[1,2,4]Triazolo[1,5-a]pyrimidine-2-sulfonamide, N-(2,6-difluorophenyl)-5-methyl-

Repeated dose toxicity: oral

Administrative data

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1st October 1987 - 18th May 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: FIFRA Guideline No. 83-5.

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EEC Directive 87/302/EEC: Combined Chronic Toxicity /Carcinogenicity Test

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: MAFF Guideline: Combined Chronic Toxicity/Carcinogenicity Study

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N-(2,6-difluorophenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidine-2-sulfonamide

EC Number:

619-383-6

Cas Number:

98967-40-9

Molecular formula:

C12H9F2N5O2S

IUPAC Name:

N-(2,6-difluorophenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidine-2-sulfonamide

Constituent 2

Reference substance name:

2',6'-difluoro-5-methyl[1,2,4]triazolo[1,5-a]pyrimidine-2-sulfonanilide

IUPAC Name:

2',6'-difluoro-5-methyl[1,2,4]triazolo[1,5-a]pyrimidine-2-sulfonanilide

Constituent 3

Reference substance name:

N-(2',6'-difluorophenyl)-5-methyl[1,2,4]triazolo[1,5-a]pyrimidine-2-sulfonamide

IUPAC Name:

N-(2',6'-difluorophenyl)-5-methyl[1,2,4]triazolo[1,5-a]pyrimidine-2-sulfonamide

Details on test material:

- Name of test material (as cited in study report): XRD-498
- Molecular formula: C12H9F2N5O2S
- Molecular weight: 325.3 Daltons

- Physical state: white powder
- Analytical purity:99.7%

- Lot/batch No.: AGR 240043

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratory (Kingston, NY)
- Age at study initiation: approximately five weeks
- Housing: suspended, stainless steel cages with wire-mesh floors
- Diet (e.g. ad libitum): Purina Certified Rodent Chow #5002 available ad libitum
- Water (e.g. ad libitum): tap water was available ad libitum

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: Purina Certified Rodent Chow #5002

Details on oral exposure:

The most probable routes of human exposure to XRD-498 may be via ingestion of foodstuffs containing low levels of residues or from accidental ingestion or dermal contact during field application or manufacture. Thus, formulation with feed was selected as a means of administering the test material to rats in this study.

XRD-498 has been shown to be stable in basal rodent chow for at least 37 days. Diets were prepared weekly during the first 13 weeks and at least once every two weeks for the remainder of the study by serially diluting a premix (test material-feed concentrate) according to the SOP of the Subchronic/Chronic Toxicity Section. This method of diet preparation has been demonstrated to produce a homogeneous distribution of XRD-498 in the diets.

Initial concentrations of test material in feed were calculated from pretest body weights and feed consumption data. Thereafter, the most recent body weight and feed consumption data were used to adjust the concentration of the test material in feed to maintain the dose levels on a mg/kg body weight/ day basis. Concentrations of test material were adjusted weekly for the first 13 weeks and at 4-week intervals thereafter. Reference samples (l/dose/sex/mix plus premix) were retained and stored at ambient temperature in a manner consistent with the sample retention policy of the laboratory.

Analyses of the test diets to verify the concentration of the test material were performed at the start of the study and approximately every three months thereafter. The analytical concentrations of XRD-498 in feed were found to be in acceptable agreement with targeted concentrations.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

2 years

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 500 or 1000 mg/kg/ day
Basis:
actual ingested

No. of animals per sex per dose:

Fifty Fischer 344 rats/sex/dose level were provided diets formulated to provide 0, 100, 500 or 1000 mg/kg/ day XRD-498 for up to two years. Ten additional rats/ sex/ dose level were randomly designated at the beginning of the study as a satellite group which were necropsied after approximately 12 months of exposure.

Control animals:

yes, plain diet

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
All animals were observed cageside at least once daily during the workweek for morbidity, mortality, availability of feed and water, and treatment-related effects. These observations included an evaluation of the skin, fur, and mucous membranes, respiration, nervous system, and behavior pattern. An additional observation was made on each day of the workweek, as well as twice daily on weekends and holidays, by animal care personnel for morbidity, mortality, and the availability of feed and water.

DETAILED CLINICAL OBSERVATIONS:
In addition, animals were examined for the presence of palpable masses and other remarkable clinical observations prior to the start of the study, after 6 months of dosing and monthly after 12 months of dosing. The date of the initial observation, progression or disappearance of any masses and/or clinical observations were noted. The final diagnoses of any changes, however, were based upon the gross and histopathological examinations.

