluorescent emissions 

of DNA (blue with 360 nm and orange with 540 nm ultraviolet illumination); the minimum size limit was 

approximately one-twentieth the diameter of the NCE cell. In addition, the percentage of PCEs among the 

total erythrocyte population was determined. 

Log transformation of the NCE data, testing for normality by the Shapiro-Wilk test, and testing for 

heterogeneity of variance by Cochran’s test were performed before statistical analyses. The frequency of 

micronucleated cells among NCEs was determined by analysis of variance using the SAS GLM procedure. 

The NCE data for each dose group were compared with the concurrent solvent control using a Student’s 

t-test. The frequency of micronucleated cells among PCEs was analyzed by the Cochran-Armitage trend 

test, and individual dose groups were compared to the concurrent solvent control by Kastenbaum-Bowman’s 

binomial test. The percentage of PCEs among total erythrocytes was determined by an analysis of variance 

on ranks (classed by sex), and individual dose groups were compared with the concurrent solvent control 

using a t-test on ranks. 
2tii Salicylazosulfapyridine, NTP TR 457 

MOUSEBONE MARROW CHROMOSOMAL ABERRATIONS TEST-PROTOCOLS 

SfandQrd Protocol:A dose range-finding study was performed in the absence of adequate toxicity 

information from the literature, and the high dose was limited by toxicity to 1,000 mg/kg. 

Salicylazosulfapyridine was tested for induction of Abs in mouse bonemarrow cells using a harvest time of 

1.7hours. 

Male B6C3Fl mice (eight animals per dose group) were administered salicylazosulfapyridine once by corn 

oil gavage (Table E5). Solvent control mice were administered corn oil by gavage. The positive control, 

dimethylbenz(a)anthracene, was intraperitoneally injected. The mice were subcutaneously implanted with a 

BrdU tablet (McFee et al., 1983) 18 hours before the scheduled harvest. (For a standard 17-hour exposure 

time, this required BrdU implantation to precede treatment with salicylazosulfapyridine by 1 hour.) The use 

of BrdU allowed selection of the appropriate cell population for scoring. (Abs induced by chemical 

administration are present in maximum number at the first metaphase following treatment; they decline in 

number during subsequent nuclear divisions due to cell death.) Two hours before sacrifice, the mice 

received an intraperitoneal injection of colchicine in saline. The animals were killed 17 hours after 

salicylazosulfapyridine treatment (1 8 hours after BrdU dosing). One or both femurs were removed and the 

marrow was flushed out with phosphate-buffered saline .(pH 7.0). Cells were treated with a hypotonic salt 

solution, fixed, and dropped onto chilled slides. After a 24-hour drying period, the slides were stained with 

Giemsa and scored (Goto et al., 1978). 

Fifty first-division metaphase cells were scored from each of eight animals per treatment group. Responses 

were evaluated as the percentage of aberrant metaphase cells, excluding gaps. The data were. analyzed 'by a 

trend test (Margolin et al., 1986). 

Nonstandard Protocol: Because positive results were observed in micronucleus experiments that employed 

multiple dosing protocols (standard for a micronucleus test), and because the standard single-dose rat and 

mouse bone marrow chromosomal aberrations protocol yielded negative results, salicylazosulfapyridine was 

tested for induction of Abs in a nonstandard, triple-treatment protocol that used three gavage administrations 

separated by 24-hour intervals in an attempt to mimic the same exposure environment that produced the 

positive micronucleus test results (Table E9). The highest dose used in this experiment was 4,000 mg/kg. 

RAT AND MOUSEBONE MARROW MICRONUCLEUSTEST,PROTOCOL 

Preliminary-range-findingstudies were performed for the initial mouse bone marrow micronucleus test. 

j Factors affecting dose selection included chemical solubility, toxicity, and the extent of cell cycle delay 

induced by salicylazosulfapyridine exposure. Based on the results of the range finding studies, male 

B6C3Fl mice were administered salicylazosulfapyridine by corn oil gavage twice, at 24-hour intervals 

(Table E6). The total dosing volume was 0.4 mL. Solvent control animals received 0.4 mL corn oil, and 

the positive control mice received injections of dimethylbenz(a)anthracene. The mice were killed 24 hours 

after the second injection, and blood smears were prepared from bone marrow cells obtained from the 

femurs. Air-dried smears were fixed and stained; 2,000 PCEs were scored for frequency of micronucleated 

cells in each of five animals per dose group. 

Doses of salicylazosulfapyridine used in subsequent micronucleus tests in male Fisher 344/N rats 

(Table E10) 'and male and female B6C3Fl mice (Table E8) were based on the accumulated information 

availabie from all toxicity studies performed with salicylazosulfapyridine. Salicylazosulfapyridine was 

administered by gavage in these additional micronucleus tests, and the number of gavage treatments 

administered to each dose group vafied from one to three; multiple treatments were administered at 24-hour 

intervals. Bone marrow sampling was performed 24 hours after the final dosing, and slide preparation and 

scoring were performed as described above. 
The results were tabulated as the mean of the pooled results from all animals within a treatment group, plus 

or minus the standard error of the mean. The frequency of micronucleated cells among PCEs was analyzed 

by a statistical software package that tested for increasing trend over exposure groups using a one-tailed 

Cochran-Armitage trend test, followed by pairwise comparisons between each exposure group and the 

control group (Margolin et al., 1990). In the presence of excess binomial variation as detected by a 

binomial dispersion test, the binomial variance of the Cochran-Armitage test was adjusted upward in 

proportion to the excess variation. In the micronucleus test, an individual trial is considered positive if 

1) the trend test P value is ~0.025, or 2) the P value for any single exposure group is sO.O25/N, where 

N=the number of exposure groups. A final call of positive for micronucleus induction is preferably based 

on reproducibly positive trials (as noted above). Ultimately, the final call is determined by the scientific 

staff after considering the results of statistical analyses, reproducibility of any effects observed, and the 

magnitudes of those effects. 

WTW mrnToewom ANALYSIS moTocoL 

This specialized micronucleus test was conducted at SITEK Research Laboratories (Rockville, MD) 

(Table E7). Male B6C3F, mice (five per treatment group) were fasted for 12 hours prior to treatment. 

They were administered salicylazosulfapyridine in corn oil by gavage three times at 24-hour intervals. 

Solvent control animals were administered corn oil by gavage, and two groups of positive control mice were 

used. One group received triethylenemelamine, a clastogen which produces mainly kinetochore-negative 

micronuclei, and the other group received vincristine sulfate, an aneuploidogen which produces micronuclei 

that contain kinetochores. Both these positive control chemicals were dissolved in deionized distilled water 

and administered by a single intraperitoneal injection. 

Mice were killed 24 hours after the final treatment, and bone marrow was flushed from the femurs into 

tubes containing fetal bovine serum. The marrow preparation was pelleted by centrifugation and passed 

through a cellulose column to isolate the erythrocytes (Romagna and Staniforth, 1989). The separated 

erythrocytes were cytocentrifuged onto slides and fixed immediately in ethanol. Slides were immersed in 

phosphate-buffered saline and processed for indirect immunofluorescence as described by Gudi et al. 

(1990). Briefly, slides were overlaid with antikinetochore-specific antibodies, rinsed in phosphate-buffered 

saline and stained with a fluorescent antibody tag (fluorescein isothiocyanate-conjugatedgoat anti-human 

IgG). Counterstaining with propidium iodide was performed to allow visualization of the micronuclei. This 

procedure allows differentiation of the PCE and NCE populations as well as simultaneous visualization of 

micronuclei and kinetochores. PCEs appear pale red and NCEs appear light green. Kinetochores are bright 

yellow dots in redstained micronuclei. 

Two thousand PCEs were scored for frequency of micronucleated cells in each of five mice per dose group. 

Data were analyzed as described previously. 

WIESrnTS 

Salicylazosulfapyridine (33-6,666 pg/plate) was not mutagenic inS. typhimurium strains TA97, TA98, 

TA100, or TA1535 when tested in a preincubation protocol with and without induced rat or hamster liver 

S9 metabolic activation enzymes (Table El). It did not induce SCEs (Table E2) or Abs (Table E3) in 

cultured Chinese hamster ovary cells, with or without induced rat liver S9 (Bishop et al., 1990). In contrast 

to the negative results obtained in vitro, results from in vivo genotoxicity tests with salicylazosulfapyridine 

yielded generally positive results. 
262 Salicylazosulfapyridine,NTP TR 457 

Salicylazosulfapyridine gave a strongly positive response in a micronucleus assay conducted with peripheral 

blood smears obtained from mice used in the 13-week toxicity study (Table E4; Bishop et al;, 1990). 

Significant increases in the frequency of micronucleated PCEs were observed in male and female mice 

administered 2,700 mg salicylazosulfapyridine/kgbody weight. Significantly elevated micronucleus 

frequencies. were also observed inNCEs in male and female mice administered675, 1,350, or 

2,700 mg/kg. .A dose-related increase in the percentage of PCEs was also observed in all dosed groups of 

males and females, indicating that erythropoiesis was stimulated in animals treated with 

sa1.icylazosulfapyridine. 

Salicylazosulfapyridine was subsequently tested for induction 

.. 

of Abs in bone marrow .. cells of male mice 

following single gavage administrations of 250 to 1,000 mg/kg (Table E5; B shop et al., 1990). These 

doses, selected on the basis of results from a preliminary range-finding study, were less than all but the 

lowest dose tested in the 13-week study. No significant increases in Abs were noted in the two trials 

conducted. 

Subsequent micronucleus tests with salicylazosulfapyridine confirmed the positive response observed in the 

initial 13-week peripheral blood test. A small but significant increase inPCEs was observed in the bone 

marrow of male mice administered 250 to 2,000 mg salicylazosulfapyridine/kgbody weight in corn oil by 

gavage, twice at 24-hour intervals; samplingwas performed 24 hours after administration of the second 

dose (Table E6; Bishop et al., 1990). 

A specialized micronucleus test was performed with salicylazosulfapyridineto investigate the aneuploidyinducing potential of the compound, implied by the positive results from micronuclei assays (which detect 

both numerical and structural chromosomal aberrations) and the negative results from thein vitro and 

in vivo Abs assays (which detect only structural damage). In this micronucleus/kinetochore-stainingassay 

(Table E7; Witt et al., 1992b), salicylazosulfapyridine induced dose-related increases in the number of total 

micronucleated PCEs; in addition, the micronuclei that were induced were predominantly kinetochorepositive (contained a fluorescent signal indicating the presenceof a kinetochore and presumably, therefore, 

an entire chromosome rather than a fragment). Salicylazosulfapyridine also induced a significant increase in 

kinetochore-negative micronuclei, but only at the highest dose tested in the secondtrial; the majority of 

induced micronuclei stained positive for the presence of a kinetochore. These results imply that 

salicylazosulfapyridine produces aneuploidogenic rather than clastogenic chromosomal effects. 

Because positive results were observed in micronucleus experiments that employed multiple dosing 

protocols (standard for a micronucleus assay), and because results from the single-dose chromosomal 

aberrations protocol were negative, salicylazosulfapyridine was tested for induction of micronuclei and 

chromosomal aberrations in nonstandard protocols that were designed to measure the influence of treatment 

schedules. Table E8 presents results from a series of bone marrow micronucleus tests performed with male 

and female mice. Significant increases in micronucleatedPCEs were observed in each of two trials in 

which female mice were dosed with salicylazosulfapyridine three times by gavage. Male mice were 

administered salicylazosulfapyridine either by single (Trial 1) or triple (Trial 2) gavage protocols. Results 

of the first experiment were negative, whereas results obtained in the second trail using the triple-treatment 

protocol were positive, thus underscoring the relationship between dosing schedule and detection of a 

genotoxic response. 

Salicylazosulfapyridine was further testedfor induction of chromosomal aberrations in a nonstandard, triple 

treatment protocol that used three gavage administrations separated by 24-hour intervals in an attempt to 

mimic the same exposure environment that produced the positive micronucleus test results. The highest 

dose used in this experiment was 4,000 mg/kg. Again, no induction-of Abs by salicylazosulfapyridine was 
noted in mouse bone marrow cells (TableE9). These results further support the hypothesis that 

salicylazosulfapyridine is aneuploidogenic. 

Because the carcinogenicity of salicylazosulfapyridine was being evaluatedby the NTP in both rats and 

mice, a micronucleus test was also performed in male rats using the triple-treatment protocol (Table E10). 

The overall results were consideredto be equivocal because the weakly positive response obtained in the 

first trial with doses as great as2,700 mg/kg did not repeatin the second trial when doses as great as 

3,000mgkg were tested. Thus, there appears to be a species difference in response to bone marrow 

erythrocyte micronucleus induction bysalicylazosulfapyridine. 

. . 
264 Salicylazosulfapyridine, NTP TR 457 

TABLEEl . % . Mutagenicity of Salicylazosulfapyridine inSalmonella @phimunuma % '

Revertants/plateb 

-s9 - +ratS9' ', 

10% 30% 10% 30% 

TA100 0 94 f 6.7 96 f 4.6 100 f 8.7 124 f 9.0 132 f 4.7 

33 105 f 10.4 105 f 2.0 

100 101 f 4.8 97 f 4.8 102 f 2.4 118 f 6.6 96 f 3.2 

333 78 f 1.8 94 f 6.4 107 f 4.7 101 f 8.5 113 f 8.5 

1,Ooo 93 f 1.2 101 f 4.4 117 f 9.5 137 f 8.7 116 f 5.9 

3,333 87 f 3.8 94 f 4.3' 103 f 13.8 101 f 3.8' 117 f 5.3 

6,666 84 f 4.4' 102 f 7.0' 102 f 7.0' 

Trial summary Negative Negative Negative Negative Negative 

Positive controld 1,276 f 14.0 1,264 f 64.3 2,153 f 47.9 1,038 f 35.8 787 f 16.7 

TA1535 0 13 f 1.2 14 f 0.9 11 f 2.8 14 f 1.5 14 f 0.6 

33 10 f 2.4 11 f 1.7 

100 16 f 1.2 13 f 1.5 14 f 4.1 15 f 1.2 13 f 2.5 

333 15 f 2.0 9 f 0.7 15 f 2.6 10 f 2.0 12 f 0.9 

1,Ooo 19 f 3.2 12 f 2.0 11 f 0.7 10 f 0.0 14 f 1.7 

3,333 16 f 0.7 12 f 0.3' 11 f 3.2 11 f 1.9' 12 f 1.5 

6,666 17 f 1.9' 17 f 2.1' 17 f 0.9' 

Trial summary Negative Negative Negative Negative . Negative 

Positive control 1,063 f 12.5 153 f 9.5 461 f 16.3 117 f 11.7 165 f 4.4 

TAW 0 89 f 7.0 138 f 3.8 144 f 6.1 148 f 9.5 138 f 16.2 

33 144 f 12.2 154 f 3.6 

100 88 f 3.8 143 f 9.8 146 f 11.9 119 f 7.0 140 f 12.5 

333 85 f 6.0 ' 129 f 4.7 130 f 2.1 151 f 3.0 158 f 3.8 

1,Ooo 83 f 1.5 149 f 4.4 134 f 4.9 140 f 3.4 163 f 1.5 

3,333 86 f 5.5 113 f 10.8' 140 f 4.2 111 f 7.2' 143 f 7.8 

6,666 83 f 2.8' , 91 f 4.4' 109 f 6.5' 

" 

' 

Trial summary Negative Negative Negative Negative Negative 

Positive control 542 f 20.5 904 f 14.5 1,039 f 17.2 736 f 29.6 489 f 24.3 

TA98 0 25 f 2.0 39 f 3.8 32 f 1.2 33 f 3.5 31 + 3.2 

33 28 f 6.1 31 f 5.2 

100 27 f 3.6 35 f 1.5 28 f 2.0 38 f 2.6 30 f 1.5 

333 29 f 2.9 35 f 3.4 34 f 4.7 43 f 1.5 25 f.3.9 

1,OoO 24 f 3.4 40 f 1.7 37 f 0.7 44 f 1.8 31 f 3.0 

3,333 24 f 2.0 21 f 1.'5' 29 f 3.1 29 f 3.2 17 f 0.9 

6,666 10 f 1.9 17 f 1.2' 21 f 1.3' 

Trial summary Negative . Negative Negative Negative Negative 

Positive control 1,292 f 65.1 1,720 f 32.0 2,086 f 18.8 1.210 f 11.3 699 f 8.4 

Shldy performed at Microbiological Associates, Inc. The detailed protocol and these data are presented in Zeiger ef al. (1988). 

