Registration Dossier

Diss Factsheets

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - guideline study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report date:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3150 (90-Day Oral Toxicity in Non-rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: M.A.F.F. Notification 12 Nousan, guideline No 8147 (2000)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): N-[[4-[(Cyclopropylamino) carbonyl]phenyl]sulfonyl]-2-methoxybenzamide
- Analytical purity: 97.4%
- Lot/batch No.: 08466/0013

Test animals

Species:
dog
Strain:
Beagle
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Marshal Farms USA, Inc, North Rose, New York, USA
- Age at study initiation: 9 months
- Weight at study initiation: 7.5 - 10.3 kg (males), 6.2 - 8.3 (females)
- Fasting period before study: not applicable
- Housing: Individually housed in stainless steel runs
- Diet: Purina Certified Canine Diet Etts 5006-3; presented to the animals for 3-6 hours/day, beginning 11 days prior to study initiation and continuing throughout the study. Prior to this time, food was vailable for ad libitum consumption.
- Water (ad libitum): Tap water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 29
- Humidity (%): 30 - 70
- Air changes (per hr): averaged 12.08
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: corn oil along with acteone
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): the test substance was mixed in the feed at the designated nominal concentrations.
- Storage temperature of food: under freezer conditions, until presented to the animals
A sample of each batch of feed mixed was taken and retained in the freezer until the study was completed.

VEHICLE
- Amount of vehicle (if gavage): corn oil, at 1% by weight of the diet, along with acetone, was used as a vehicle to suspend the test substance prior to being mixed in the diet. The control diet was prepared the same as the treated diet, excluding only the test substance.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
High performance liquid chromatographic/mass spectrometer/mass spectrometer method was used to measure the test substance concentrations in dog ration for the study. The homogeneity of the test substance in the diet and the stability of the active ingredient of the test substance in feed, stored at room temperature for 7 days and freezer temperature for 35 days, was determined: the test substance was found to be homogenously distributed in the feed and was stable at both room temperature and freezer conditions. The concentration of the active ingredient in the feed was verified for study weeks 1, 2, 3, 7, and 11 for all dose groups. The mean concentrations for the study were 97 to 98% of the nominal levels. %RSD (Relative standard deviation) values ranged from 3.65 to 5.98%.
Duration of treatment / exposure:
90 - 92 days
Frequency of treatment:
daily, 7 days/week
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
2500, 7500, 15000 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
75, 221, 416 mg/kg bw/ day (males)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
79, 221, 341 mg/kg bw/ day (females)
Basis:
actual ingested
No. of animals per sex per dose:
4
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: selection of these dose levels was based on a 28 day feeding study in the Beagle dog (M-254188-01-1, see chapter 7.5.1), which used doses of 3200, 10000, and 30000 ppm. In this previous study, there were microscopic lesions in the kidney of males and females in the 30000 ppm dose group and in males in the 10000 ppm dose group.
Positive control:
None

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily
- Cage side observations included: clinical signs of toxicity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at study initiation and weekly thereafter on all animals

BODY WEIGHT: Yes
- Time schedule for examinations: individual body weights were measured weekly throughout the study. Body weights were also taken immediately prior to necropsy to allow for calculation of organ-to-body weight ratios.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes, individual food consumption measured daily throughout the study.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: following the acclimation period and prior to initiation of dosing.
- Dose groups that were examined: ophthalmic examinations were conducted on all animals, and also on all animals sacrificed just prior to termination of the study.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: pre-exposure and during study weeks 5, 9, and 12.
- Anaesthetic used for blood collection: no, via jugular venipuncture
- Animals fasted: Yes, animals were fasted overnight prior to the collection of blood.
- How many animals: all animals.
- Parameters examined: Hematocrit (HCT), Hemoglobin (HGB), Leukocyte count (WBC), Erythrocyte count (RBC), Platelet count, Blood clotting measurements (Thromboplastin time, Prothrombin time), Leukocyte differential count, Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin conc. (MCHC), Mean corpuscular volume (MCV), Reticulocyte count, Blood cell morphology, Red Blood Cell Distribution (RDW), Hemoglobin Distribution Width (HDW)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: pre-exposure and during study weeks 5, 9, and 12.
- Animals fasted: Yes, animals were fasted overnight prior to the collection of blood.
- How many animals: all animals.
- Parameters examined: Electrolytes: Calcium (calc), Chloride (Cl), Phosphorus (Phos), Potassium (K), Sodium (Na); Enzymes: Alkaline Phosphatase (ALK), Creatine phosphokinase (CK), Lactic acid dehydrogenase (LD), Alanine aminotransferase (ALT/SGPT), Gamma Glutamyl transferase (GGT); Other: Albumin (ALB), Creatinine (Creat), Urea nitrogen (Urea-N), Total Cholesterol (Chol), Globulins (Glob), Glucose (gluc), Total bilirubin (T-Bili), Total protein (TP), Triglycerides (Trig), Uric Acid (Uric-A), Albumin/Globulin ratio (A/G)