BODY WEIGHT, FOOD CONSUMPTION AND FOOD EFFICIENCY:
Body weight data was recorded and feed consumption and feed efficiency data were calculated for all animals at weekly intervals for the first 13 weeks of the dosing period and monthly thereafter. The equation used to calculate feed efficiency was as follows:
Feed Efficiency (grams feed consumed/kg/ day) = (grams feed consumed/ day) / (kg body weight)

OPHTHALMOSCOPIC EXAMINATION:
Ophthalmic examinations were conducted on all animals prior to the start of the study and at the scheduled necropsy using a pen light illumination and moistened slide/ fluorescent light techniques, respectively.

HAEMATOLOGY:
Hematologic determinations were conducted on all surviving rats of the satellite groups of rats following approximately 6 and 12 months of the dosing period and on the first 10 and 20 surviving rats/sex/dose level of the 24-month sacrifice animals following approximately 18 and 24 months of the dosing period, respectively. Blood samples were obtained by orbital sinus puncture from fasted, methoxyflurane anesthetized animals and immediately mixed with EDTA to prevent clotting. Hematocrit (HCT), hemoglobin concentration (HGB), erythrocyte count (RBC), total leukocyte count (WBC) and platelet count (PLAT) were determined on all samples. In addition, blood smears were prepared from all animals from which samples were collected, stained with Wright's stain and examined. Blood smear examinations consisted of differential leukocyte counts of 100 cells and an assessment of RBC,WBC and PLAT morphology.

CLINICAL CHEMISTRY:
Clinical biochemical determinations of the activities of alkaline phosphatase (AP), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and creatine phosphokinase (CPK) and the concentrations of urea nitrogen (UN), creatinine (CREAT), total protein (TP), albumin (ALB),globulin (GLOB), glucose (GLUC), cholesterol (CHOL), triglycerides (TG), total bilirubin (TBILI) and electrolyte levels (Na, K, P, CI, Ca) were made on serum samples drawn from all surviving rats of the satellite groups after approximately 6 and 12 months of the dosing period. The same parameters were evaluated for the first 10 and 20 surviving rats/sex/dose group of the 24-month sacrifice group following approximately 18 and 24 months of the dosing period, respectively. Samples were drawn by orbital sinus puncture from fasted, methoxyflurane-anesthetized animals.

URINALYSIS:
Urine samples were obtained by spontaneous urination from the first 10 nonfasted surviving rats/sex/dose group 1 to 2 weeks prior to the 6- and 18-month blood collections, and 12- and 24-month sacrifices. The specific gravity and a semiquantitative analysis of pH, bilirubin, glucose, protein, ketones, blood and urobilinogen was determined for each sample. In addition, the color and appearance of individual urine samples were determined and the presence of microsediment in a pooled sample from each group was evaluated.

Sacrifice and pathology:

All surviving rats of the interim sacrifice groups were fasted overnight and necropsied following 12 months of dosing. All remaining surviving rats were also fasted overnight and necropsied after approximately 24 months of the dosing. Terminal body weights were recorded, animals were anesthetized with methoxyflurane, and blood was collected via orbital sinus puncture.

The trachea was exposed and clamped prior to decapitation to prevent aspiration of blood. Eyes were examined in situ by visual inspection of the cornea, lens and other internal components via placement of a moistened glass slide on the corneal surface using a fluorescent light. A complete gross examination was performed by a veterinary pathologist. Weights of the brain, heart, adrenals, liver, kidneys, cecum (full and empty), ovaries and testes were recorded and the organ weight to final body weight ratios calculated for all animals. Lungs were distended to an approximately normal inspiratory volume with neutral, phosphate-buffered 10% formalin solution by tracheal instillation using a hand-operated syringe. The nasal cavity was flushed by a similar method via the pharyngeal duct. Representative samples of tissues and any masses or lesions were preserved in formalin. A similar procedure was followed for rats which died or were sacrificed in a moribund condition except that blood samples, terminal body weight and organ weight data were not collected.

Tissues were prepared for light microscopic evaluation by standard procedures, sectioned at approximately 6 µm and stained with hematoxylin and eosin. A complete histologic evaluation of all tissues with the exception of auditory sebaceous gland were made on all control and high-dose animals and of all animals that died or were sacrificed in a moribund condition during the study. In addition, the liver, kidneys, lungs and cecum, from all low and intermediate dose group rats; the pancreas from low and intermediate dose group males; the adrenal gland from low and intermediate dose group females; and all lesions observed at necropsy were also examined microscopically.

Grading of histopathologic findings was done to reflect the severity of specific lesions when considered necessary to;
1) evaluate the contribution of a specific lesion to the health status of the animal,
2) evaluate exacerbations of common naturally occurring lesions as a result of the test material, and
3) examine dose-response relationships for treatment-related effects.
Very slight and slight conditions reflect findings in excess of the "normal textbook appearance" of an organ or tissue, but were of minimal severity, and involved less than 10% of the parenchyma. This type of change would not be expected to significantly affect the function of the specific organ/tissue involved nor have a significant affect on the overall health of the animal. Moderate conditions were of sufficient severity that the function of the organ/ tissue may have been adversely affected, but not to the point of organ failure. The health status of the animal mayor may not have been affected, depending on the organ/tissue involved and the type of alteration. Severe conditions were those extensive enough to result in organ failure and may have been life-threatening.

Statistics:

Descriptive statistics only (means and standard deviations) are reported for feed consumption, feed efficiency and white blood cell differential counts. Body weights, organ weights, clinical chemistry data, appropriate hematologic data and urinary specific gravity were evaluated by Bartlett's test for equality of variances. Based on the outcome of Bartlett's test, exploratory data analysis was performed by a parametric or nonparametric analysis of variance (ANOVA), followed respectively by Dunnett's test or the Wilcoxon Rank-Sum test with a Bonferroni correction for multiple comparisons. Statistical outliers were identified by a sequential test, but routinely excluded only from feed consumption and feed efficiency statistics. Outliers were not excluded from other analyses.

The nominal alpha levels used and test references are as follows:
Bartlett's test (Winer, 1971) α = 0.01
Parametric ANOV A (Steel and Torrie, 1960) α = 0.10
Nonparametric ANOVA (Hollander and Wolfe, 1973) α = 0.10
Dunnett's test (Winer, 1971) α = 0.05 two-sided
Wilcoxon Rank-Sum test (Hollander and Wolfe, 1973) α = 0.05 two-sided
(Bonferroni correction - Miller, 1966)
Outlier test (Grubbs, 1969) α = 0.02 two-sided

(continued in further information on materials and methods)

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
No treatment-related changes in the clinical appearance or behavior of animals ingesting XRD-498 were observed during the dosing period. All clinical changes noted were considered to be spontaneous in nature and consistent with the age and strain of rat used in the study or were related to accidental physical injuries.
Animals were observed to die from a number of disease states during the dosing period. Most early deaths occurred in male and female rats diagnosed as having Fischer rat leukemia. No statistically identified differences occurred between the mortality of male and female rats ingesting XRD-498 and controls. While male rats ingesting 500 mg/kg/ day XRD-498 and all treatment groups of females appeared to have died at a faster rate than controls during the last 3-4 months of the dosing period, overall, mortality lacked any dose-response over this or any other time period. Any differences observed in the mortality of dose groups were due to random variability and not ingestion of the test material.

BODY WEIGHT AND WEIGHT GAIN , FOOD CONSUMPTION AND FOOD EFFICIENCY
No significant changes in the body weights, feed consumption or feed efficiency of animals ingesting XRD-498 relative to control rats were observed during the dosing period.

HAEMATOLOGY
A slight, statistically identified decrease in RBC count and HCT was observed in female rats ingesting 1000 mg/kg/day XRD-498 for approximately 6 or 24 months relative to controls. However, these changes were not associated with histologic alterations in the bone marrow of affected animals were similar to or within historical control values for the laboratory and were not interpreted as treatment-related. No other relative differences in hematological parameters were noted in female rats nor in any parameters measured in males.

CLINICAL CHEMISTRY
A very slight, yet statistically identified, increase in serum CREAT levels was observed in male rats ingesting 1000mg/kg/day XRD-498 for 12 months or 2 years and in intermediate and/or high dose group females following 6, 18 and 24 months of dosing. In every case differences consisted of a single unit of measure (0.1 mg/ dl) and lacked any histopathological correlate. The TP and CHOL levels of high dose group males were also slightly lower than those of controls following 12 and 18 months of dosing, respectively. Finally, the level of TBILI was observed to be slightly decreased in high dose group males following 2 years of dosing. These latter changes in TP, CHOL and TBILI also lacked any histopathological correlates and, along with the observed changes in CREAT levels, were considered not to represent biologically or toxicologically significant alterations.