Revertants are presented as mean f standard error from three plates. 

Slight toxicity 

The ~ ~~ wsitive controls in the absence of metabolic activation were sodium azide (TA100 and TA1535). 9-aminoacridine (TAW), and r ~~ 

4-Ntro-o-phenylenediamine(TA98). The positive control for metabolic activation with all Strains was 2-aminOanthraCene. 
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266 Salicylazosulfapyridine, NTP TR 457 

TABLEE3 

Induction of Chromosomal Aberrations in Chinese Hamster Ovary Cells bySalicylazosulfapyridinea 

-

DoseTotal 

(Ccg/rnL) Cells 

-

No. of 

Abs 

Absl 

Cell 

Cellswith 

Abs (%) (Icg/mL) 

Dose 

Cells 

Total 

Abs 

+s9 

No. of 

Cell. 

Absl 

Abs (%) 

Cellswith 

Summary: Negative 

Harvesttime: 12.0 hours 

Summary:Negative 

Harvesttime: 13.0hours 

Dimethylsulfoxide Dimethylsulfoxide 

0.01 20 1 0.5 200 4 0.02 1.5 

Mitomycin-C 

0.125 200 54 0.27 21.5 

Cyclophosphamide 

5.0 200 29 0.15 13.5 

0.250 50 33 0.66 34.0 7.5 50 23 0.46 42.0 

Salicylazosulfapyridine Salicylazosulfapyridine 

300 200 0 0.00 0.0 300 200 1 0.01 0.5 

500 200 1 0.01 0.5 500 200 1 0.01 0.5 

1,ooo 200 2 0.01 1.o 1,ooo 200 0 0.00 0.0 

P=0.182b P=O.%5 

a Study performed at Environmental Health Researchand Testing, Inc. The detailed protocoland these data are presented in Bishop et al. 

(1990). Abs=aberrations. 

Significance of percent cells with aberrationstested by the linear regression trend test versus log of the dose 
267 

Male 

Salicylazosulfapyridine 

Ob 1.71 f 0.32 1.07 f 0.08 2.33 f 0.14 10 

675 2.27 f 0.42 2.46 f 0.17** 3.94 f 0.16** 10 

1,350 3.42 f 0.45 2.94 f 0.22" 4.35 f 0.21** 10 

2,700 3.66 f 0.41** 2.78 f 0.20" 5.23 f 0.24** 10 

P=O.001C P<O.OOld P<0.001e 

Salicylazosulfapyridine 

0 1.45 f 0.21 0.86 f 0.01 1.62 f 0.15 10 

675 2.30 f 0.41 1.93 f 0.18** 2.51 f 0.20" 10 

1,350 2.48 f 0.54 2.24 f 0.19" 2.76 f 0.17". 10 

2,700 2.72 f 0.36* 2.04 f 0.129' 3.14 f 0.28'" 10 

P=O.O315 P <0.001 P<O.o001 

* P=O.O26 

'* PiO.01 

a Study performed at USDA, Western Regional Research Center. The detailed protocol and these data are presented in Bishop et al. (1990). 

PCE=polychromatic erythrocyte; NCE=normochromatic erythrocyte. At least 2,o00 PCEs and 10.00 NCEs were scored for 

micronuclei per animal. Data are presented as mean f standard error. 

Corn oil (solvent) control 

Cochran-Armitage trend test 

Linear contrasts from analysis of variance 

e Analysis of variance 
268 Salicylazosulfapyridine, NTP TR 457 

TABLEE5 .: 

InductionofChromosomalAberrationsinBoneMarrowCellsofMaleMiceTreatedOnce . ' 

with Salicylazosulfapyridine by Gavage: Standard Protocola 

. . Dose Total ' ' AbslCell Cells with 

Compound (Wkg) Abs ' (withoutgaps) Absb (%) 

(without gaps) 

-

Trial 1 .. 

Corn oilc 0.003 0.250 f 0.25 

Dimethylbenz(a)anthracened 100 0.030 2.750 f 0.92 

Salicylazosulfapyridine 250 0.008 0.750 f 0.37 

. . 

500 0.018 1.750 f, 1.49 

1 ,Ooo 0 0.00 f 0.00 

P=0.637e 

Trial 2 

Corn oil 5 0.013 1.250 f 0.53 . . 

Dimethylbenz(a)anthracene 100 24 0.060,,' , 5.000.f 1.51.. 

, *. 

,,' 

Salicylazosulfapyridine 

1*Ooo 

500 

250 

0 

3 

4 0.010 

0 

0.008 

1.000 f 0.65 

0.750 f 0.37 

0.00 f 0.00 , 

, . 

P=O.984 

a Study performed at Environmental Health Research and Testing, Inc. The detailed protocol and these data are presented in Bishop e?ul. 

(1990). Fifty firstdivision metaphase cells were scoredin each of eight animals in each dose group. Abs=chromosomal aberrations. 

Data are presented as mean f standard error. 

Solvent control 

Positive control 

e One-tailed trend analysis (Margolin et nl., 1986);significant at P<0.025 
269 

Dose (mgkg) Micronucleated Cells/n.W Mice per Dose 

Trial B Trial 2 Trial 3 

corn oilb 1.4 f 0.3 1.1 f 0.4 0.6 f 0.2 

Dimethylbenz(a)anthracene' 

12.5 1.8 f 0.2 

100 29.5 f 4.4 34.5 f 4.0 33.3 f 4.9 

Salicylazosulfapyridine 

250 2.1 f 0.7 1.2 f 0.3 

500 2.2 f 1.1 2.0 f 0.8 

2.9 f 0.8 2.0 f 0.6 1.8 f 0.2' 

2900 1.8 f 0.6' 

1.00 

P=0.770d P=O.Ol P=.O32

Q Significantly different (PrO.01) from the solvent control a Study performed at Environmental Health Research and Testing, Inc. The protocol and these data are presented in Bishop et al. (1990). 

PCE=polychromatic erythrocyte: 2.00 PCEs were scored in each of five animals in each dose group. Data are presented as 

mean f standard error. 

Solvent control 

Positive control 

Significance of micronucleated cells11.000 PCEs tested by the one-tailed trend test; significant atPr0.025(Margolin et al., 1990). 
270 Salicylazosulfapyridine,NTP TR 457 

TABLEE7 

Frequency of Kinetochore Positive and Kinetochore Negative Micronuclei in Polychromatic Bone Marrow 

Cells of Male Mice Treated Three Times with Salicylazosulfapyridine by Gavagea 

Compound Dose 

(mg/kg) 

Trial 1 

Triethylenemelamineb 

1.o 

Vincristine sulfate' 

0.125 

Salicylazosulfapyridine 

Od 

1.875 

2,721 

3,649 

Trial 2 

Triethylenemelamine 

1.o 

Vincristine sulfate' 

Total 

100.2 f 4.94 

83.0 f 2.91 

1.6 f 0.19 

4.5 ,f0.52 

5.3 f 0.34 

6.3 f 0.68 

p<0.001e 

63.1 f 6.54 

Kinetochore 

Negative 

96.3 f 4.68 

6.7 f 0.94 

1.6 f 0.19 

2.2 f 0.20 

2.8 f 0.34 

2.1 f 0.29 

P=O.122 

54.0 f 4.16 

Kinetochore 

Positive 

3.9 f 0.58 

76.3 f 3.00 

0.0 

2.3 f 0.44 

2.5 f 0.27 

4.2 f 0.58 

P<O.001 

. ,_ : 

9.1 f 2.63 j r 

0.125 79.9 f 6.67 

Salicylazosulfapyridine 

1.243.9 76.0 f 5.62 

0 1.1 f 0.29 0.8 f 0.26 0.3 f 0.12

1.289 7.2 f 0.20 1.3 f 0.26 5.9 f..O.33 

2,797 9.1 f 0.19 1.3 f 0.26 7.8 f 0.26 

5,634 10.7 f 0.15 2.7 f 0.60 8.0 & 0.22 . 

P<O.001 P<O.005 P<O.001 

a Study performed at SITEK, Inc. The detailed protocol and these data are presented by Win et nl. (1992b). PCE-polychromatic 

erythrocyte;2,OOO PCEs were scored in each of five animals in each dose group. Data are presented as mean f standard error. 

Clastogenic positive control ' Aneuploidogenic positive control 

corn oil (solvent) control 

e Trendtest 
Mice per Dose 

Tdd 2 

"e 

Dimethylbenz(a)anthracene' 

12.5 3.8 f 0.588 

1 X Gavageb 

5.3 f 0.82* 

3 X Gavage 

5 ,. 

Salicylazosulfapyridine 

Od 0.9 f 0.29 1.5 f 0.42 5 

1,ooo 0.3 f 0.12 4.3 f 0.62' 5 

2.W 0.9 f 0.29 4.7 f 0.70' 5 

4,ooo 0.8 f 0.26 3.9 f 0.33' 5 

P=0.38ge P=O.O10 

Female 

3 X Gavage 3 X Gavage 

Dimethylbenz(a)anthrene 

12.5 3.8 f 0.77' 1.5 f 0.5Sf 5 

Salicylazosulfapyridine 

0 0.4 f 0.29 1.0 f 0.27 

5.4 f 0.80' 5.2 f 0.54' 

6.7 f 0.588 7.5 f 0.988 

4.8 f 0.78 5.7 f 0.96' 

P<O.Ol P<O.ool 

* Significantly different(P<O.Ol) from the solvent control a Study performed at Environmental Health Research and Testing, Inc. The protocolis presented in Shelby et al. (1993). 

PCE=polychromaric erythrocyte;2.W PCEs were scored in each of five animals in each dose group. Data are presented as 

mean f standard error. 

This trial was performed using a single gavage administration of salicylazosulfapyridine insteadof the triple treatment protocol used in the 

other trials presented in this table. 

Positive control 

Corn oil (solvent) control 

e Significance of micronucleated cells/l,C00 PCEs tested by the one-tailed trend test (Margolin et nf., 1990). 

Positive control failure; experiment consideredvalid due to response registered in the treated groups. 
;!72 Salicylazosulfapyridine,NTP TR 457 

‘FABLE E9 . . Induction of Chromosomal Aberrations in Bone MarrowCellsof Male Mice Treated 

with Salicylazosulfapyridine by Gavage: Nonstandard Protocola 

Compound 

Trial 1 

I)imethylbenz(a)anthraceneC 

Salicylazosulfapyridined 

Trial 2 

Dimethylbenz(a)anthracene 

SaIicyIazosuIfapyridineg 

* 

Dose CellsAbdCell Total Abs .. 

(m@g) (without gaps) 

150 12.5 0.09 

Oe 0.8 0.00 

1,Ooo 1.8 0.01 

2,Ooo 1.9 0.01 

4,Ooo 1.1 0.01 

100 18.8 0.17 

0 1.3 0.00 

1,Ooo 1.1 0.00 

2,Ooo 0.3 0.00 

4,Ooo 0.8 0.00 

Significantly different(P<O.001) from the solvent control 

(without gaps) 

Absb(%) 

with 

8.00 f 1.51* 

0.75 f 0.37 

1.00 f0.53 

1.25 f 0.53 

0.50 f 0.33 

, 

P=0.4Mf 

15.25 f 3.14* 

0.00 f 0.00 

0.25 f 0.23 

0.50 f 0.33 

0.50 f 0.33 

P=O.925 

. . 

.. 

a Study performed at Environmental Health Research and Testing, Inc. Fifty first-division metaphase cells were scored in each of eight 

animals in each dose group. Abs=chromosomal aberrations. 

Data are presented as mean f standard error. 

Positive control 

Results from singledose gavage treatment 

e Corn oil(solvent)control 

Trend test 

Results from tripledose gavage treatment 
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276 Salicylazosulfapyridine,NTP TR 457 

TABLEF1 

Organ Weights and Organ-Weight-to-Body-Weight Ratios forRats in the 16-Day Gavage Study 

of Salicylazosulfapyridinea -

n 

Male 

Necropsy body wt 

Brain 

Absolute 

Relative 

Heart 

Absolute 

Relative 

R.Kidney 

Absolute 

Relative 

Liver 

Absolute 

Relative 

Lung 

Absolute 

Relative 

R. Testis 

Absolute 

Relative 

Thymus 

Absolute 

Relative 

Female 

Necropsy body wt 

Brain 

Absolute 

Relative 

Heart 

Absolute 

Relative 

R. Kidney 

Absolute 

Relative 

Liver 

Absolute 

Relative 

Lung

Absolute 

Relative 

Thymus 

Absolute 

Relative 

Vehicle Control 

5 

215 f 5 

1.803 f 0.016 

8.41 f 0.14 

0.718f 0.011 

3.35 f 0.04 

0.787f 0.025 

3.67f 0.09 

10.067f 0.282 

46.87f 0.81 

1.181 f 0.108 

5.48 f 0.43 

1.217f 0.028 

5.67 f 0.13. 

0.455 f 0.027 

2.12 f 0.10 

147 f 3 

1.714f 0.031 

11.64f 0.17 

0.535 f 0.025 

3.62f 0.13 

0.533 f 0.013 

3.62f 0.06 

5.573 f 0.051 

37.87f 0.75 

0.936f 0.023b 

6.42f 0.17b 

0.362f 0.013 

2.45 f 0.06 

675 mgkg 

5 

204 f 4 

1.770f 0.022 

8.69f 0.20 

0.708f 0.025 

3.47f 0.12 

0.762f 0:030’ 

3.73f 0.10 

8.653f 0.218** 

42.39f 0.41 

1.158 f 0.050 

5.68 f 0.25 

1.182f 0.036’ 

5.79f 0.11 

0.404f 0.020 

1.98 f 0.09 

133 f 2** 

l.’& f 0.025 

12.58f 0.25* 

0.491f 0.016 

3.71f 0.09 

0.547f 0.009 

4.13 f 0.08** 

5.182f 0.072 

39.14f 0.66 

0.823 f 0.025** 

6.23f 0.28 

0.306 f 0.019 

2.32f 0.16 

I 

1,350 mgkg 

5 

160 f 4**’ 

1.715 f 0.015** 

10.76f 0.25** 

0.572f 0.017** 

3.59 f 0.10 : 

0.691 f 0.030* 

4.33 f 0.20** 

6.832f 0.303** 

42.93 f 2.33 , 

0.930f 0.030* 

5.83f 0.17 

1.076f 0.033** 

6.75f 0.25** 

0.236f 0.017** 

1.47 f 0.09** 

129 f 4** 

1.638f 0.037 

12.73 f 0.15** 

0.494f 0.026 

3.84f 0.18 

0.543 f 0.011 

4.22f 0.10** 

5.510 f 0.320 

42.73 f 1.72* 

0.863 f 0.022** 

6.72f 0.19 

0.269 f 0.023** 

2.08 f 0.16 

. 

2,700 mg/kg 

5 

147 f 4** 

1.680f 0.022** 

11.49f 0.28** 

0.510 f 0.014** 

3.48 f 0.06 

0.637f o.oio** 

4.35 f 0.08** 

7.298f 0.254** 

49.78f 1.14 

0.921 f 0.056* 

6.28& 0.33 

1.028f‘0.@7** . 7.01 f 0.03** 

, 

-0.133f 0.004** 

0.91 f 0.03** : 

, . 

111 f 3** 

1.584f 0.015** 

14.36 f 0.35** .) 

0.443 f 0.033 

4.00f 0.23 

0.539f 0.018 

4.88 f 0.12** 

6,019f 0.277 

54.34f 1.51** 

0.755 f 0.009** 

6.84f 0.14 

0.132 f 0.020** 

1.21 f 0.21** 

* Significantly different(PsO.05) from the control group by Williams’ or Dunnett’s test 

** PsO.01 

a Organ weights (absolute weights) and.body weights are given in grams; organ-weight-to-body-weight ratios (relative weights) are given as 

mg organ weightlg body weight (mean f standard error). 

n=4 
Vehicle Control 84 mglkg 868.8 mgkg 337.5 mglkg 

n 10 10 10 10 

Male 

Necropsy body wt 357 f 7 344 f 4 345 f 5 341 f 6 

Brain 

Absolute 1.968 f 0.014 1.952 f 0.015 1.964 f 0.015 1.941 f 0.020 

Relative 5.53 f 0.10 5.68 f 0.08 5.71 f 0.11 5.70 f 0.09 

Heart 

Absolute 0.994 f 0.016 1.004 f 0.008 1.005 f 0.017 1.022 f 0.027 

Relative 2.79 f 0.04 2.92 f 0.04 2.92 f 0.05 3.00 f 0.07* 

R. Kidney 

Absolute 1.075 f 0.023 1.073 f 0.025 1.094 f 0.027 1.089 f 0.028 

Relative 3.01 f 0.03 3.12 f 0.07 3.18 f 0.07 3.19 f 0.07 

Liver 

Absolute 13.779 f 0.247 13.266 f 0.272 13.389 f 0.313 12.875 f 0.267 

Relative 38.66 f 0.48 38.57 f 0.73 38.84 f 0.63 37.73 f 0.51 

LungAbsolute 1.692 f 0.043 1.617 f 0.047 1.733 f 0.057 1.647 f 0.038 

Relative 4.74 f 0.08 4.70 f 0.11 5.02 f 0.10 4.83 f 0.12 

R. Testis 

Absolute 1.485 f 0.028 1.505 f 0.016 1.501 f0.018 1.467 f 0.035 

Relative 4.17 f 0.06 4.38 f 0.06 4.36 f 0.10 4.30 f 0.10 

Thymus 

Absolute 0.323 f 0.016 0.283 f 0.013 0.291 f 0.012 0.286 f 0.024b 

Relative 0.91 f 0.05 0.82 f 0.04 0.84 f 0.03 0.84 f 0.07b 

Female 

Necropsy body wt 199 f 3 190 f 2** 189 f 2** 188 f 2** 

Brain 

Absolute 1,817 f 0.020 1.792 f 0.019 1.763 f 0.015 1.814 f0.017 

Relative 9.14 f 0.11 9.44 f 0.11 9.37 f 0.14 9.67 f 0.15* 

Heart 

Absolute 0.660 f 0.014 0.643 f0.011 0.639 f 0.011 0.661 f 0.014 

Relative 3.32 f 0.05 3.39 f 0.06 3.39 f 0.06 3.52 f 0.07 

R. Kidney 

Absolute 0.616 f 0.010 0.621 f 0.009 0.605 f 0.009 0.660 f 0.011** 

Relative 3.09 f 0.03 3.27 f 0.04* 3.21 f 0.05* 3.52 f 0.04** 

Liver 

Absolute 6.134 f 0.151 6.141 f 0.135 6.173 f 0.165 6.467 f 0.176 

Relative 30.81 f 0.55 32.32 f 0.54 32.72 f 0.68* 34.41 f 0.73** 

Lung

Absolute 1.178 f 0.046 1.190 f 0.037 1.188 f 0.024 1.147 f 0.036 

Relative 5.91 f 0.20 6.26 f 0.16 6.30 f 0.11 6.11 f 0.21 

Thymus 

Absolute 0.241 f 0.008 0.253 f 0.011 0.238 f 0.011 0.252 f 0.008 

Relative 1.21 f 0.04 1.33 f 0.06 1.26 f 0.05 1.34 f 0.04 

* Significantly different (PsO.05)from the control group by Williams' or Dunnett's test 

** PsO.01 

a Organ weights (absolute weights) and body weights are given in grams; organ-weight-to-body-weight ratios (relative weights) are given as 

mg organ weight/g body weight (mean f standard error). 

n=9 
278 Salicylazosulfapyridine,NTP TR 457 

TABLEF3 

Organ Weights and Organ-Weight-to-Body-Weight Ratios for Male Rats 

at the 6-Month Interim Evaluation in the Stop-Exposure Evaluationof Salicylazosulfapyridinea 

-

n 

Necropsy body wt 

R.Kidney 

Absolute 

Relative 

Liver 

Absolute 

Relative 

Spleen 

Absolute 

Relative \ 

R. Testis 

Absolute 

Relative 

Thyroid Gland 

Absolute 

Relative 

Vehicle Control 

10 

3% f 9 

1.335 f 0.026 

3.37 f 0.05 

14.775 f 0.518 

37.26 f 0.88 

0.676 f 0.022 

1.7l.f 0.05 

1.442 f 0.023 

3.65 f 0.09 

0.012 f 0.001 

0.03 f 0.00 

337.5 mgkg 

10 

382 f 9 

1.339 f 0.038 

3.50 f 0.06 

14.165 f 0.461 

37.04 f 0.71 

0.656 f 0.018 

1.72 f 0.02 

1.449 f 0.026 

3.80 f 0.06 

0.013 f 0.001 

0.03 f 0.00 

a Organ weights (absolute weights)and body weights are given in grams; organ-weight-to-body-weight ratios (relative weights) are given as 

m g organ weight/g body weight (mean f standard error). Differences from the vehicle control we,re not significant by Williams’ or 

Dunnett’s test. 
"@ 

n 10 10 10 10 10 

Necropsy body wt 491 f 8 478 f 10 485 f 8 459 f 8" 488 f 11 

R. Kidney

Absolute 1.639 f 0.061 1.556 f 0.060 1.586 f 0.035 1.590 f 0.029 1.563 f 0.028 

Relative 3.33 f 0.10 3.25 f 0.10 3.27 f 0.07 3.47 f 0.05 3.21 f 0.07 

Liver 

Absolute 16.660 f 0.544 15.488 f 0.483 15.895 f 0.399 15.713 f 0.370 15.125 f 0.421" 

Relative 33.89 f 0.92 32.42 f 0.84 32.76 f 0.57 34.24 f 0.69 30.97 f 0.408' 

Spleen 

Absolute 0.803 f 0.026 0.787 f 0.033 0.739 f 0.031 0.727 f 0.015 0.747 f 0.022b 

Relative 1.63 f 0.04 1.65 f 0.07 1.52 f 0.06 1.58 f 0.02 1.52 f 0.CQ3 

L. Testis 

Absolute 1.641 f 0.055 1.660 f 0.068 1.794 f 0.131 1.842 f 0.167 1.690 f 0.035 

Relative 3.34 f 0.08 3.47 f 0.11 3.71 f 0.28 4.01 f 0.35 3.47 f 0.09 

R. Testis 

Absolute 1.559 f 0.040 1.634 f 0.072 1.739 f 0.177 1.743 f 0.157 1.750 f 0.207 

Relative 3.17 f 0.06 3.43 f 0.17 3.58 f 0.35 3.79 f 0.32 3.64 f 0.50 

Thyroid Gland 

Absolutk 0.025 f 0.003 0.018 f 0.003 0.016 f 0.001' 0.022 f 0.002 0.020 f 0.002 

Relative 0.05 f 0.01 0.04 f 0.00 0.03 f 0.00' 0.05 f 0.00 0.04 f 0.00 

Female 

Necropsy body wt 265 f 4 261 f 4 253 f 4 237 f 8"' 

R.Kidney 

Absolute 0.800 f 0.017 0.839 f 0.021 0.829 f 0.019 0.908 f 0.038" 

Relative 3.02 f 0.07 3.22 f 0.06" 3.28 f 0.05' 3.83 f 0.08" 

Liver 

Absolute 7.986 f 0.188 8.121 f 0.136 8.132 f 0.352 8.023 f 0.300 

Relative 30.12 f 0.62 31.17 f 0.50 32.10 f 1.23 33.92 f 0.98'' 

Spleen 

Absolute 0.467 f O.Wb 0.507 f 0.041 0.454 f 0.01 lb 0.431 f O.OISb 

Relative 1.78 f 0.03b 1.95 f 0.17 1.79 f 0.03b 1.81 f O.07b 

Thyroid Gland 

Absolute 0.016 f 0.001 0.014 f 0.001 0.014 f 0.001 0.014 f 0.001 

Relative 0.06 f 0.00 0.05 f 0.00 0.06 f 0.00 0.06 f 0.00 

* Significantly different (Pr0.05) from the control group by Williams' or Dunnett's test "PiO.01 

a Organ weights (absolute weights) and body weights are given in grams; organ-weight-to-body-weight ratios (relative weights) are given as 

mg organ weight/g body weight (mean f standard error). 

n=9 
280 Salicylazosulfapjlidine, NTP TR 457 

TABLEF5 

Organ Weights and Organ-Weight-to-Body-Weight Ratiosfor Mice in the 16-Day Gavage Study 

of Salicylazosulfapyridinea 

Male 

n 

Necropsybodywt ' 

Brain 

Absolute 

Relative 

Heart 

Absolute 

Relative 

R.Kidney 

Absolute 

Relative 

Liver 

Absolute 

Relative -

Lung 

. Absolute 

Relative .. 

R. Testis 

Absolute 

Relative , 

Thymus 

Absolute 

Relative , 

Female 

n 

Necropsy body wt 

Brain 

Absolute 

Relative 

Heart 

Absolute 

Relative 

R. Kidney 

Absolute 

Relative 

Liver * 

Absolute 

Relative 

Lung 

Absolute 

Relative 

Thymus . 

Absolute 

Relative 

* 

VehicleControl 675 mgkg 1,350 mg/kg 

5 5 5 

23.6 f 0.4 23.0f 0.4 24.2f 0.5 

0.455 f 0.004 0.452 f 0.004' 0.464f 0.008 

19.27f 0.33 19.62 f 0.20 ' 19.19 f 0.34 

0.122 f 0.002 0.117 f 0.004 0.136 f 0.003* 

5.19 f 0.07 5.10f 0.15 5.61 f 0.07 

0.211 f 0.012 0.205f 0.008 0.217f 0.004 

8.93 f 0.37 8.92f 0.27 .8.97f 0.08 

1.263 f 0.043 1.254f 0.048 1.378f 0.064 

'53.45f 1.19 54.41 f 1.42 56.91 f 2.24 

0.208f 0.015 0.204 f 0.017 0.219f 0.006 

8.79f 0.63 8.83f 0.67 9.06f 0.27 

0.099 f 0.003 0.091 f 0.006 0.099 f 0.003 

' 4.18f 0.13 3.96 f 0.21 4.09 f 0.12 

0.048 f 0.006 0.046 f 0.002 0.049 f 0.006 

2.01 f 0.24 1.99 f 0.10 2.03 f 0.21 

5 4 5 

19.6 f 0.3 20.8f 0.2 20.1 f 0.3 

0.458 f 0.010 0.456 f 0.008 0.459 f 0.006 

23.43 f 0.72 21.95 f 0.49 22.80 f 0.40 

0.108f 0.005 0.105f 0.002 0.114f 0.004 

5.52f 0.30 5.05 f 0.11 5.64f 0.15 

0.159 f 0.003 0.157 f 0.002 0.157 f 0.004 

8.12 f 0.25 7.57 f 0.16 7.78 f 0.14 

1.060f 0.040 1.194f 0.027 1.154f 0.030 

54.17f 2.15 57.46f 1.11 57.30 f 1.59 

0.184 f 0.007 0.210f 0.013 0.222 f 0.019 

9.40 f 0.50 10.09f 0.61 10.98f 0.88 

0.071 f 0.004 0.065 f 0.003 0.071 f 0.003 

3.61 f 0.22 3.14 f 0.15 3.55 f 0.19 

Significantly different(Pr0.05)from the control group by Dunnett's test 

2,700 mg/kg 

5 

24.1 f 0.4 

0.464f 0.004 

19.24f 0.46 ' 

0.122f 0.003 

5.04f 0.16 

0.201 f 0.006 

8.34f 0.18 ' 

1.395 f 0.058 

57.74 f 1.83 

0.201 f 0.005 

8.35 f 0.22 

0.100f 0.001 

4.16f 0.11 

0.042f 0.003 

1.75 f 0.12 

4 

20.3f 0.7 

0.450 f 0.007 

22.29f 0.82 

0.112f 0.005 

5.56 f 0.37 

0.153 f 0.003 

7.59f 0.27 

1.228 f 0.084 

60.43 f 2.43 

0.207 f 0.009 , 

10.22f 0.47 

0.063 f 0.003 

3.10 f 0.10 

a Organ weights (absolute weights) and body weights are given in grams; organ-weight-to-body-weight ratios (relative weights) are given as 

mg organ weightlg body weight (mean f standard error). 
60'0 T LI'Z 

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,282 Salicylazosulfapyridine,NTP TR 457 

‘rmm~7 

Organ Weights and Organ-Weight-to-Body-Weight RatiosFor Mice at the 15-Month Interim Evaluation 

,in the 2-YearGavage Study of Salicylazosulfapyridinea 

. . > . 

Vehicle Control 675 mgkg 1,350 mg/kg 2,700 m%kg 

n 10 10 10 10 

Male 

Necropsy body wt 53.9 f 1.1 52.8 f 1.5 54.8 f 1.5 44.9 f 1.3?* , 

R. Kidney 

. . 

Absolute 0.424 f 0.018 0.372 f 0.008* 0.397 f 0.017 0.359 f 0.011** 

Relative 7.87 f 0.30 7.09 f 0.21 7.26 f 0.26 8.05 f 0.30 

Liver 

Absolute 2.328 f 0.106 2.609 f 0.199 2.920 f 0.174* 2.305 f 0.062 

Relative 43.02 f 1.15 49.62 f 3.98 53.16 f 2.45* 51.55 f 1.19* 

Spleen 

Absolute 0.100 f 0.008. 0.103 f 0.008 0.117 f 0.005 0.119 f 0.006 

Relative 1.86 f 0.17 1.95 f 0.14 2.14 f 0.10 2.67 f 0.15** 

R. Testis 

Absolute 0.113 f O.kb 0.113 f 0.003 0.112 f 0.002 0.108 f 0.005 

Relative 2.11 f O.OSb 2.14 f 0.06 2.07 f 0.07 2.44 f 0.14 ’ 

Thyroid Gland 

Absolute 0.003 f O.OO0 0.004 f 0.m 0.004 f. 0.m 0.004 f 0.m 

Relative 0.06 f 0.01 0.07 f 0.01 0.07 f 0.01 0.08 f 0.01 

Female 

Necropsy body wt 53.0 f 2.1 51.9 f 2.3 48.1 f 2.0 42.8 f 1.4** 

R. Kidney 

Absolute 0.264 f 0.008 0.265 f 0.007 0.273 f 0.006 0.280 f 0.012 

Relative 5.02 f 0.19 5.19 f 0.25 5.76 f 0.29* 6.56 f 0.23** 

Liver 

Absolute 1.805 f 0.247 1.895 f 0.089 2.034 f 0.055 2.125 f 0.070 

Relative 33.68 f 3.19 36.60 f 0.88 42.62 f 1.40** 49.79 f 1.30** 

Spleen 

Absolute 0.102 f 0.010 0.114 f 0.003 0.164 f 0.008** 0.191 f 0.009** 

Relative 1.92 f 0.16 2.24 f 0.13 3.47 f 0.25** 4.48 f 0.20** 

Thyroid Gland 

Absolute 0.004 f 0.m 0.004 f 0.m 0.003 f O.oo0 0.003 f O.OO0 

Relative 0.08 f 0.01 0.07 f 0.01 0.07 f 0.01 0.08 f 0.01 

* Significantly different (PgO.05) from the control group by Williams’ or Dunnett’s test 

** PsO.01 

a Organ weights (absolute weights) and body weights are givenin grams; organ-weight-to-body-weight ratios (relative weights) are given as 

mg organ weightlg body weight (mean f standard error). 

n=9 

284 Salicylazosulfapyridine, NTP TR 457 

TABLEG1 

Clinical Chemistry Datafor Rats in the 16-Day Gavage Studyof Salicylazosulfapyridinea 

* . 

Vehicle Control mglkg 675 1,350mg/kg 2,700mg/kg

n 5 5 5 5 

Male 

Thyroid-stimulating hormone (nglmL) 401 f 38 398 f 35 567 f 44 2,919 f 351** 

Triiodothyronine (ngldL) 67 f 3 53 f 1 57 f 3 18 f 2** 

Thyroxine (pgldL) 2.7 f 0.3 2.2 f 0.2 1.3 f 0.1* 0.8 f 0.1** . 

Female .. 

Thyroid-stimulating hormone(nglmL) 595 f 240 393 f 17 685 f 96 2,852 f 836** 

Triiodothyronine (ngldL) 72 f 6 54 f 4 69 f 5 36 f 8* 

Thyroxine (pgldL) 1.8 f 0.0 1.4 f 0.1 1.5 f 0.2 1.0 f 0.1** 

* Significantly different (PsO.05)from the controlgroup by Dun” test 

** PsO.01 

a Mean f standard error. Statistical tests wereperformed on unroundeddata. 
“e 

n 

Hematology 

Hematocrit (46) 

Hemoglobin (g/dL) 

Erythrocytes (1061pL) 

Reticulocytes(1061pL) 

Mean cell volume(fL) 

Mean cell hemoglobin(pg) 

Mean cell hemoglobin 

concentration (gIdL) 

Leukocytes (103/pL) 

Segmented neutro hils (IdIpL)

Lymphocytes (10 PIpL) 

Monocytes (1dlpL) 

Eosinophils (1031pL) 

Methemoglobin (gIdL) 

Clinical Chemistry 

Urea nitrogen (mg/dL) 

Creatinine (mg/dL) 

Glucose (mg/dL) 

Total protein (g/dL)

Albumin (g/dL) 

Alanine aminotransferase (IUIL) 

Alkaline phosphatase (IUIL) 

Aspartate aminotransferase (IUIL) 

Thyroid-stimulating hormone (ngImL) 

Triiodothyronine (ngIdL) 

Thyroxine (pgIdL) 

Urinalysis 

Volume (mLI16 hr) 

Specific gravity 

10 

46.4 f 0.4 

16.2 f 0.1 

9.15 f 0.05 

0.10 f 0.0P 

50.7 f 0.3 

17.8 f 0.1 

35.0 f 0.2 

8.83 f 0.34 

1.15 f 0.12 

7.51 f 0.35 

0.12 f 0.03 

0.05 f 0.02 

0.80 f 0.08 

18.9 f 0.7 

0.55 f 0.03 

152 f 3 

6.7 f O.lb 

4.8 f 0.1 

92f8 

257 f 12 

153 f 16 

528 f 43 

Ef2 

3.6 f 0.1 

2.9 f 0.6 

1.043 f 0.004 

10 

46.5 f 0.3 

16.2 f 0.1 

9.10 f 0.08 

0.12 f 0.01 

51.1 f 0.3 

17.8 f 0.1 

34.9 f 0.1 

9.58 f 0.40 

1.26 f 0.17 

8.12 f 0.38 

0.14 f 0.02 

0.06 f 0.03 

0.90 f 0.11 

17.7 f 1.0 

0.60 f 0.04 

150 f 4 

6.9 f 0.2b 

5.0 f 0.2 

73 f 3 

249 f 11 

121 f 7 

543 f 33 

87 f 3 

3.2 f 0.2 

2.7 f 0.4 

1.045 f 0.003 

10 

46.8 f 0.3 

16.4 f 0.1 

9.07 f 0.05 

0.09 f 0.01 

51.6 f 0.3 

18.1 f 0.1* 

35.0 f 0.1 

9.47 f 0.27 

1.19 f 0.11 

8.13 f 0.34 

0.12 f 0.04 

0.04 f 0.02 

0.87 f 0.10 

18.4 f 1.0 

0.51 f 0.03 

156 f 4 

7.0 f 0.1 

4.9 f 0.1 

72 f 5 

251 f 12 

124 f 11 

597 f 26 

78 f 3** 

3.1 f 0.1** 

4.3 f 0.5 

1.040 f 0.004 

10 

46.8 f 0.3 

16.4 f 0.1 

9.01 f 0.07 

0.12 f 0.02 

52.0 f 0.2** 

18.2 f 0.2* 

35.0 f 0.3 

9.78 f 0.38 

1.17 f 0.11 

8.40 f 0.37 

0.14 f 0.04 

0.06 f 0.03 

0.95 f 0.09 

18.0 f 0.9 

0.53 f 0.02 

149 f 3 

7.1 f O.Zb 

5.0 f 0.1 

81 f 8 

266 f 9 

134 f 12 

789 f 64** 

72 f 4** 

2.6 f 0.2** 

3.7 f 0.5 

1.041 f 0.003 
286 Salicylazosulfapyridine,NTP TR 457 

'FABLE62 

]Hematology, Clinical Chemistry,and Urinalysis Datafor 'Ratsin the 13-Week Gavage Study 

of Salicylazosulfapyridine(continued) . 

~~ 

168.8 mg/kg 337.5 mg/kg 

10 10 

44.4 f 0.4 "44.3 f 0.5 

16.2 f 0.2 15.8 f 0.2 

8.01 f 0.09 7.98 f 0.07 

0.07 f 0.01 0.08 f 0.02' 

55.3 f 0.2 55.5 f 0.2 

20.3 f 0.4 19.9 f 0.1 .. 

36.6 f 0.5 35.8 f 0.3 

6.84 f 0.42 .. 7.12 f 0.41 

0.86 f 0.07 . ' 0.65 f 0.07 

5.86 f 0.41 6.30 f 0.39 

0.06 f 0.02 0.09 f 0.02 

0.04 f 0.02 0.08 f 0.03 

1.03 f 0.09 1.07 f 0.17 

. ~ I 

14.6 f 0.6 15.6 f 0.7 

0.40 f 0.02 0.45 f 0.05 

147 f 4 148 f 3 

6.6 f 0.1 6.5 f 0.1 

4.7 f 0.2 4.6 f 0.1 

50 f 4 58 f 5 

224 f 16 212 f 13 

95 f 7 109 f 6 

1,221 f 195 989 f 166 

72 f 4 69 f 2 

2.2 f 0.1 1.8 f 0.1** 

4.84 f 1.50 7.77 f 1.35 

1.028 f 0.004 1.026 f 0.005 

. 

. 

. 

' 

" 

Female 

n 

Hematology 

Hematocrit (X) 

Hemoglobin (gIdL) 

Elythrocytes(106/pL) 

lteticulocytes(IO~I~L) 

Mean cell volume (fL) 

Mean cell hemoglobin (pg) 

Mean cell hemoglobin 

concentration (gIdL) 

Leukocytes (103/pL) 

Segmented neutro hils (103/pL) ?p Lymphocytes (10 IpL) 

Monocytes (103/pL) 

~ ~ i ~ ~ m ~ (~o~I~L) n
 

Methemoglobin (gIdL) 

Clinical Chemistry 

Urea nitrogen (mgIdL) 

Creatinine (mgIdL) 

Glucose (mgIdL) 

Total protein (gIdL) 

Albe (gIdL) 

Alanine aminotransferase (IUIL) 

Alkaline phosphatase (IUIL) 

Aspartate aminotransferase (IUIL)

Thyroid-stimulatinghormone (ngImL) 

Triiodothyronine (ngIdL) 

Thyroxine (pgIdL) 

Urinalysis 

Volume (mL/16 hr) 

Specific gravity 

-

Vehicle Control 

10 

45.0 f 0.3 

16.2 f 0.2 

8.09 f 0.06 

0.08 f 0.02' 

55.8 f 0.2 

20.0 f 0.3 

3519 f 0.4 

6.83 f 0.17 

0.79 f 0.09 

5.92 f 0.17 

0.08 f 0.02 

0.03 f 0.01 

0.85 f 0.13 

14.3 f 0.9 

0.44 f 0.02 

148 f 2 

6.7 f 0.1 

4.9 f 0.1 

50 f 3 

246 f 13 

95 f 6 

941 f 187 

75 f 3 

2.3 f 0.1 

4.52 f 1.31 

1.029 f 0.004 

.84mgkg 

10 

44.4 f 0.6 

16.1 f 0.3 

7.98 f 0.12 

0.08 f 0.01' 

55.6 f 0.2 

20.2 f 0.3 

36.4 f 0.4 

7.35 f 0.31 

0.60 f 0.08' 

6.35 f 0.28' 

0.11 f 0.02' 

0.08 f 0.03' 

0.86 f 0.11 

15.9 f 0.8 

0.42 f 0.03 

147 f 3 

6.4 f 0.1 

4.5 f 0.1* 

62 f 4 

252 f 20 

116 f 9 

552 f 70 

72 f 4 

2.1 f 0.1* 

4.06 f 1.23 

1.035 f 0.006 

* Significantly different (PsO.05j'from the control groupby Dum's or Shirley's test 

** P?;O.Ol 

a Mean f standard error. Statistical tests were performed on unrounded data. 

n=8 ' n=9 
- - 

- - - 

n 10 10 10 10 10 

Thyroid-stimulating hormone(nglmL) 

6-Month interim evaluation 110 

15-Month interim evaluation 1 io 

2-Year study 2 f oc 

Triiodothyronine(ng/dL) 

6-Month interim evaluation 80 f 6 - 68 f 6

15-Month interim evaluation a4f9 - 68 f 8d 

2-Year study 121 f 4 120 f 8d 115 f 10 127 f 6 118 f 7 

Reverse triiodothyronine(nglmL) 

6-Month evaluation interim 0.224 f 0.010 - - - 0.242 f 0.020

15-Monthinterimevaluation 0.164 f 0.011 0.172 f 0.010 

study 2-Year ’ 0.0111 f 0.017 0.074 f 0.012 0.087 f 0.019 0.042 f 0.014d 0.078 f 0.020

Thyroxine (pg/dL) 

6-Month interim evaluation 3.4 f 0.3 - - - 3.1 f 0.1 

15-Month interim evaluation 2.7 f 0.1 - - 2.7 f 0.2 -

2-Year study 4.3 f 0.3 3.7 f 0.2 3.7 f 0.3 3.3 f 0.1** 3.6 f 0.3 

2-Year study 

Thyroid-stimulating hormone 

2fO 2 f od 2f Id 1 f od 

Triiodothyronine(ng/dL) 93 f 7 106 f 3 loo f 4 108 f 7 

Reverse miodothyronine 

(nglmL) 0.029 f 0.m 0.055 f 0.013 0.063 f 0.015 0.062 f 0.018 

Thyroxine(pg/dL) 2.8 f 0.1 2.7 f 0.2 2.6 f 0.2 2.9 f 0.3 

** Significantly different (Pr0.01) from the control group by Shirley’s test 

a Mean f standard error. Statistical tests were performed on unrounded data. 

Not measured at this dose level 

n=8 

n=9 

I 
288 Salicylazosulfapyridine,NTP. TR 457 

I 

TABLE64 

Clinical Chemistry Datafor Mice in the 16-Day Gavage Studyof Salicylazosulfapyridinea 

Vehicle Control 675'mglkg 1,350mglkg 2,700 mglkg 

Made 

n 5 5 5 5 

Thyroid-stimulating hormone (nglmL) lab 275 f 73' 283 f 61' 246 f 37' 

Triiodothyronine (ngldL) 67 f 1 73 f 2 68 f 5 62 f 4 

Thyroxine (pgldL) 2.7 f 0.2 2.4 f 0.1 2.4 f 0.3 2.3 f 0.2 

Female 

n 5 4 5 - 4 

Thyroid-stimulating hormone(nglmL) 232 f 7Sd 123 f 17' 242 f 41e 142 f 25' 

Triiodothyronine (ngldL) 70 f 6 68 8 62 f 6 65 f 4 

Thyroxine (pgldL) 

a Mean f 

3.9 f 0.4 3.5 f 0.4 3.1 f 0.2 2.9 f 0.2 

standard error. Statistical tests were performed on unrounded data. Differences from the vehicle control group are not 

significant by Dun" or Shirley's test. 

n= 1; no standard error calculated ' n=3 

n=2 

e n=4 i
Vehicle Control 

Male 

n 10 

Hematology 

Hematocrit (%) 45.8 f 1.0 

Erythrocytes (106/pL) 9.57 f 0.14 

Reticulocytes (106/pL) 0.08 f 0.02 

Nucleated erythrocytes(103/pL) 0.02 f 0.01 

Mean cell volume (fL) 48.0 f 0.5 

Leukocytes (103/pL) 4.97 f 0.70 

Segmented neutro hils (103/pL) 1.05 f 0.21 ?p Lymphocytes (10 lpL) 3.67 f 0.48 

Monocytes (~o~/~L) 0.03 f 0.02 

Eosinophils (103/pL) 0.22 f 0.06 

Methemoglobin (g/dL) 0.71 f 0.19b 

Clinical Chemistry 

Urea nitrogen (mgldL) 24.7 f 1.5 

Creatinine (mg/dL) 0.34 f 0.05 

Glucose (mg/dL) 217 f 10' 

Total protein (gldL) 5.0 f 0.2 

Albumin (gldL) 3.6 f 0.0 

Alanine aminotransferase (IUIL) 20 f 2' 

Alkaline phosphatase (IUIL) 58 f 1 

Aspartate aminotransferase (IUIL) 52 f 4h 

Thyroid-stimulating hormone (nglmL) 333 f 23d 

Triiodothyronine (ng/dL) 84 f 3 

Thyroxine (pgIdL) 3.9 f 0.2 

Urinalysis 

Volume (mL116 hr) 1.2 f 0.1 

Specific gravity 1.023 f O.Wf 

. . 

675,mg/kg 

10 

45.7 f 0.9 

9.37 f 0.17 

0.15 f 0.03 

0.03 f 0.02 

48.6 f 0.3 

5.73 f 0.61 

1.39 f 0.22 

4.10 f 0.50 

0.05 f 0.02 

0.17 f 0.05 

0.99 f 0.2oc 

25.9 f 2.1f 

0.37 f 0.04' 

189 f 9f 

5.0 f 0.1' 

3.7 f. O.lf 

25 f 2' 

60 f 2f 

51 f 4f 

232 f 20*f 

72 f 49 

2.8 f 0.2" 

1.0 f 0.2f 

1.039 f 0.003*h 

1,35QmJkg 

10 

45.1 f 0.6 

9.15 f 0.11 

0.18 f 0.05* 

0.02 f 0.01 

49.2 f 0.4 

6.40 f 0.60 

1.33 f 0.21 

4.78 f 0.44 

0.08 f 0.02 

0.18 f 0.04 

0.70 f 0.12d 

27.1 f 1.8 

0.40 f 0.04 

202 f 9 

5.0 f 0.1 

3.8 f 0.1 

27 f 2** 

69 f 3**f 

52 f qf 

283 f 36h 

87 f 4' 

2.6 f 0.1** 

0.6 f 0.1* 

1.051 f 0.003*** 

2,7W mgkg 

10 

44.9 f 0.5 

9.23 f 0.10 

0.31 f 0.04" 

0.01 f 0.01 

48.6 f 0.3 

9.03 f 0.50** 

2.17 f 0.34' 

6.41 f 0.31** 

0.11 f 0.03 

0.31 f 0.08 

0.85 f 0.12e 

'20.5 f 1.4 

0.32 f 0.03 

205 f 13 

5.1 f 0.1 

3.7 f 0.1 

31 f 4** -

64 f 1** 

65 f 12 

304 f 34c 

79 f 8' 

2.7 f 0.1** 

1.2 f 0.1 

1.044 f 0.004** 
290 Salicylazosulfapyridine,NTP TR 457 

TABLEG5 

Hematology, Clinical Chemistry, and Urinalysis Datafor Mice in the 13-Week Gavage Study 

of Salicylazosulfapyridine(continued) 

Vehicle Control mglkgmglkg mg/kg 1,350 2,700 675

Female 

n 10 10 10 10 

Hematology 

Hematocrit (96) 47.7 f 0.5 44.9 f 1.0* 45.7 f 0.4 45.8 f 0.7 

Hemoglobin (gIdL) 16.2 f 0.2 15.4 f 0.3 15.8 f 0.2 15.8 f 0.2 

Mean cell hemoglobin (pg) 

Mean cell volume (a) Reticulocytes (106/pL) 

Erythrocytes (1061pL) 

17.0 f 0.2 

50.0 f 0.4 

0.10 f 0.01 

9.54 f 0.09 

17.3 f 0.1 

50.2 f 0.3 

0.16 f O.M*' 

8.93 f 0.17* 

17.2 f 0.2 

49.8 f 0.4 

0.22 f om**

9.18 f 0.08 

17.6 f 0.1** 

50.9 f 0.3 

0.20 f 0.03*% 

8.98 f 0.15* 

Mean cell hemoglobin 

concentration (gIdL) 34.0 f 0.2 34.3 f 0.3 34.6 f 0.2 34.5 f 0.2 

Segmented neutro hils (1dIpL) 

Leukocytes (l#/pL) 

0.69 f 0.10 

5.37 f 0.49 

0.80 f 0.08 

5.27 f 0.39 

0.74 f 0.08 

6.60 f 0.44 

1.08 f 0.26 

8.26 f 0.40** 

Lymphocytes (ldpL) 4.46 f 0.45 4.25 f 0.35 5.57 f 0.42 6.81 f 0.46** 

Monocytes (1031pL) 0.05 f 0.01 0.04 f 0.02 0.10 f 0.02 0.11 f 0.04 

Eosinophils (~o~I~L) 0.16 f 0.04 0.17 f 0.04 0.18 f 0.04 0.25 f 0.04 

Methemoglobin (gIdL) 0.70 f 0.13B 0.82 f O.llf 1.12 f 0.09**h 1.47 f 0.21**b 

Clinical Chemistry 

Urea nitrogen (mgIdL) 24.9 f 0.9 24.4 f 2.2 23.6 f 1.7 24.3 f 1.8 

Total protein (gIdL) 

Glucose (mgIdL) 

Creatinine (mgIdL) 0.40 f 0.03' 

5.4 f 0.1 

190 f 1$ 

5.6 f 0.2 

189 f gf 

0.30 f 0.03 

5.5 f 0.1 

176 f 9 

0.34 f 0.04f 

5.4 f 0.1 

194 f 12 

0.40 f 0.06f 

Albumin (g/dL) 4.2 f 0.1 4.2 f 0.1 4.0 f 0.1 4.1 f 0.1 

Alanine aminotransferase (IUIL) 26 f 2 38 f 10 36 f 9f 36 f 3 

Alkaline phosphatase (IUIL) 109 f 3 119 f 5' 112 f 4 113 f 3 

Thyroid-stimulating hormone(ngImL) 

Aspartate aminotransferase (IUIL) 

320 f 16e 

69 f gf 

280 f 26h 

67 f 13 

256 f 13* 

67 f 11' 

255 f 16' 

78 f 7 

Thyroxine (pgIdL) 

Triiodothyronine (ngIdL) 

3.9 f 0.1 

79 f 3 

3.3 f o.2*f 

86 f 4f 

3.3 f 0.1** 

82 f 2 

< 2.7 f 0.2** 

87 f 3 

Urinalysis 

Volume (mLI16 hr) 1.0 f 0.2 0.8 f 0.1 0.8 f 0.1 0.8 f 0.1 

Specific gravity 1.019 f O.Wf 1.034 f 0.004* 1.038 f 0.003**f 1.036 f 0.004**f 

~~ ~~~ ~ ~ ~ ~~~ ~ ~~~ ~~~~~~~ 

* Significantly different(PsO.05) from the control groupby DUM'S or Shirley's test 

** PiO.01 

a Mean f standard error. Statistical tests were performed on unrounded data. Special study animals were used for the thyroid-stimulating 

hormone, triiodothyronine, and thyroxine analyses. 

b n=3 ' n=5 

n=6 

e n=4 ' n=9 

n=8 

n=7 
i 

1,350 rng/kg 

9 

-

483 f 80 

-

188 4 11 

-

0.166 * 0.018 

-

8.6 f 0.4h 

155 f 12** 

159 f 8 

0.148 f 0.013f 

7.8 f 1.6h 

2,700 rng/kg 

10 

287 f 65* 

327 f 100d 

183 f 8 

206 f lof 

0.173 f 0.008 

0.136 f 0.007*g 

7.2 f 0.4 

9.9 f l.oe 

241 f 40 

175 f 12 

0.150 f 0.017‘ 

7.2 f 0.5 

. -

n 

Male 

Thyroid-stimulating hormone(ng/mL) 

15-Month interim evaluation 

2-Year study 

Triiodothyronine (ngldL) 

15-Month interim evaluation 

2-Year study 

Reverse triiodothyronine (nglmL) 

15-Month interim evaluation 

2-Year study 

Thyroxine (pgldL) 

15-Month interim evaluation 

2-Year study 

Female 

2-Year study 

Thyroid-stimulating hormone (nglmL) 

Triiodothyronine (ngldL) 

Reverse triiodothyronine (nglmL) 

Thyroxine (pgldL) 

* Significantly different(PsO.05) from the control group by DUM’Stest 

** PsO.01 

a Mean f standard error. Statisticaltestswereperformed on unrounded data. 

Not measured at this dose level 

n=7 

n=6 

e n=9 

n=8 

g n=5 

n=10 

n=4 

Vehicle Control 

10 

463 f 54 

470 f 142‘ 

171 f 5e 

179 f 12e 

0.193 f 0.007 

0.316 f 0.084d 

7.3 f 0.3 

9.2 f 0.8 

422 f 82 

146 f 8 

0.203 f 0.027d 

6.1 f 0.3 

rng/kg 675

10 

-b 

493 f 72 

-

200 f 13e 

-

0.128 f 0.009**d 

-

9.1 4 0.7 

299 f 59f 

168 f 13 . 

0.222 f 0.041’ 

6.9 f 0.7 
292 Salicylazosulfapyridine, NTP TR 457 

! 

294 Salicylazosulfapyridine,NTP TIR 457 

TABLEH1 

Summary of ReproductiveTissue Evaluations and Estrous Cycle Characterization forRats 

inthe13-WeekGavageStudyof Salicylazosulfapyridinea , . 

Vehicle Control ‘ 8 4 mdkg 168.8 mg/kg 337.5 mgkg

Male 

n 10 10 10 10 

Weights (g) 

Necropsy body wt 357 f 7 344 f 4 345 f 5 341 f 6 

R.cauda 0.146 f 0.005 0.145 f 0.004 0.139 f 0.005 0.142 f 0.004 

R.epididymis 0.444f 0.007 0.455 f 0.010 0.438 f 0.005 0.430 f 0.010 

R.testis 1.485 f 0.028 1.505 f 0.016 1.501 f 0.018 1.467 f 0.035 

’ Epididymal parameters spermatozoal 

Motility (46) 84.92 f 2.09 75.68 f 1.20** 71.86 f 4.08** 71.34 f 2.55** 

Concentration 

(1OVg cauda epididymal tissue) 808.1 f 46.1 778.0 f 51.2 758.4 f 54.2 675.4 f 54.0 

Abnormal (R) 0.720 f 0.090 0.860 f 0.095 0.900f 0.054 1.080 f 0.120 

Female 

n 10 10 10 10 

Necropsy body wt 199 f 3 190 f 2** 189 f 2** 188 f 2** 

Estrous cycle length (days) ,430 f 0.25 5.11 f 0.20b 4.89 f 0.20b 4.89 f 0.26b 

Estrous stage (96of cycle) 

Diestrus 37.1 30.0 35.7 34.3 

Proestrus 17.1 21.4 20.0 17.1 

Estrus 24.3 27.1 27.1 25.7 

Metestrus 21.4 21.4 17.1 22.9 

** Significantly different(PgO.01) from the vehicle control group by Shirley’s test (epididymal spermatozoal motility)or Williams’ or 

Dunnett’s test (weights) 

a Weights, epididymal spermatozoal parameters, and estrous cycle lengths are presented as mean f standard error. Statistical tests were 

performed on unrounded data. Differences from the vehicle control group for epididymal spermatozoal concentrationand abnormality are 

not significant by Dunn’s test. By multivariate analysis of variance, dosed females do not differ significantly from the vehicle control 

females in relative length of time spent in the estrous stages. 

Estrous cycle was longer than7 days or was unclearin 1of 10 animals. 
295 

n 

Weights (g) 

Necropsy body wt 

R. cauda 

R. epididymis 

R. testis 

Epididymal spermatozoal parameters 

Motility (X) 

Concentration 

(106/gcauda epididymal tissue) 

Abnormal (X) 

n 

Necropsy body wt 

Estrous cycle length (days) 

Estrous stage(X of cycle) 

Diestrus 

Proestrus 

Estrus 

Metestms 

Vehicle Control . 

10 

30.2f 0.5 

0.017 f 0.001 

0.048 f 0.001 

0.115 f 0.003 

82.02f 2.43 

1,318f 92 

1.14 f 0.17 

10 

26.1 f 0.5 

4.89f 0.20' 

18.6 

22.9 

41.4 

17.1 

: ,675 mgkg 

9 

29.8 f 0.4b 

0.016f 0.001 

0.044 f 0.001** 

0.112f 0.002 

81.61 f 1.41 

1,169f 79 

1.11 f 0.12 

10 

26.2 f 0.3 

4.88 f 0.30d 

34.3 

20.0 

27.1 

18.6 

1,35Qmgkg 

10 

29.2 f 0.4 

0.014f 0.001** 

0.041 f 0.001** 

0.109 f 0.004 

77.06 f 2.20 

1,367f 125 

1.34f 0.17 

10 

26.0f 0.3 

4.89 f 0.31' 

32.9 

18.6 

27.1 

21.4 

2,700 mgkg 

10 

29.9 f 0.5 

0.015 f O.OO0 

0.042 f 0.001** 

0.111 f 0.002 

77.68 f 1.66 

1,319f 116 

1.48 f 0.28 

10 

26.1 f 0.4 

5.13 f 0.30d 

28.6 

20.0 

30.0 

21.4 

Q*Significantly different(P~0.01)from the vehicle control group by Williams' or Dunnett's test 

a Weights, epididymal spermatozoal parameters, and estrous cycle lengths are presented as mean f standard error. Statistical tests were 

performed on unrounded data. Differences from the vehicle control group for epididymal spermatozoal parameters are not significant by 

DUM'S test. By multivariate analysis of variance, dosed females do not differ significantly from the vehicle control females in relative 

length of time spent in the estrous stages.

n=lO 

Estrous cycle was longer than7days or was unclear in 1 of 10animals. 

Estrous cycle was longer than7days or was unclear in2 of 10 animals. 
296 Salicylazosulfapyridine,NTP TR 457 

. . 
305 
298 Salicylazosulfapyridine,NTP TR 457 

CHEMICAL CHARACTERIZATIONAND DOSE 

FORMULATION STUDIES 

PlROCUREMEW AND CHARACTERIZATION OF SALICYLAZOSULFAPYRIDINE 

Salicylazosulfapyridine was obtained from Salisbury Labs, Inc. (Charles City, IA) in one lot (1089), which 

was used during the 16-day and 13-week studies. For the 2-year studies, one lot (61838) was obtained from 

Pharmacia, Inc. (Piscataway, NJ). Identity, purity, and stability 'analyses were conducted by the analytical 

chemistry laboratory and confirmed by the study laboratories. Reports on analyses performed in support of 

the salicylazosulfapyridine studies are on fde at the National Institute of Environmental Health Sciences. 

Both lots of the chemical, an orange powder, were identified as salicylazosulfapyridine by infrared, 

ultraviolethisible, and nuclear magnetic resonance (NMR) spectroscopy. All spectra were consistent with 

the literature spectra (Florey, 1976; Nygird et al., 1966) of salicylazosulfapyridine. The infrared and NMR 

spectra are shown in Figures I1 and 12. The observed melting point of 247" to 248" C for lot 1089 was 

consistent with the literature reference'(MerckIndex, 1983). 

The purity of each lot was ,determined by elemental analyses, Karl Fischer water analysis, functional group 

titration, 'thin-layer chromatography (TLC), and high-performance liquid chromatography (HPLC). , For

functional group titration.of lots 1089 and 61838, samplesof salicylazosulfapyridine'weredissolved in 

pyridine, titrated with aqueous 0.1 N sodium hydroxide and monitored potentiometrically using a 

combination pH/mV electrode filled with 3M or 4 M potassium chloride. Samples were also dissolved in 

dimethylformkide and titrated with 0.1 N tetrabutylammonium hydroxide in 2-propanol and monitored 

potentiometrically using a glass indicating electrode versus a calomel reference electrode filled withM1 

tetrabutylammonium chloride 'in methanol. TLC for lot 1089 was performed on Silica Gel 60 F-254 plates 

with two solvent systems: 1) methyl ethyl ket0ne:acetone:water (16: 16:l) and 2) chloroform:n-butanol: 

acetone:glacial acetic acid(4:1:1:1). Salicylic acid was used as a reference standard. The plates were 

examined with visible and ultraviolet light (254 and 366nm) and with a spray of potassium iodoplatinate 

reagent. TLC for lot 61838 was performed on Silica Gel 60A K6F 60 plates with two solvent systems: 

1) methyl ethyl ket0ne:acetone:water (16: 16: 1) 2)andchloroform:isopropanol:acetone:glacial acetic acid 

(4.: 1: 1: 1). Salicylic acid was used as a reference standard. The plates were examined with visible and 

ultraviolet light (254 and 366 nm) and with a spray.of ferricyanide-ferric chloride reagent. HPLC for 

lot 1089 was performed with a Waters Nova Pak C,, column using ultraviolet light detection (254 and 

365 nm) and an isocratic solvent system of1% phosphoric acid in water: 1'% phosphoric acid in methanol

(4555) at a flow rate of 1 mL/minute. HPLC for lot 61838 was performed with the same system usedfor 

lot 1089 except with a solvent ratioof 53:47. 

Elemental analyses for carbon, hydrogen, nitrogen, and sulfur agreed with theoretical valuesfor both lots. 

Karl Fischer water analysis indicated0.50% f 0.04% water in lot 1089 and 0.5% f 0.1% water in 

lot 61838. Functional group titration for lot 1089 by two methods indicated purities of 99.3% f 0.6% and 

100.6% f 0.8%:~ Functional group titration for lot 61838 bytuio methods indicated purities of 

100.6% f 0.3% and 101.0% f 0.4%. For lot 1089, TLC by each system indicated a major spot and a 

trace inhpurity. For lot 61838, TLC indicated a major spot with a trace andtwo slight trace impurities by 

system 1. System 2 indicated a major spot withtwo trace impurities and a slight trace impurity. Using a 

detection wavelength of254 nm, HPLC revealed a major peak and.three impurities with a combined areaof 

2.3% relative to the major peak area for lot 1089.At a detection wavelength of 365nm, two impurities 

with a combined area of 1.9% relative to the major peak were observed for lot 1089. HPLC using a 

detection wavelength of254 nm resolved a major peak andtwo impurity peaks with a combined area of 

1.7% relative to the major peak area for lot 61838. When 365nm was used .as the detection.wavelength, 

three impurity peaks were observed with a combined areaof 1.2% relative to the major peak for lot 61838. 
The overall purity was determined to be approximately 96% to 97% for lot 1089 and approximately 98%

for lot 61838. 

The analytical chemistry laboratory analyzed lot 1089 to determineif it met United States PharmacopeiaXX 

(USP) purity requirements. The complete battery of USP analyses was performed as a supplement to the 

chemical characterization of salicylazosulfapyridine. The chemical met the USP requirements for the 

identification tests, which included infrared and visible absorption spectroscopy. The loss on drying was 

0.30% f 0.01%, which met the USP requirement. The residue on ignition was 0.18% f 0.04%, which 

met the USP requirement. The chemical met the USP requirements for the chloride, sulfate, and heavy 

metals tests. The USP purity assay indicated that lot 1089 had a purity of 96.5 % f 1.7%, calculated on the 

anhydrous basis, relative to the USP salicylazosulfapyridine reference standard. This purity was consistent 

with USP requirements. 

Each lot was concomitantly analyzed with the USP standardfor salicylazosulfapyridine content using the 

HPLC system described for the purity analyses except witha solvent ratio of 4258 (lot 1089) or 5050 

(lot 61838). Relative to the USP standard, lot 1089 was determined to contain 96.3% f 0.7% 

salicylazosulfapyridine, and lot 61838 was found to contain 97.0% f 0.9% salicylazosulfapyridine. 

Stability studies of the bulk chemical for lot 1089 were performed by the analmcal chemistry laboratory. 

HPLC was performed using the system describedfor the purity studies except witha solvent ratio of 4258, 

valerophenone as an internal standard, and detection at 254nm. These studies indicated that 

salicylazosulfapyridine was stable as a bulk chemical for 2 weeks when stored protected from lightat 

temperatures up to 60" C. Stability was monitored by HPLC and potentiometric titration prior to the start 

of the 16-day and 13-week studies, twice during the 13-week studies, and at least every 4 months by HPLC 

only during the 2-year studies. To ensure stability, the bulk chemical was stored in sealed amber plastic 

bags at 5 O C during the 16-day and 13-week studies and in metal cans protected from light at room 

temperature during the 2-year studies. 

rnPrnlrlION mL4iwILrrslIs;OF DOSE IFomAlrlIas; 

The dose formulations were preparedby mixing salicylazosulfapyridine with corn oil to give the required 

concentrations (Table 11). The dose formulations were prepared weekly during the 16-day. studies and 

every 2 weeks during the 13-week and 2-year studies and storedfor up to 3 weeks at 0" f 5 " C in clear 

(16-day studies) or amber glass (13-week and 2-year studies) bottles. 

The corn oil vehicle used in the studies was analyzed every1 to 3 months for peroxide levels. All analyses 

determined that the peroxide levels were within the specified limits except for two lots (2-year studies) that 

were replaced as soon as possible. 

Homogeneity studies of the500 mg/kg dose formulation were performed by the analytical chemistry 

laboratory. Extracts were prepared by mixing samples with N,N-dimethylformamide. Aliquots of the 

extracts were mixed with5 mL of internal standard solution (2-bromo-4,6-dinitroaniline,3 mg/mL in 

methanol) and 3 mL of deionized water, and then diluted to 250 mL with HPLC mobile phase. HPLC was 

performed with a Brownlee RP-18 (100X 4.6 MM ID) column using ultraviolet detection (365nm) and a 

mobile phase solvent system of methano1:water:phosphoric acid (60.538.5: 1) at a flow rate of 

1.O mL/minute. Homogeneity was confirmed. 

Stability studies of the 60 mg/g dose formulation were performed using the HPLC system described for the 

homogeneity studies. Stability was confirmed for 3 weeks when stored in the dark at room temperature and 

for 3 hours when stored open to air and light. 
30 Salicylazosulfapyridine, NTP TR 457 

Periodic analyses of the dose formulationsof salicylazosulfapyridine were conducted at the study 

laboratories using HPLC. The dose formulations were analyzed at the beginning of the 16-day studies, and 

all formulations (4/4) were within 10% of the target concentrations (Table 12). During the 13-week studies, 

the dose formulations were analyzed at the beginning, midpoint, and endof the studies, and all formulations 

(lfi/15) were within 10% of the target concentrations (Table 13). During the 2-year studies, the dose 

fo~mulations were analyzed every 6 to 12 weeks. Of the dose formulations analyzed for the 2-year studies, 

98% (103/105) were within 10% of the target concentrations. The dose formulations not within 10% of the 

target concentrations were remixedand found to be within 10%- of the target'concentrations (Table 14): 

Results of thi periodic referee analyses performedby the analytical chemistry laboratory were in agreement 

with the results obtained by the study laboratory (Table15); : , . . . 

, 

... . 
i-x 

t 

DO6 
7.94-8-02 

i 

0. SI I 

1. os 

0.98 

0.99 

3 

I 

I 

I 

e. 95 

Not lntmpratmd 

0.97 I 

I. 07 I 

e. 01 e 

P

m 

t 

Eb 

* S.lV.Qt 

-2 -

0 PPM 

.. 

. . 5 
R@B)W&QW 

Salicylazosulfapyridine (finely Same as 16day studies Same as 16day studies 

powdered) was slowly addedto corn oil 

in a graduated, light-protected 

Erlenmeyer flask. The corn oil was 

continuously stirred atroom ‘ 

temperature and medium speed by a 

magnetic stir bar. Stirring was 

continued for up to 5 minutes, until the 

suspension appeared homogeneous. 

The mixture was then blended in a 

Brinkman Polytron blender for 

3 minutes. 

Same as 16day studies 61838 

3 weeks 3 weeks 

StQE3ge@Ql“iQlW 

Stored in sealed glass containers Same as 16-day studies Same as 16day studies 

protected from light at 0” f 5” C 

Study ILXTL~QU~J~QH~~ 

EGBrG Mason Research Institute EGBrG Mason Research Institute Southern Research Institute 

(Worchester,MA) (Worchester. MA) (Birmingham,AL) 

Refem 1L”Xat~~ 

Midwest Research Institute Same as 16day studies Same as 16day studies 

(Kansas City, MO) 
:304 Salicylazosulfapyridine,NTP TR 457 

‘TABLE I2 

Results of Analyses of Dose Formulations Administered to Rats and Mice ’ 

in the 16-Day Gavage Studies of Salicylazosulfapyridine 

DatePreparedDateAnalyzedConcentration 

Target Determined 

Concentration 

% Difference 

from Target 

(mgl@ (mg/gla 

17 September 1985 September 1985 72 73.42 +2 

141 141.95 +1 

269 273.10. +2 

496 497.35 0 

10 October 1985b 72 . 73.29 +2 

141 142.26 +1 

269 272.08 +1 

496 506.74 +2 

a Results of duplicate analyses. Dosing volume for rats = 5 mL/kg; 141 mglg = 675 mglkg; 269 mglg = 1,350 mglkg; 496 mglg = 

2,700 mglkg. Dosing volume for mice = 10 mLlkg; 72 mglg = 675 mglkg; 141 mglg = 1,350 mglkg; 269 mglg = 2,700 mglkg. 

bd-room samples 
PS+ 

6+ 

0 

I+ 

I+ 

I+ 

1-

I -

I+ 

0 

ZI+ 

I+ 

I+ 

I+ 

0 

0 

Z+ 

8+ 

0 1+ 

I+ 

ZSS'6SZ 

9'LPI 

89'81 

9'69Z 

8'IPI 

66'ZL 

LS'ZL I I '9E 

60'81 

6E'ZL 

EZ'9E 

E6'LI 

ZP'ZL 

08'9E 

PE'81 

8L'ZL 

PS'9E 

12'81 

19'EL 

SZ'6E 

P6'61 

66.11. 

69'SE 

EZ'LI 

9.692 

8'WI 

O'ZL 

P'69Z 

8'WI 

O'ZL 

O'ZL 

P'9E 

2'81 

O'ZL 

P'9E 

2'81 

O'ZL 

P'9E 

2'81 

O'ZL / P'9E 

2'81 

O'2L 

P'9E 

2.81 

O'ZL 

P'9E 

2'81 

9867 amf EZ 

9861 KWI E1 
306 

'i 

ThBLE ~) . I3 

Results of Analyses of Dose Formulations Administered to Rats and Mice 

in the 13-Week Gavage Studies of Salicylazosulfapyridine (continued) 

Determined Target

ConcentrationConcentration Analyzed Date Date Prepared

(whi9 

Mice (continued) 

13 May 1986 14May 1986 

140.8 

72.0 

269.4 

11 June 198ab 72.0 

140.8 

269.4 

23 June 1986 24June 1986 72.0 

140.8 

269.4 

12 August 1986b 72.0 

140.8 

269.4 

a Results of duplicate analyses. Dosing volume for rats = 5 mLlkg; 18.2mglg = 

72.0mglg = 337.5mglkg. Dosing volume for mice = 10mL/kg; 72.0mglg = 

269.39mglg = 2,700mglkg. 

~nimal room samples 

% Difference 

from Target 

(mg/& 

72.78 +1 

141.8 +1 

273.6 +2 

72.94 +1 

142.5 +1 

273.8 +2 

72.39 +1 

142.6 +1 

274.6 +2 

72.57 +1 

139.6 -1 

270.3 0 

84 niglkg; 36.4mglg = 168.8mglkg; 

675 mglkg; 140.75mplg = 1,350mglkg; 
z+ 

z+ 

P+ 

P+ 

I+ 

Z+ 

z+ 

2-

E+ 

Z+ 

Z0 

I+ 

ZI+ 

01-

0 

I -

I+ 

I -

I+ 

Z+ 

$+ 

P+ 

L+ 

I+ 

I+ 

I+ 

I+ 

I+ 

E -

I -

9-

I -

8.PL 

9'PL 

O8E 

061 

O'PL 

OSL 

S'LE 

081 

S'SL 

8'PL 

8'SE 

E'81 

E'PL 

Z'ZL 

O'LE 

P'91 

L'EL 

8'ZL 

I 'LE 

Z'8I 

OVL 

Z'SL 

O'8E 

061 

E'8L 

6'9E 

P'81 

P'PL 

O'PL 

8'9E 

8'LI 

1'81 

9'LI 

Z'81 

E'81 

9'9E 

P'EL 

P'EL 

f'8I 

9'9E 

VEL 

P'EL 

9'9E 

P'EL 

P'EL 

E'81 

E'81 

9'9E 

P'EL 

VEL 

6861 aW 8-L 6861 aunf 9 

E'81 

9'9E 

VEL 

P'EL 

6861 I!JdV PI-E1 6861 l!JdVI I 

€31 

9'9E 

P'EL 

P'EL 

E'81 

9'9E 

VEL 

qE'81 

,E'81 

pE'81 

E31 

9'9E 

P'EL 

P'EL 
308 Salicylazosulfapyridine,NTP TIR 457 

T~IBLEI~ ' 

Rt%ultsof Analyses of Dose Formulations Administeredto Rats and Mice 

in the 2-YearGavage Studies of Salicylamsulfapyridine(continued) 

ConcentrationConcentration Analyzed Date Date Prepared

Rats (continued) 

7 November 1989 

16 January 1990' 

27 February 1990 

24 April 1990 

3 July 1990 

11 September 1990 

, 6 November 1990 

8-9 November 1989 

29-30 November 1989e 

16-17 January 1990 

27-28 February 1990 

24-25 April 1989 

15 May 

5 July 1990 

6-7 November 1990 

29 November 1590' 

Determined Target % Difference

from Target 

mg/& (mg/& 

18.3 19.0 +4 

36.6 36.4 -1 

73.4 72.2 -2 

73.4 73.0 -1 

18.3 20.6 +13 

36.6 37.2 +2 

73.4 90.8 +24 

73.4 97.8 +33 

18.3 19.4 +6 

36.6 38.5 +5 

73.4 76.8 +5 

73.4 76.0 +4 

18.3 18.4 +1 

36.6 36.8 +1 

73.4 73.8 +1 

73.4 74.0 +1 

18.3 18.3 0 

36.6 36.4 -1 

73.4 73.8 +1 

73.4 74.4 +1 -

18.3 19.0 +4 

36.6 49~8 +36 

13.4 82.9 +13 

18.3 19.8 +8 

36.6 37.4 +2 

73.4 73.4 0 

73.4 72.8 -1 

18.3 17.5 -4 

36.6 33.4 -9 

73.4 71.5 -3 

73.4 70.8 -4 

18.3 18.5 +1 

36.6 37.7 +3 

73.4 74.6 +2 

18.3 18.4 +1 

36.6 36.6 0 

73.4 73.2 0 
EI -

I -

ZEZI+ 

Z+ 

Z+ 

0 

Z+ 

I+ 

I+ 

Z+ 

Z+ 

I+ 

Z+ 

Z+ 

0 

I -

EI+ 

I -

Z+ 

0 

Z+ 

ZI -

I+ 

0 

I+ 

0 

0 

982 

9PI 

EL 

682 

EPI 

Z'ZL 

962. 

osI 

6'PL 

p6Z 

os1 

P'PL 

96Z 

osI 

Z'SL 

96Z 

os1 

9'PL 

967. 

SPI 

P'TL 

962 

9PI 

O'SL 

E6Z 

osI 

Z'ZL 

762 

891 

9'EL 

IP'PL 

L'EL 

S'EL 

8

'E6Z 

6'9PI 

P'EL 

8'E6Z 

6'WI 

P'EL 

8'E6Z 

6'991 

P'EL 8'E61 

6'9PI 

P'EL 

8'E6Z 

6'WI 

P'EL 8'E6Z 

6'9PI 

P'EL 8'E62 

6'9PI 

P'EL 

8'E6Z 

6'991 

P'EL 

8'E6Z 

6'WI 

P'EL 

8'E6Z 

6'WI 

P'EL 

VEL 

P'EL 

P'EL 

6861 PdV PI-E1 
310 Salicylazosulfapyridine,NTP TR 457 

:rmBLE 14 

Results of Analyses of Dose Formulations Administered to Rats and Mice 

in the 2-YearGavage Studies of Salicylazosulfapyridinine (continued) 

oncentrationConcentration Analyzed Target Prepared fromDate Date

Mice (continued) 

16January 1990 

18January 1990f 

19January 1 w 

27 February 1990 

24 April 1990 

3 July 1990 

11 September 1990 

6November 1990 

15 January 1991 

12March 1991 

7May 1991 

2July 1991 

30 July 1991 

16-17January 1990 

19January 1990 

19 January 1990 

27-28February 1990 

24-25April 1990 

15 May 1990e 

5 July 1990 

11-13 September 1990 

6-7November 1990 

29 November 1990e 

15 January 1991 

12March 1991 

7-9May 1991 

2July 1991 

31 July-1 August 1991 

19-20August 1991e 

Determined Target

(mg/& 

73.4 

146.9 

293.8 

146.9 

146.9 

73.4 

146.9 

293.8 

73.4 

I - -146.9 

293.8 

73.4 

146.9 

293.8 

73.4 

146.9 

293.8 

73.4 

146.9 

293.8 

73.4 

146.9 

293.8 

73.4 

146.9 

293.8 

293.8 

293.8 

293.8 

293.8 

293.8 

293.8 

bg/& 

76.2 

83.2 

304 ' 

174 

150 

73.4 

147 

294 

74.4 

147 

292 

82.6 

158 

294 

72.4 

145 

289 

70.0 

146 

302 

74.1 

148 

287 

74.3 

148 

291 

289.2 

292.6 

292.1 

286.7 

292.7 

291.7 

% Difference 

+4 

-43 

+3 

+18 

+2 

0 

0 

0 

+1 

0 

-1 

+ 13 

+8 

0 

-1 

-1 

-2 

-5 

-1 

+3 

+1 

+1 

-2 

+1 

+1 

-1 

-2 

0 

-1 

-2 

0 

-1 
aac 
312 Salicylazosulfapyrid, NTP TIR 457 

TABLEI5 

Results of Referee Analyses of Dose FormulationsAdministered to Rats and Mice in the 13-Week 

and 2-YearGavage Studies of Salicylazosulfapyride 

Date Prepared (mg/& 

Target Concentration Study. 

Laborato$ 

Referee 

Laboratoryb 

13-WeekStudies (EG&GMason) . . . 

23 June 1986 

24 March 1986 

72.0 

269.4 

72.39 

269.6 

72.5 f 0.3 

269 f 1 

%Year Studies(southernResearchInstitute) ' . .. 

' Rats 

6 June 1989 

6 December 1988 

73.4 

18.3 

72.2 

17.8 

73.7 f 0.8 

18.1 f 0.4 

Mice 

27 February 1990 

21 December 1988 

293.8 

146.9 

294 

148 

292.1 f 0.9 

143 f 1 

a Results of duplicateanalyses 

Results of triplicate analyses (meanf standard error) 

3114 

. . TABLEJ1 , v 

Ingredients of NIH-07 Rat and MouseRationa . . , , . , -

Ingredientsb Percent by Weight 

around #2 yellow shelled corn 24.50 

G.round hard winter wheat 23.00. 

Soybean meal (49% protein) 12.00 

Fish meal (60% protein) 10.00 

Wheat middlings 10.00 

Dried skim milk 5.00 

Alfalfa meal (dehydrated, 17% protein) 4.00 

Corn gluten meal(60% protein) 3.00 

soy oil 2.50 

Dried brewer’s yeast 2.00 

Dry molasses 1SO 

Dicalcium phosphate 1.25 

Ground limestone 0.50 

salt 0.50 

Premixes (vitaminand mineral) 0.25 

a NCI, 1976; NM, 1978 

Ingredients were ground to pass through a U.S. Standard Screen No. 16 before beii mixed. 

TABLE52 

Vitamins and Mineralsin NlH-07 Rat and Mouse Rationa r . .

Amount . Source 

Vitamins 

A 5,500,OOOIu Stabilized vitaminA palmitate or acetate 

4,600,OOOIu D-activated animal sterol D3 2.8 g Menadione K 3

d-a-Tocopheryl acetate 20,OOO Iu II Choline 560.0 g Choline chloride 

Folic acid 2.2 g 

Niacin 30.0 g 

d-Pantothenic acid 18.0 g d-Calcium pantothenate 

Riboflavin 3.4 g 

Thiamine 10.0 g Thiamine mononitrate 

B12 ‘ 4,OOOcg

Pyridoxine . 1.7 g Pyridoxine hydrochloride 

’ B’lotin 140.0 mg d-Biotin

Minerals 

Iron 120.0 g Iron sulfate 

Manganese 60.0 g Manganous oxide 

Zinc 16.0 g Zinc oxide 

Copper 4.Og Copper sulfate

Iodine 1.4 g Calcium iodate 

Cobalt 0.4 g Cobalt carbonate 

a Per ton (2,OOO lb) of’fhshed product 

., 
Nutrient 

Protein (% by weight) 

crude fat (96by weight) 

Crude fiber (% by weight) 

Ash (% by weight) 

bhAccidls (% of tobl BieU) 

Arginine 

Cystine 

Glycine 

Histidine 

Isoleucine 

Leucine 

Lysine 

Methionine 

Phenylalanine 

nireonine 

Tryptophan

Tyrosine 

Valine 

Essential Fat$ Acids (% @ftotal diet) 

Linoleic 

Linolenic 

Vi&lEllhlS 

Vitamin A (Iullcg) '. 

Vitamin D (IUlkg) 

a-Tocopherol (ppm) 

Thiamine (ppm)

Riboflavin (ppm) 

Niacin (ppm) 

Pantothenic acid (ppm) 

Pyridoxine (ppm) 

Folic acid (ppm) 

Biotin (ppm) 

Vitamin B12 (ppb) 

Choline (ppm) 

wmerals 

Calcium (46) 

Phosphorus (I) 

Potassium (Z) 

Chloride (k) 

Sodium (%) 

Magnesium (96) 

Sulfur (I) 

Iron (ppm)

Manganese (ppm) 

Zinc (ppm) 

Copper (Ppm)

Iodine (ppm) 

Chromium (ppm) 

Cobalt (ppm) 

23.23 f 0.67 

5.24 f 0.19 

3.51 f 0.40 

6.45 f 0.20 

1.287 f 0.084 

0.306f 0.075 

1.160f 0.050 

0.580 f 0.024 

0.917 f 0.034 

1.972f 0.052 

1.273f 0.051 

0.437 f 0.115 

0.994f 0.125 

0.896f 0.055 

0.223 f 0.160 

0.677 f 0.105 

1.089 f 0.057 

2.389 f 0.233 

0.277 f 0.036 

6.808 f 1,864 

4,450f 1,382 

36.92f 9.32 

18.67 f 2.20 

7.92 f 0.93 

100.95f 25.92 

30.30f 3.60 

9.25 f 2.62 

2.51 f 0.64 

0.267 f 0.049 

40.14f 20.04 

3,068f 314 

1.20f 0.10 

0.94f 0.03 

0.887 f 0.067 

0.526f 0.092 

0.315f 0.034 

0.168f 0.008 

0.274 f 0.063 

356.2f 90.0 

92.24f 5.35 

58.14f 9.91 

11.50f 2.40 

3.70f 1.14 

1.71 f 0.45 

0.797 f 0.23 

21.80

- 24.30 

4.60

- 5.60 

2.60

- 4.30 

6.11

- 6.94 

1.100

- 1.3% 

0.181

- 0.40 

1.060

- 1.220 

0.531

- 0.608 

0.867

- 0.%5 

1.850- 2.040 

1.200

- 1.370 

0.306

- 0.699 

0.665 - 1.110 

0.824

- 0.985 

0.107

- 0.671 

0.564 - 0.794 

0.962

- 1.170 

1.830

- 2.570 

0.210

- 0.320 

4,180 - 12,140 

3,ooO - 6.300 

22.5

- 48.9 

15.0

- 28.0 

6.10

- 9.00 

65.0

- 150.0 

23.0

- 34.6 

5.60 - 14.0 

1.80

- 3.70 

0.19

- 0.35 

10.6

- 65.0 

2.40 - 3.430 

1.00

- 1.54 

0.85

- 1.00 

0.772

- 0.971 

0.380

- 0.635 

0.258

- 0.370 

0.151

- 0.180 

0.208

- 0.420 

255.0

- 523.0 

81.70- 99.40 

46.10

- 81.60 

8.0% - 15.39 

1.52

- 5.83 

0.85 - 2.09 

0.490

- 1.150 

36 

36 

36 

36 

10 

10 

10 

10 

10 

10 

10 

10 

10 

10 

10 

10 

10 

9 

9 

36 

4 

9 

. . 36 

10 

9 

10 

10 

10 

10 

10 

9 

36 

36 

8 

8 

10 

10 

10 

10 

10 

10 

10 

10 

9 

6 
316 Salicylazosulfapyridine,NTP TR 457 

TABLE54 

Contaminant Levels in NIH-07 Rat and Mouse Rationa 

Contaminants 

Arsenic (ppm) 

Cadmium (ppm) 

]Lead (ppm) 

,Mercury (ppm)

Selenium (ppm) 

Aflatoxins (ppb)' 

Nitrate nitrogen (ppmId 

Nitrite nitrogen (ppmId 

BHA (ppm)e

BHT (ppm)e 

Aerobic plate count (CFUlg) 

Coliform (MPNIg) 

Escherichia coli (MPNlg) 

Salmonella (MPNlg) 

Total nitrosoamines (ppb)f 

N-Nitrosodimethylamine (p b)f P N-Nitrosopyrrolidine (ppb) 

Pesticides (ppm)

a-BHC 

P-BHC 

y-BHC 

&BHC 

Heptachlor 

Aldrin 

Heptachlor epoxide 

DDE 

DDD 

DDT 

HCB 

Mirex 

Methoxychlor 

Dieldrin 

Endrin 

Telodrin 

Chlordane 

Toxaphene 

Estimated PCBs 

Ronnel 

Ethion 

Trithion 

Diazinon 

Methyl parathion 

Ethyl parathion

Malathion 

Endosulfan I 

Endosulfan I1 

Endosulfan sulfate 

~ 

.. . . 

Mean'.* Standird. ' 

Deviationb 

0.31 f 0.19 

0.10f 0.05 

0.28f 0.21 

0.03f 0.19 

0.38f 0.22 

<5.0 

14.50f 5.17 

0.19f 0.16 

1.75 f 1.64 

1.50 f 1.34 

43,434f 28,288 

4.08f 4.42 

3.00 

Negative 

7.57f 2.62 

5.70f 2.28 

1.87f 1.02 

<0.01 

<0.02 

<0.01 

<0.01 

<0.01 

<0.01 

<0.01 

<0.01 

<0.01 

<0.01 

<0.01 

<0.01 

<0.05 

<0.01 

<0.01 

<0.01 

<0.05 

<0.1 

<0.2 

<0.01 

<0.02 

<0.05 

<0.1 ' 

<0.02 

<0.02 

0.25 f 0.24 

<0.01 

<0.01 

<0.03 

Rmze 

0.06- 0.70 

0.05 - 0.2 

0.10- 1.00 

0.02- 0.11 

0.05- 1.21 

2.90- 24.0 

0.10- 0.70 

1 .00- 10.0 

1.00- 8.00 

-

Number of Samples 

36 

36 

.36 ' 

36 

36 

35 

36 

36 

36 

36 

4,100- 120,OOO 35 

3.00- 23.00 36 

<3.00 36 

36 

3.60- 16.50 36 

2.60- 13.00 36 

1.00- 4.30, 36 

31 

31 

31 

31 

31 

31 

31 

31 

31 

31 

31 

31 

31 

31 

31 

31 

31 

31 

31 

31 

31 

31 

31 

31 

31 

0.05 - 1.00 36 

31 

31 

31 

a CFU=colony forming units (excludes the high value of 710,OOOobtained on batches produced on2October 1991).MPN=most probable

number, BHC =hexachlorocyclohexane or benzene hexachloride 

For values less than the limitof detection, the detection limit is given as the mean. 

No aflatoxin measurement was recorded for thelot milled 2October 1989. 

Sourcesof contamination: alfalfa, grains, and fish meal 

e Sources of contamination: soy oiland fish meal 

All values were corrected for percent recovery. 
~ O D S ................................... ........................... 3318 
,318 salicylazosulfapyridine, NTP-TR457 

SENTImL ANIMAL PROGRAM 

METHODS 

Rodents used in the Carcinogenesis Program of the National Toxicology Programare produced in optimally 

clean facilities to eliminate potential pathogens that may affect study results. The Sentinel AnimalProgram 

is part of the periodic monitoring of animal health that occurs during the toxicologic evaluation of chemical 

compounds. Under this program, the disease state of the rodents is monitored via serologyon sera from 

extra (sentinel) animalsin the study rooms. These animals and the study animals are subject to identical 

environmental conditions. The sentinel animals come from the same production source and weanling.. 

groups as the animals used for the studies of chemical compounds. 

Serum samples were collected from randomly selected rats and mice during the 2-year studies. Blood from 

each animal was collected and allowed to clot, and the serum was separated. The samples were processed 

appropriately and sent to Microbiological Associates, Inc. (Bethesda, MD), for determination'of antibody 

titers. The laboratory serology methods and viral agents for which testing' was performed are tabulated. 

below; the times at which blood was collected during the studies arealwlisted. 

RATS 

ELISA 

Mycoplasma arthritidis 

Mycoplasma pulmonisPVM (pneumonia virusof mice) 

RCV/SDA (rat coronavirusl 

sialodacryoadenitis virus) 

Sendai 

Hemagglutination Inhibition 

H-1 (Toolan's H-1 virus) 

KRV (Kilham rat virus) 

M[ICE 

ELISA 

Ectromelia virus 

EDIM (epizootic diarrhea of infant mice) 

GDVII (mouse encephalomyelitis virus) 

LCM (lymphckytic choriomeningitis virus) 

MVM (minute virusof mice) 

Mouse adenoma virus 

Mouse adenoma virus- FL 

MHV (mouse hepatitis virus) 

M. arthritidis 

M. pulmonis 

PVM 

Reovirus 3 

Sendai 

6 months, study termination (males) 

6 months, study termination (males) 

6, 12, 14, 15, and 18 months, study termination 

6, 12, 14, 15, and 18 months, study termination 

6, 12,14,15, and 18 months, study 

termination 

6, 12, 14, 15, and 18 months, study tekination 

6, 12, 14, 15, and 18 months, study termination 

6, 12, and 18 months, study termination 

18 months, study termination 

' 6, 12, and 18 months, study termination 

6, 12, and 18 months, study termination 

6 months ' . 

6 and 12 months 

18 months, study termination (males) 

6, 12, 18, and 23 months, study termination 

6 months 

6 months 

6, 12, and 18 months, study termination 

6, 12, 18, and 23 months, study termination 

6, 12, 18, and 23 months, study termination 
MCE(continued) .I 

-. , 

Immunofluorescence Assay 

EDIM 6 and 12 months 

GDVII 18 months 

MVM .. 12 and 18 months' 

Mouse adenoma virus - FL Study termination 

MHV Study termination 

Reovirus 3 23 months, study termination (males) 

Hemagglutination Inhibition 

MVM

K (papovavirus) 

Study termination 

6, 12, and 18'months, siudy termination 

Polyoma virus 6, 12, and 18 months, study termination , .. , 

.. 

Results of serology tests are presented in Table Kl . 

IIlltel'Val 

Rats 

6 Months 

12Months 

14Months 

15 Months 

18 Months 

Study termination 

Mice 

6 Months 

12Months 

18 Months 

23 Months 

Study termination 

, 

Incidence of Antibody 

in Sentinel hiids 

1/10 

0110 

. 0/1

o/1 

018 

0110 

0110 

018 

0111 

loll0 

9/10 

8/10 

Positive Serologic 

Reaction for 

M.arthiitidha 

None positive 

None positive 

None positive 

None positive 

None positive 

None positive 

None positive 

None positive 

MHV 

MHV (ELISA) 

MHV (Immunofluorescence assay) 

a Further evaluation of the sample positive for M.arthrifidis by immunoblot and Western blot procedures indicated that the 'positive titer may 

have been due to cross reaction with antibodies of nonpathogenic Mycoplasma or other agents. Only one sample was positive and there 

were no clinical findingsor histopathologic changesiofM.arthritidis infection in the animal with the positive titer. Accordingly, the 

M.arthritidis-positive titerwas consideredto be a false positive. 
Salicylazosulfapyridine, NTP TR 457 

\ 
wn 
322 Salicylazosulfapyridine,NTP TR 457 

. . CHEMICAL DISPOSITION 

OF SALICYLAZOSULFAPYRIDINE 

IN F344RATS.AND B6C3Fl MICE . .. 

OBJECTIVE 

The primary objective of these studies was to investigate the pharmacokinetics of salicylazosulfapyridine in 

male F344 rats and B6C3F, mice following iritravenous admihistration of Salicylazosulfapyridine(5 mg/kg) 

and in male and female B6C3F1 mice following gavage dosing (67.5, 675, 1',350, or 2,700 mg/kg); To '' 

determine if male and female mice form the she metabolites of salicylazosulfapyridine; the metabolites 

were identified in plasma collected following gavage administration.' 

CHEMICALS 

Chemicals were obtained from the following sources: salicylazosulfapyridine(2-hydroxy-5-[[4-[(2-pyridylamino) sulfonyl] phenyl] azo] benzoic acid, CAS No. 599-79-1) from the NTP Chemical Repository 

(Research Triangle Park, NC); sulfapyridine from Sigma (St. Louis, MO); and sodiumphosphate'(dibasic 

or monobasic) and methanol from Fisher Scientific CO. (Fair Lawn, NJ). N-Acetylsulfapyridine standard 

was synthesized by acetylation of sulfapyridine with acetic anhydride and purified by recrystallization.. The 

structure was confirmed by mass spectrometry. All reagents used in this experiment were analytical grade, 

high-performance liquid chromatography '(HPLC) grade, or the best available' pharinaceutical grade.. . 

ANIMALS , I 

1 . 

Rats: Male, jugular-vein cannulated F344 rats were supplied by Hilltop Lab Animals, Inc. (Scottdale, PA). 

The weight range of the animals at the start of the study was 188 f 5.89 .g. Upon receipt, the rats were 

housed in a temperature-controlled, 12-hour lightldark cycle facility and were acclimatedfor 1 week prior 

the start of the study, Rats were given tap w9er Ad feed (Teklad 4% Mouse/Rat Diet, HarlanTeklad, 

Madison, WI)ad libitum. 

, . 

Mice: Male and female B6C3Fl mice (24f 3 g) were supplied by the NIEHS (Research Triangle Park, 

NC). At the start of the study they were 8 to 10 weeks old. Upon receipt, the mice were kept in a 

temperature-controlled, 12-hour lightldark cycle facility and were 'acclimated for 1 week prior to the start of 

the study. Mice were given tap water and feed (Teklad 4% Mouse/Rat Diet, Harlan Teklad, Madison, WI) 

ad libitum. Mice were fasted for 12 hours prior to gavage administration of salicylazosulfapyridine. 

hMINISTRATION OF TEST COMPOUNDS 

Ruts: Salicylazosulfapyridine(5 mg/kg) was dissolved in distilled, deionized water (pH 9-10, adjusted with 

5 N NaOH) and administered intravenously to four male F344 rats. 

Mice: Salicylazosulfapyridine was dissolved incorn oil and administered by gavage to groups of male and 

female mice at doses of 67.5, 675, 1,350, and 2,700 mg/kg. For the intravenous administration, 

salicylazosulfapyridine was dissolved in distilled, deionized water (pH 9-10, adjusted with 5 N NaOH) and 

administered via the tail veinat a dose of 5 mg/kg (0.2 to 0.3 mL/mouse) over approximately5 to 

10 seconds. 
COLLIECTI[ONOF BLOOD SmlIDILIEs . .. 

Rats: Blood samples were collected from the capulated jugular vein into heparinized syringes at 0.03, 

0.08,0.17, 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 12;and'.24.hours following intravenous administration. Four rats 

were evaluated at each time point. The blood was centrifuged at,5,OOO X g for 5 minutes, and the plasma 

was transferred to an Eppendorf tube and stored at -20"C until analyzed. The samples were usually 

processed within 3 days. 

Mice: Mice were anesthetized with carbon dioxide, and blood samples were collected from the inferior 

vena cava into heparinized syringes prior to (0 hours) and at 0.25,0.5, 1, 3, 4, 6, 8, and 12 hours 

following intravenous administration and at 0.5, 1, 2, 3, 4, 6, 8, 12, and 24 hours following gavage 

administration. Four to seven mice were evduated at each time point. The blood was centrifuged at 

5,000X g for 5 minutes, and the plasma was transferred to an Eppendorf tube and stored at -20°C until 

analyzed. The samples were usually processed within 3 days. 

SrnlIDLIE ANALYSIIS 

Wavelength scanning of salicylazosulfapyridine in methanol/0.025 M (pH 2.5) phosphate buffer 

(64336, v:v).showed two absorption peaks at 364 nm and 244 nm in the ultraviolet range. Since 360 nm 

specifically represents the integrity of azo linkage, 360 nm was used for the detection of the parent 

compound. 

The plasma samples were mixed with two volumes of methanol and were allowed to stand in an ice bath for 

15 minutes to precipitate protein. After centrifugation at 13,000 X g at 4" C for 15 minutes, an aliquot of 

the supernatant was analyzed using HPLC. Salicylazosulfapyridine and its metabolites were separated and 

quantitated on a Phenomax 250 x 4.6 mm Ultracarb C,* reverse-phase column (7 ,urn) coupled to a 

Spectra-Physics Model 100 UV detector. Mobile phase system 1 consisted of methanol and 0.025 M 

(pH 2.5) phosphate buffer (64:36, v:v) and was used for salicylazosulfapyridine analysis. Separations were 

performed at room temperature at a rate of 1 mL/minute. Mobile phase system 2 was composed of 

methanol and 0.02 M (pH 7.0) phosphate buffer (20:80, v:v) and was used for sulfapyridine and 

N-acetylsulfapyridine analyses. The metabolites were separated at room temperature at a flow rate of 

2 mL/minute. A Spectra-Physics Model 100 UV detector was used for detection of salicylazosulfapyridine 

and its metabolites (360 nm for salicylazosulfapyridine and 254 nm for the metabolites, respectively). The 

detection limits in plasma were 0.32 nmol/mL for salicylazosulfapyridine, 0.5 nmol/mL for sulfapyridine, 

and 1 .O nmol/mL for N-acetylsulfapyridine. 

The results of these studies are presented in Tables L1 through L3. 
3241 Salicylazosulfapyridine,NTP TR 457 

TABLELl 

Pharmacokinetic Parameters Following Intravenous Administration of5 mg/kg Salicylazosulfapyridine 

in Male F344Rats 

Flat A 1.558 0.445 15.36 0.82 0.37 0.52 

Flat B 2.126 0.326 17.85 0.70 0.29 0.33 

Flat C 0.845 0.820 19.88 0.63 0.36 0.75 

Flat D 1.329 0.521 28.19 0.45 0.37 0.33 

Mean 1.465 0.528 20.32 0.65 0.35 0.48 

SEM 0.266 0.105 2.78 0.08 0.02 0.099 

a k=Elimination rate constant

b t,,=Elimiition half-life E AUC=Area under the concentration-time curve' 

CLs=Clearance e MRT=Mean residence time ' V,=Volume of distribution 

TABLEL2 

Comparison of Pharmacokinetic Parameters Following Intravenous Administration' I.' .

of 5 mg/kg Salicylazosulfapyridine in MaleF344Rats and B6C3Fl Micea - kb t*nc AUCd CL!f ' MRT' vag

fir-') @r) @Me hr) (Lbhr) (hr) (Lk)

Kats 1.47 0.53 20.32 0.65 r 0.35 0.48

Mice 1.28 0.54 9.21 1.36 0.45 1.07 

a The data were calculated from the plasma concentration-time curves where each point represents the mean of four to six animals from the 

' mouse intravenous study and of four animals from the rat study. 

k=Elimination rate constant 

t,,=Elimination half-life 

AUC =Area under the concentration-time curve

'

e CLS=Clearance 

MRT=Mean residence time 

V,=Volume of distribution 
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WIG9I 
DEPARTMENT OF SPECIAL FOURTH-CLASS RATE

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