URINALYSIS: Yes
- Time schedule for collection of urine: pre-exposure and during study weeks 5, 9, and 12
- Metabolism cages used for collection of urine: no data
- Animals fasted: No
- Parameters examined: Appearance, Volume (UVol), Specific gravity / osmolality (Sp.Gr.), pH, Sediment (microscopic), Protein (Pro), Glucose (Glu), Ketones (Ket), Bilirubin (Bil), Blood (Bld), Nitrite (Nit), Urobilinogen (Uro), Leukocytes (U-Leu)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. Animals were euthanised at the end of the study by intravenous injection of Fatal-Plus (Vortech Pharamceuticals, Dearborn, Michigan, USA). A complete gross examination was performed on all animals, consisting of a systematic gross examination of each animal's general physical condition, body orifices, external and internal organs and tissues. Organs/tissues examined: Cecum, Colon, Duodenum, Esophagus, Gall bladder, Ileum, Jejunum, Liver, Pancreas, Rectum, Salivary glands, Stomach, Larynx, Lung, Nasopharynx, Nose, Pharynx, Trachea, Aorta, Bone marrow, Heart, Lymph node (mesenteric, retropharyngeal), Spleen, Thymus, Cervix, Epididymides, Fallopian tube (oviduct), Kidneys, Mammary gland, Ovaries, Prostate, Testes, Ureter, Urinary bladder, Uterus, Vagina, Adrenal gland, Thyroid (with parathyroid), Brain (multiple sections), Eyes, Nerve (optic, sciatic), Pituitary, Sciatic nerve, Spinal cord (cervical, thoracic, lumbar), Bone (rib/cc jct, sternum), Gross lesions, Muscle, Physical Identifier, Skin
HISTOPATHOLOGY: Yes, target organs (i.e., kidney, ureter, and urinary bladder) and gross lesions from all dose groups were examined histologically, while all other tissues were examined histologically only from the control and high-dose groups. The physical identifier (i.e., ear tattoos) and vagina were not examined histologically. Tissues were processed routinely and stained with hematoxylin and eosin.
Other examinations:
Organ weight: Liver, Lung, Heart, Spleen, Thymus, Epididymides, Kidneys, Ovaries, Prostate, Testes, Uterus, Adrenal gland, Thyroids, Brain, Pituitary
Statistics:
Statistical significance was determined at p≤0.05 for all tests with the exception of Bartlett's test, in which a probability value of p≤0.001 was used. All tests were two-tailed, except for gross and histopathologic lesion evaluations which were one-tailed. Continuous data were analyzed by Bartlett's test for homogeneity. If the data were homogeneous an ANOVA was performed, followed by Dunnett's t-test on parameters showing a significant effect by ANOVA. If the data were non-homogeneous a Kruskal-Wallis ANOVA was performed, followed by the Mann-Whitney U-test to identify statistical significance between groups. Frequency data, that were examined statistically, were initially analysed by a Chi-Square procedure. If there was statistical significance using the Chi-square test, each treatment group was compared to the control group using a Fisher's Exact test.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
15000 ppm: one early, treatment-related, death of a female animal occurred on Day 35 of the study; 7500 ppm: compound-related decrease in urine specific gravity in females.
Mortality:
mortality observed, treatment-related
Description (incidence):
15000 ppm: one early, treatment-related, death of a female animal occurred on Day 35 of the study; 7500 ppm: compound-related decrease in urine specific gravity in females.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
15000 ppm: compound-related decrease in body weight in the females. No effect on the body weight of the males.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
15000 ppm: a compound-related reduction in animals of both sexes were observed.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
15000 ppm: high WBC in early death female animal; mild, but compound-related anemia in females
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
15000 ppm: elevated urea nitrogen and serum creatinine in both sexes; elevated serum phosphorus in females
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
15000 ppm: compound-related findings: elevated urine volume in males; decrease in urine specific gravity in both sexes; decreased pH in females
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
not compound-related
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
15000 ppm: compound-related effects: calculi in the renal pelvis and renal pelvis dilatation in both sexes; calculi in the ureter and urinary bladder of males; dilated ureters in females
Details on results:
CLINICAL SIGNS AND MORTALITY
15000 ppm: one early death occurred on Day 35 of the study. A female animal was sacrificed-in-extremis due to clinical signs of impending death. This death was considered to be treatment-related due to the kidney calculi and abscessation.
The only compound-related clinical sign was "thin", which was noted for two females near the end of the study and yellow powder in the urine of two males.
No treatment-related findings were observed in the low- and mid-dose group.

BODY WEIGHT AND WEIGHT GAIN
15000 ppm: A compound-related decrease in body weight in the females (weight gain of 458 g for the control group and a loss in weight of -874 g for the high-dose group during the study). No effect on body weight for the males was observed.
The termination body weights were statistically decreased in the high-dose group females and considered to be compound-related (decreased 27% compared to controls).
No treatment-related findings were observed in the low- and mid-dose group.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
15000 ppm: A compound-related reduction in food consumption for males and females was observed (-14 and -15% for males and females, respectively, compared to controls).
No treatment-related findings were observed in the low- and mid-dose group.