URINALYSIS
A statistically significant decrease in the specific gravity of the urine of male rats ingesting 1000 mg/kg/day XRD-498 relative to controls was observed following 6, 12 and 18 months of dosing. An increased amount of blood was also noted to be present in the urine of a number of these animals following 6 and 12 months dosing. Despite the lack of significant changes in urinary parameters following 2 years of dosing, these changes were considered to be treatment-related and were attributed in males to the presence of histologic changes in renal tissues (see below). A statistically identified deaease in urinary specific gravity was also noted in high dose group females, but only following 12 months of dosing. This latter change was not associated with any renal histopathologic changes and was not considered to represent an adverse treatment-related effect. No other significant changes were observed in any other parameter measured in either sex of treated rat.

ORGAN WEIGHTS
The absolute and relative weights of ceca, with or without ingesta, from high dose group male and female rats were elevated relative to controls following 12 and 24 months of dosing. The weights of full ceca from both sexes of rats, and of empty ceca from females, ingesting 500 mg/kg/day XRD-498 were also elevated at both time points. Cecal weight changes were not associated with any histologic change in cecal tissues. Rather, increases in cecal weights appeared to have resulted from a normal physiological adaptation to inaeases in cecal contents. The statistically-identified inaease in the absolute weights of empty ceca from female rats ingesting the 100 mg/kg/day XRD-498 following 12 months of dosing was due to the higher body weights of these animals relative to controls. No other relative changes in organ weights were observed.

GROSS PATHOLOGY
A number of high dose group male rats ingesting 1000 mg/kg/ day for 12 and 24 months (4/10 and 14/50, respectively) had a dilated renal pelvis(es) (bilateral and/or unilateral). Several high dose male rats were also observed to have sand-like calculi in the renal pelvis following 2 years of dosing. In addition, the ceca of a number of high dose group males and females and a few intermediate dose group rats were visibly enlarged following 12 and 24 months of dosing. All other gross pathologic observations were interpreted to be associated with spontaneous processes and were not treatment-related.

HISTOPATHOLOGY: NON-NEOPLASTIC
Histologic examination of tissues revealed treatment-related changes in the kidneys of male rats ingesting 1000 mg/kg/ day XRD-498 for 12 or 24 months. A few high dose group males were observed to have a minimal, focal necrosis and hyperplasia of the epithelium in the region of the renal pelvis and/or a minimal, focal necrosis of the renal papilla following 12 months dosing. Unilateral or bilateral atrophy of the renal papilla(e), hyperplasia of the epithelium overlying the papilla(e), and mineralization of the pelvic epithelium were observed in high dose group males following 2 years dosing. A single high dose group male and a single intermediate dose group male also had acute unilateral necrosis of the renal papilla following 2 years of dosing. However, this latter change was not statistically identified as being significantly different from controls and lacked dose-response. These factors plus the acute nature of this change following a chronic treatment of these animals with the test material do not suggest a treatment-related effect. No treatment related changes were observed in the kidneys of treated female rats.
No treatment-related changes were observed in the ceca of treated animals upon microscopic examination suggesting that any weight changes observed in intermediate and/or high dose group male and female rats were normal adaptive changes related to increased amounts of ingesta present in this organ.
In addition to the renal lesions observed in male rats, the incidence of a number of other histologic lesions found in treated groups of rats following 2 years of dosing were statistically identified as being different from control groups. The occurrence of these lesions was interpreted as not being treatment-related because they were:
1) decreased relative to controls (adrenal gland medullary hyperplasia, parasites of the colon and rectum, atrophy of pancreatic acini, mineralization of renal pelves - females);
2) secondary to other disease processes or alterations (atrophy of mesenteric adipose tissue, presence of ROC in sinusoids of the mediastinal lymph nodes); or
3) within the range of values normally occurring in similarly aged Fischer 344 rats in this laboratory,
lacked dose-response and/ or were observed to be of greater severity in lower dose group rats than in high dose group animals (atrophy of pancreatic acini, hepatic bile duct hyperplasia). The incidence of cataracts was also observed to be different between control and treated groups of rats, albeit not statistically identified. The incidence of this latter observation was also within historical control values and for these reasons was not interpreted to be biologically or toxicologically significant.

HISTOPATHOLOGY: NEOPLASTIC
No treatment-related increases in the incidence of tumors were observed in either sex of rats ingesting XRD-498. The incidence of pancreatic islet adenomas and of mammary fibroadenomas in high dose group male and female rats, respectively, were somewhat higher than in concurrent-control animals. However, these differences were not identified as being statistically significant and were well within historical control values of this laboratory.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

500 other: mg/kg bw/day (actual ingested)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Atrophy of the renal papilla(e), hyperplasia of the epithelium overlying the papilla(e) and/or mineralization of the renal pelvic epithelium

2

Dose descriptor:

NOAEL

Effect level:

1 000 other: mg/kg bw/day (actual ingested)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No dose related adverse effects observed.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Dosing

Analyses of the test material used to prepare test diets demonstrated that it was stable under the storage conditions used during the conduct of the study. The concentrations of XRD-498 found upon analysis of diets provided rats during the dosing period were in very close agreement with targeted concentrations of the test material. This fact, plus the periodic adjustment of the concentration of test material in the feed based upon the most recent feed consumption and body weight data, indicate that male and female rats were accurately administered the targeted dosages of XRD-498 during the study.

Applicant's summary and conclusion

Conclusions:

The primary target tissue of XRD-498 in the male Fischer 344 rat following chronic administration of the test material was identified as the kidney. Histopathological changes consisting of atrophy of the renal papilla(e), hyperplasia of the epithelium overlying the papilla(e) and/or mineralization of the renal pelvic epithelium were observed in approximately a third of male rats ingesting 1000 mg/kg/ day XRD-498 for up to 2 years. No treatment-related changes were observed in renal tissues of female rats ingesting a similar dosage of XRD-498. Increased cecal weights of male and female rats ingesting 500 or 1000 mg/kg/day XRD-498 were unassociated with any histopathological change in cecal tissues and were attributed to a physiological adaptation to increased levels of ingesta present in this organ. Decreases in urinary specific gravity of high dose group males, often accompanied by hematuria, following 6, 12 and 18 months of dosing appeared to be related to observed changes in renal tissues. However, the relatively minor differences noted in a few other parameters between treated and control groups of animals appeared to represent spontaneous events unassociated with treatment. No treatment-related tumorigenic response was observed in either sex of rats administered XRD-498.

The no-observed-adverse-effect level in male and female rats under the conditions of the study was 500 mg/kg/day and 1000 mg/kg/day XRD-498, respectively.

Executive summary:

Fischer 344 rats (50/sex/dose group) were provided diets formulated to provide 0, 100, 500 or 1000 mg/kg/day XRD-498 for up to two years. Satellite groups of 10 rats/sex/dose level were also dosed for approximately 12 months and then necropsied as an interim sacrifice group of animals. Data were collected on clinical appearance, body weight, feed consumption, hematologic parameters, clinical chemistry parameters, urinalysis parameters, organ weights and gross and histologic appearance of tissues.

No treatment-related changes were observed in the clinical appearance, mortality rates, body weights or feed consumption of treatment groups of rats relative to controls. Likewise, no clearly defined treatment-related changes were observed in hematologic and clinical chemistry parameters measured in treated rats. In contrast, decreases in the specific gravity of urine from high dose group males, occasionally accompanied by hematuria, following 6, 12 and 18 months of dosing appeared to be related to histologic changes in the kidneys of these animals.

The only significant treatment-related changes observed upon gross and histological examination of tissues were in the kidneys of high dose group male rats following 12 or 24 months dosing. A few of these animals were observed at necropsy to have a roughened surface in the papillary region of the kidneys and/or dilated renal pelves with or without calculi. Histologic examination of this tissue revealed atrophy of the renal papilla(e), hyperplasia of the epithelium overlying the papilla(e) and/or mineralization of the pelvic epithelium. Examination of tissues at necropsy also revealed that the ceca of a number of male and female rats ingesting 500 or 1000 mg/kg/day XRD-498 were visibly enlarged and were heavier than those of control rats. However, these latter changes were unassociated with any histopathological change in cecal tissues and were attributed to a physiological adaptation to increased levels of ingesta present in this tissue. No treatment-related tumorigenic response was observed in either sex of rats administered XRD-498.

The no-observed-adverse-effect level in male and female rats under the conditions of the study was 500 mg/kg/day and 1000 mg/kg/day XRD-498, respectively.