HAEMATOLOGY
In one early death high-dose group female on study Day 35, very high WBC (57.8 x 10³/mm³) was observed and thought to be due to kidney changes described microscopically (abscess).
Furthermore, a mild anemia was observed in the females of the 15000 ppm dose group, that was characterized by statistically significant decrease in erythrocyte counts (5.18 vs. 7.05 x 10E+06/mm³ in controls), hemoglobin (12.5 vs. 16.9 g/dL in controls), and hematocrit (38 vs. 48.1% in controls) at termination and was thought to be compound-related.
There were no compound-related changes in male dogs in any treatment group. There were some statistically significant differences in MCV (73.4 vs. 68.2 µm³ in controls), MCHC (32.9 vs. 35.1 g/dL in controls) in high-dose group females and in RDW (11.9 vs. 12.9% in controls), HDW (1.57 vs. 1.73 g/dL in controls) in high-dose males at the end of the treatment period, but these findings were not considered to be biologically significant.
No treatment-related findings were observed in the low- and mid-dose group.

CLINICAL CHEMISTRY
15000 ppm: Elevated urea nitrogen in both sexes at one month, two month, and termination (35 vs. 17 and 85 vs. 16 mg/dL in males and females, respectively, compared to controls at termination) was observed. Additionally, elevated serum creatinine in males and females was noted at one month, two month, and termination (1.6 vs. 1 and 2.8 vs. 1 mg/dL in males and females, respectively, compared to controls at termination). Serum phosphorus was increased in females at termination (7.3 vs. 3.6 mg/dL in controls). These effects were assumed to be compound-related.
No treatment-related findings were observed in the low- and mid-dose group.

The few scattered statistically significant changes in clinical chemistry parameters that were not considered to be compound-related since the changes were small, not dose-dependent, and/or not biologically significant were chloride, alkaline phosphatase, total protein, albumin, phosphorus (mid-dose group), and globulin in females and total bilirubin, gamma glutamyltransferase, and total protein in males.

URINALYSIS
15000 ppm: Elevated urine volume (non-statistical) at one-month, two-month, and term intervals in male dogs was observed (815.3 vs. 137 mL in controls at termination). A decrease in urine specific gravity in both sexes at termination was also noted (about 2%). Furthermore, a decreased pH in the high-dose group females at one- (6.2 vs. 8.4 in controls) and two-month (6.3 vs. 9 in controls) intervals was observed.
7500 ppm: In females, a decrease in urine specific gravity was observed (about 2%).
No treatment-related findings were observed in the low-dose group.

ORGAN WEIGHTS
15000 ppm: The absolute brain (62.6 vs. 69.1 g) and heart (47.2 vs. 64.5 g) weights were statistically significant decreased in females but the relative organ weights were not affected (brain: 0.842% of bw vs 0.888% of bw; heart: 0.841% of bw vs 0.845% of bw), so this was thought to be due to the decreased body weights and therefore not compound-related. The thymus weight was reduced in three of four high-dose group female dogs (absolute organ weights: 8.529 g vs 3.936 g; relative organ weights: 0.112% of bw vs 0.065 % of bw).
No treatment-related findings were observed in the low- and mid-dose group.

GROSS PATHOLOGY
Compound-related changes were all limited to the urinary system of the animals of the 15000 ppm dose group:
1. Calculi in the renal pelvis of males and females (M: 0, 0, 0, 3; F: 0, 0, 0, 4 (gender, 2500, 7500, 15000 ppm))
2. The renal pelvis was dilated in one dog of each sex (M: 0, 0, 0, 1; F: 0, 0, 0, 1 (gender, 2500, 7500, 15000 ppm)).
3. Calculi were seen in the ureter and urinary bladder of one of four males.
4. Ureters were dilated in one of four female dogs.
No treatment-related findings were observed in the lo - and mid-dose group.

HISTOPATHOLOGY: NON-NEOPLASTIC
15000 ppm: Compound-related changes were noted in the urinary system of both male and female dogs. The inflammatory changes, renal pelvis epithelial hyperplasia and necrosis and associated changes in the ureters and urinary bladder correlated with the gross observations at necropsy of calculi in the kidneys, ureters and urinary bladder.
No treatment-related findings were observed in the low- and mid-dose group.

Numerous incidental microscopic observations were noted in control and treated dogs. The thymic atrophy and involution in high-dose group females were due to the statistically significant body weight decrease and not compound-related, as it was not present in other dose groups or any male groups. Minimal mineralization of the renal papilla of the kidney was noted in most control and treated dogs.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
221 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: corresponding to 7500 ppm for males and females
Dose descriptor:
LOAEL
Effect level:
416 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Clinical signs, body weight changes, food consumption, clinical chemistry, urinalysis, gross pathology, histopathology; corresponding to 15000 ppm
Dose descriptor:
LOAEL
Effect level:
341 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Clinical signs, body weight changes, food consumption, clinical chemistry, urinalysis, gross pathology, histopathology; corresponding to 15000 ppm

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion